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Targeting Underlying Inflammation in Carcinoma Is Essential for the Resolution of Depressiveness | 412ac2da-3d0f-4572-b520-4b82115a944f | 10000718 | Internal Medicine[mh] | Treating patients with cancer is challenging. In modern clinical practice and research on behavioral changes in patients with oncologic problems, there are several one-sided approaches to this problem. Oncologists are concerned in great detail with localization of the primary oncologic process, pre- and post-operative care, protocols for chemotherapy and radiation therapy, and monitoring for recurrence. However, it is quite common for psychiatrists to be involved in some of the phases of integrative treatment. Mental predisposition could be discussed in the etiology of various carcinomas . Mental disturbances could be a consequence of the patient’s awareness of the illness onset and its possible impact on the patient’s overall quality of life, or they may follow somatic perturbations and be an impact of the various therapies applied . Mental disorders could also induce cancer recurrence . In the new therapeutic strategies for the treatment of neuropathic pain as an oncological complication, it was very interesting to draw a parallel between the changes in the acute and chronic phases of pain and mental disorders management . Inflammatory processes, both acute and chronic, are a hallmark of both oncological and mental disorders . The exacerbation of somatic disorders and mental illnesses could reflect acute inflammation, whereas prolonged processes are related to chronic inflammation . The question is if these landmarks could induce depressive symptomatology, and if so, to what extent and whether these phenomena should be treated as simple non-comorbid depression. In the up-to-date literature, there are a lot of useful pointers on the relationship between carcinoma and inflammation and between depression and inflammation. This review article aimed to compare and integrate these complex interactions in the same context of carcinoma and depression comorbidity. Further, we will try to use this information to potentially improve the clinical approach and discuss the importance of the resolution of inflammation as a new treatment strategy in the cooccurrence of carcinoma and depression.
Inflammation represents the systemic host response to tissue damage. It is usually caused by injury, ischemia, infection, or chemical exposure . Additionally, inflammation plays an important role in tissue repair, regeneration, and remodeling . The inflammatory response involves the recruitment and action of the immune response . Inflammation occurs in two stages, acute and chronic inflammation . Acute inflammation is a part of innate immunity initiated by immune cells and lasts for a short time . It serves as a defense against infection, tissue damage, and allergens. Receptors of innate immunity recognize the structures of microorganisms (pathogen-associated molecular patterns—PAMPs), but also molecules that are released from damaged host cells . These molecules are called danger-associated molecular patterns (DAMPs) and represent proteins or nucleic acids that are not normally found outside the cell. The most important DAMPs include chromatin-associated protein high-mobility group box 1 (HMGB1), adenosine triphosphate (ATP), uric acid (UA), deoxyribonucleic acid (DNA), and degraded extracellular matrix (ECM)-like heparan sulfate and hyaluronan. PAMPs and DAMPs are recognized via pattern recognition receptors (PRRs) . The term “alarmin” is today used as a synonym for DAMP . In acute inflammation, pro-inflammatory mediators such as acute-phase proteins, prostaglandins, leukotrienes, oxygen- and nitrogen-derived free radicals, chemokines, growth factors, and cytokines that are released by immune defense cells locally at the site of inflammation cause neutrophil infiltration . C-reactive protein (CRP), fibrinogen, and procalcitonin (PCT) are part of an innate immune response that is detectable in serum within a few hours of the initiation of inflammation . They facilitate the inflammatory process and represent hallmarks of acute inflammation . Subsequently, other cells of innate and adaptive immunity (e.g., macrophages and lymphocytes) are recruited to the inflammatory environment . In response to DAMPs, innate immune cells secrete cytokines that mediate normal cellular processes and communication between leukocytes and other cells, but also regulate the host’s response to damage . Cytokines can exert proinflammatory and anti-inflammatory effects both locally and systemically . Activated cells of innate immunity produce the most important proinflammatory cytokines: interleukin (IL)-1, tumor necrosis factor-alpha (TNF-α), IL-6, IL-12, and IL-23 . Conversely, the cells of adaptive immunity—activated T lymphocytes—produce interferon-gamma (IFN-γ) and IL-17 . Some cytokines, such as IL-1α and IL-33, act as alarmins . They are released from host cells as a result of injury or death and subsequently mobilize and activate immune cells . The resolution of acute inflammation begins when PAMPs and DAMPs are no longer present . However, if the pathogen cannot be completely eradicated or there is a constant source of self-antigens or a growing tumor that continuously disrupts tissue structure and induces the production of inflammatory cytokines, the second stage of inflammation, chronic inflammation, occurs . Long-lasting chronic inflammation can lead to many chronic diseases including cardiovascular, respiratory, neurodegenerative diseases, and cancer via dysregulation of various signaling pathways . When Rudolf Virchow described leukocytes within primary tumor tissue, a possible link between inflammation and cancer was established in the 19th century . Today, it is obvious that inflammation plays an important role in the biology of tumors. Inflammation may play an anti- or pro-tumorigenic role. Acute inflammation in neoplastic tissues is indicative of an anti-tumor immune response . In chronic inflammation, the inflammatory microenvironment facilitates cell mutations and proliferation leading to tumor development . Alteration of several signaling pathways may contribute to the development of genetic and epigenetic changes in local tissue cells . Additionally, chronic inflammation attenuates anti-tumor immunity and affects cell proliferation, death, senescence, DNA mutation, and angiogenesis . The question remains whether the inflammation is a consequence of the anti-tumor immune response or whether the tumor arose in the setting of chronic inflammation.
Pro-inflammatory peripheral biomarkers elevation, a higher risk of depression in inflammatory and autoimmune diseases, the ability of immune mediators to induce depressive symptoms, and the fact that activated microglial cells reduce levels of serotonin and generate oxidative stress (OS) molecules all point to immune system involvement in the pathogenesis of depression . Blood-brain barrier (BBB) permeability, the brain-gut axis, and the brain-fat axis bring systemic, particularly inflammatory, changes into the spotlight, not just central nervous system (CNS) disturbances in depression . Specific depressive symptomatology was explored in correlation with inflammatory changes in the periphery. Majd et al. (2020) conducted a narrative review and indicated that there is an association between neurovegetative symptoms of depression, such as sleep problems, fatigue or loss of energy, appetite changes, and inflammation . Increased inflammatory markers were measured in patients with major depressive disorder: IL-1β, IL-6, TNF-α, and CRP. Peripheral inflammation could signal the brain by leaky regions in the BBB, the cytokine transport system, and the vagus nerve. They based their conclusions on Capuron et al. (2002) , who demonstrated that IFN administration causes neurovegetative symptoms in the first two weeks, which are less responsive to antidepressant therapy, and depressed mood and cognitive symptoms later, which are responsive to antidepressants. Among other prominent theories of depression, the cytokine theory has played an important role in clinical practice . Cytokines and peripheral immune cell counts could serve as biomarkers for distinct subgroups of inflamed depression and direct further treatment . As recently noted in coronavirus disease (COVID-19), acute inflammation could be followed by behavioral changes termed “sickness behavior”, the resolution of which follows eradication of the infection, although in some cases psychotropic medications are required to resolve mental symptoms, particularly agitation . It seems that some individuals have a predisposition to an exaggerated immune response to an infectious agent that could be harmful, not protective, and also lead to a later onset of depression . The peripheral immune response is particularly exacerbated in depressive patients that are resistant to antidepressants . Resilient animals do not display exacerbated immune responses following acute and chronic stress, suggesting that positive affectivity could buffer the negative impact of stress on immunity . This hypersensitivity could be linked to the role of IL-6 as an important marker. A recently published first meta-analysis with a robust sample reported an adjusted association between IL-6 and future depression . In addition, a small prospective association between depression and IL-6 was observed in both directions. If inflammation is prolonged and chronic, it is important to consider whether symptoms meet the threshold for a diagnosis of a depressive episode and require treatment. However, the elevation of IL-6 may be associated not only with chronic inflammation but also with other pathological processes that may also be observed in depression .
The estimated high prevalence of depression in cancer patients and the insufficient data on the mechanisms by which tumors per se may alter brain functions, including mood and cognition, have engaged the preclinical research community to search for novel cancer-induced models. The main advantage of using animal models in research is the control of confounding variables that are difficult to control in the clinical setting and the ability to unravel mechanistic interactions between neural, immune, and inflammatory processes through which tumors alter brain function. Animal models provide a better explanation for the independent impact of tumor-associated biological processes on affective and cognitive symptoms, independent of cancer-associated stress and treatments. Significant behavioral changes were found in mice with implanted tumors, characterized primarily by an increase in avoidance behavior and a decrease in immobility, defensive-submissive behavior, and non-social exploration . Changes in brain plasticity as a result of disturbed neural redox homeostasis were detected in the brains of tumor-bearing mice with depressive-like behavior . Structural evidence for a depressive-like state induced in a model of mammary cell carcinoma was also observed through decreased dendritic branching of pyramidal neurons in the medial prefrontal cortex . Lipopolysaccharide is a component of gram-negative bacteria commonly used to induce a potent inflammatory response and behavioral changes that rapidly resolve within 24 h, followed by hyperalgesia . Cytokine production in the tumor microenvironment can be detectable in the general circulation of experimental models of various tumor types, as well as in brain areas responsible for mood regulation. These studies reported increased plasma levels of IL-6, IL-12, TNF-α, IL-10, and IL-1β, but also increased IL-1β, IL-10 expression of IL-1β mitochondrial ribonucleic acid in the cortex and hippocampus, and increased levels of IL-6 and TNF-α in the hippocampus . Hippocampal inflammation was related to depressive-like behavior in breast cancer mice, and also gastric-cancer-bearing mice with a significant increase in IL-6, IL-1β, reactive oxygen species (ROS), and cyclooxygenase-2 (COX2) . The model of chronic stress and smoke exposure induced depression-like behavior and lung cancer, respectively, in mice, with the synergistic effect in a combined model manifested through a more prominent inflammatory response . However, the impact of antidepressant fluoxetine was significantly attenuated under the conditions of chronic stress and LPS-induced inflammation, suggesting the role of chronic inflammation in the development of treatment-resistant depression . We could identify several important underlying cascades in the development of depressiveness induced by inflammation. Activation of inflammasomes, particularly nod-like receptor family pyrin domain containing 3 (NLRP3), may occur through DAMPs or PAMPs mediated by toll-like receptors (TLRs) and subsequently activate important intracellular pathways such as IFN I and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) . At the cellular level, repercussions of these processes could be the production of IL-1α, IL-1β, TNF-α, and IL-6, as well as the activation of microglia and the impairment of astrocytes in depression .
Somatic illnesses could be followed by mental disturbances, or mental disorders could be a typical response to illness that vanished in reconvalescence with the illness resolution or could persist after somatic illness recovery . Hart was the first to propose the concept that “sickness behavior” occurs as a short-term reaction in an acute inflammatory state and is crucial for the survival of the individual . However, when inflammation becomes chronic, as in autoimmune diseases, neurodegenerative diseases, cardiovascular diseases, diabetes and obesity, and cancer, mood symptoms predominate and can worsen the disease. Nearly 30% of cancer patients meet the criteria for a psychiatric diagnosis of depression, neurotic and stress-related disorders, adjustment disorders, sleep disorders, or delirium . The problem of insomnia is very pronounced in patients in the active and stable phase of cancer, especially when associated with a pain syndrome and distress . With regard to the onset and persistence of depressive symptomatology, it was very interesting for us to consider the overlap with pain and fatigue as symptoms of the cluster, presented as two or three concurrent and interrelated symptoms that may or may not have a common etiology and pathophysiological pathways . Dodd et al. (2001) defined pain, fatigue, and insomnia in cancer patients as a cluster . Recently, Charalambous et al. (2019) provided preliminary evidence that targeting fatigue, anxiety, and depression in patients with breast and prostate cancer may have a meaningful effect on pain as a related symptom . A proposed underlying mechanism in the pathogenesis of these symptoms includes systemic inflammation with high pro-inflammatory cytokine levels, oxidative stress, and neuroendocrine-immune alterations . Inflammation-mediated tryptophan catabolism along the kynurenine pathway might contribute significantly to the development of fatigue and depression in cancer patients . Consideration of the common neuroimmune mechanisms of chronic pain and depression and the possible corrective anti-inflammatory effect of antidepressants seem to be of greater importance in this case . Therefore, researchers have developed a model of inflammatory cytokine activity in cancer to explain the co-occurrence of pain, fatigue, and sleep disturbances (summarized in ). Sometimes it is necessary to remember that the primary goal is to eliminate pain sensations to prevent the onset of depressive symptoms. Functioning could be especially compromised with pain sensations that are also correlated with ongoing inflammation . Acute pain was also associated with acute inflammation, and chronic inflammation reflected chronic pain . Chronic pain and depression in humans are associated with persistent low-grade inflammation rather than severe systemic inflammation, with only a partially common underlying mechanism . Neuropathic pain has been shown to be associated with increases in the tryptophan-metabolizing enzyme indolamine 1,3 deoxygenase (IDO1) in the liver but not in the brain, and antagonism of the N-methyl-D-aspartate (NMDA) receptor by kynurenic acid . On the contrary, co-morbid depression was mediated downstream of spinal cord IL-1β signaling and the formation of kynurenine and its metabolites in the brain . Along with anxiety and depression, cancer-related fatigue is one of the most common symptoms in cancer patients . Fatigue and depression have similar clinical presentations . Fatigue can occur independently, be a prodromal symptom of depressive disorders, or be part of a developed depression . Fatigue is defined as a loss of energy that can affect physical, mental, or cognitive functioning and is manifested by loss of motivation, apathy, and reduced concentration and attention . The above symptoms are important characteristics of depressive mood disorder. For these reasons, it is sometimes very difficult in clinical practice to distinguish whether it is just fatigue or depression. Recently, our research group has pointed out that acute and chronic inflammation have a significant impact on fatigue and depression in patients with the inflammatory and neurodegenerative disease multiple sclerosis. We observed that peripheral inflammation was related to fatigue and postulated that brain inflammation in acute episodes could further lead to neurodegeneration and mood and cognitive changes . The new important clinical entity of paraneoplastic disorder should be considered in the context of the clinical field of autoimmune-mediated depression . Paraneoplastic neurologic syndromes (PNSs) are rare cancer-related diseases that can affect any level of the central and peripheral nervous systems . These disorders do not result from tissue invasion by the tumor, metastases, or metabolic or toxic effects of cancer therapy . PNSs are caused by an immune response directed toward neural self-antigens aberrantly expressed by neoplastic cells and marked by specific autoantibodies . Although PNSs can occur in any type of tumor, the most frequently associated malignancies include ovarian and breast cancer, small-cell lung cancer, thymoma, Hodgkin’s lymphoma, and neuroendocrine tumors . The exact immunopathogenic mechanisms for most paraneoplastic syndromes are still unclear. The autoimmune theory postulates an immune cross-reaction between antigens expressed by tumor cells (“onconeural” antigens) and neurons . The autoimmune response, initially directed against tumor cells, results in further damage to neurons that physiologically express the same antigen . The target of the immune attack can be intracellular antigens (anti-Hu, anti-Yo, anti-Ma2, anti-Ri, GAD), antigens on synaptic receptors (NMDA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, γ-aminobutyric acid receptor) or ion channels, and other cell-surface proteins (LGI 1, GQ1b) . The main effector of the immune response in PNSs associated with antibodies directed against intracellular antigens is the CD8 + cytotoxic T cell, whose action results in rapid and extensive neuronal death by cytotoxic activity . Mild signs of inflammation are commonly detected in the cerebrospinal fluid in the early phases of these disorders . Antibodies against plasma membrane antigens, such as ion channels and surface receptors, may play a pathogenic role as direct effectors in neural tissue injury. Mechanisms by which these antibodies affect the targeted cells include antigen internalization and degradation, activation of complement cascades, antibody-dependent cell-mediated cytotoxicity, and blockade of receptor function . Paraneoplastic syndromes of the CNS can be present with neuropsychiatric and cognitive symptoms, abnormal movements, new-onset epilepsy, and sleep disorders . Over the past decade, evidence has accumulated of an intriguing relationship between cancer and neurodegenerative diseases. Progression of both conditions is primarily defined by a set of molecular determinants that are complementarily dysregulated or share important underlying biological mechanisms that promote cell proliferation and apoptosis, including alarmins (discussed in detail in ). DNA, cell cycle aberrations, redox imbalance, inflammation, and immunity are closely associated with shared characteristics of cancer and neurodegenerative diseases. The question arises whether each depressive episode and these kinds of repeated excessive immune and autonomic dysregulation could also contribute to neurodegeneration.
The basic mechanism of action of conventional therapy for malignant diseases, such as radiotherapy and chemotherapy, is to induce the death of tumor cells . However, the process of tumor cell necrosis is often triggered as an accompanying phenomenon in addition to the desired apoptosis. Necrosis is followed by the release of cellular contents outside the cell. Thus, endogenous alarmins reach the intercellular space and become inducers and facilitators of inflammation . In this way, therapeutically induced tumor necrosis may be beneficial to the host . Therefore, another no less important mechanism of action of the therapy is the induction of inflammation and the strengthening of the antitumor immune response. New therapeutically induced tumor necrosis may benefit the innate antitumor immune response, as necrotic cells facilitate the maturation of antigen-presenting cells . Mature antigen-presenting cells, especially dendritic cells, induce a potent acquired antitumor response. Thus, the increase in systemic values of proinflammatory cytokines of innate immunity is accompanied by an increase in values of cytokines of acquired immunity. Chronic inflammation is present in and around most tumors, including those not causally related to an inflammatory process . The percentage of patients with inflammatory components in the tumor microenvironment varied from 28% to 63% depending on tumor type . Anti-tumor therapy is usually followed by a wave of acute inflammation that changes the intensity and course of the antitumor immune response . Although radiotherapy and chemotherapy are options for the treatment of cancer, other treatments are increasingly being explored today, such as immunotherapy . The use of monoclonal antibodies, immunomodulatory agents, modulated immunocompetent cells, or blocking antibodies for checkpoint molecules has shown significant results in cancer therapy and has fundamentally changed the approach to cancer therapy . The discovery of checkpoint molecule inhibitors was awarded the Nobel Prize . The blockade of cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and programmed cell death protein 1 (PD1) molecules with antibodies is now very topical and has also found its application in clinical practice . Research on blocking other checkpoint molecules such as T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), the cluster of differentiation 96 (CD96), natural killer receptor NKG2A is in full swing . A strong effect of the application of this type of therapy is the enhancement of both innate and acquired antitumor immune responses . This phenomenon is almost always accompanied by increased production of pro-inflammatory cytokines and momentum of inflammation in the host. These effects could be unwanted in the propagation of inflammation and consequently trigger depressive symptomatology. Since alterations of various cytokines have been established in both depression and cancer, cytokine inhibitors deserve more detailed discussion. Infliximab, a TNF antagonist, improves depressive symptomatology by decreasing CRP levels but has also shown beneficial effects in treating cancer-related fatigue . Adalimumab, another TNF-α-specific neutralizing monoclonal antibody similar to infliximab, has been shown to significantly improve depressive symptomatology in patients with various chronic diseases , but without studies in psychiatric patients. Etanercept, another TNF-α antagonist, reduced depressive-like behavior in preclinical models, but also clinical studies in patients with psoriasis and rheumatoid arthritis . Pentoxifylline, a methylxanthine drug that acts as a strong non-selective TNF-α inhibitor, has improved depressive behavior in animal models but has also shown positive results as an add-on treatment for depression . Ustekinumab, an inhibitor of IL-12 and IL-23, dupilumab, an antagonist of the receptor of IL-4, ixekizumab, an IL-17A inhibitor, and guselkumab, an IL-23 inhibitor, have all been for their antidepressant action . Although cytokine inhibitors have a more targeted effect on depression-related inflammation, these results were limited to specific patient groups. Because cytokine inhibitors are large molecules, they cannot cross the BBB, suggesting that their anti-inflammatory action is limited to peripheral TNF-α. This does not preclude their efficacy, but further studies are needed to determine their potential for treating depression in the presence of concomitant carcinoma. Conversely, re-establishing balance in the peripheral secretion of cytokines is observed after antidepressant use and the resolution of depression. The most recent pharmacological protocols for the treatment of depression in carcinoma target monoamine neurotransmitters, brain-derived neurotrophic and inflammatory factors, and glutamate and its receptors, using monoamine oxidase inhibitors, tricyclic drugs, selective serotonin reuptake inhibitors (SSRIs) and selective serotonin noradrenaline reuptake inhibitors (SNRIs), glutamatergic drugs, opioids, and benzodiazepines . In vitro, SSRIs have been shown to inhibit the release of TNF-α and NO from activated microglia, impede calcium ion influx, decrease the activation of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, and also reduce inflammatory changes . SSRIs and SNRIs decrease blood and tissue cytokines and regulate complex inflammatory pathways of NF-κB, inflammasomes, TLR4, and peroxisome proliferator-activated receptor gamma (PPAR-γ) . Liu et al. (2020) showed in their systematic review and meta-analysis that patients with depression who responded to treatment had lower baseline levels of the chemotactic factor for neutrophils IL-8 than non-responders . In addition, treatment with antidepressants decreases TNF-α and IL-5 levels. However, long-term treatment with SSRIs has been postulated to increase Th1 and decreases Th2-derived cytokines . Celecoxib, nonsteroidal anti-inflammatory drugs, minocycline, but also statins, polyunsaturated fatty acids, pioglitazone, modafinil, corticosteroids, the vitamin D2 analog i.e., paricalcitol, etc. have already been reported as classical anti-inflammatory drugs with consequent antidepressant effects . Celecoxib, a selective COX-2 inhibitor, exerts anti-depressive action by decreasing IL-6 expression and/or levels . Minocycline, the second-generation tetracycline antibiotic, can cross the BBB more efficiently than other tetracycline antibiotics. It has anti-inflammatory, antioxidant, and neuroprotective effects within the CNS by preventing the release of inflammatory cytokines such as IL-6 and TNF-α . It also inhibits neutrophil migration, degranulation, oxygen-free radical production, and NO release. Statins, known as lipid-lowering agents, have shown anti-inflammatory potency by decreasing levels of CRP and low-density lipoprotein (LDL) cholesterol, TNF-α and IFN-γ production in stimulated T cells, but also by reducing immune activation of T-helper cells . Pioglitazone, primarily used as an antidiabetic drug, acts as a PPAR-γ agonist and decreases the expression of IL-1β, IL-6, TNF-α, inducible nitric oxide synthase (iNOS), and chemoattractant protein-1 (MCP-1/CCL2) . It ameliorates depression-like behaviors by inducing the neuroprotective phenotype of microglia . The psychostimulant modafinil reduces brain inflammation by impacting monocyte recruitment and activation, T cell recruitment and differentiation, cytokine production, and glial activation . Corticosteroids, known for their anti-inflammatory properties, have also been studied for their antidepressant properties . Because of their various side effects, which depend on their dosage and duration of treatment, they should be used with caution . Paricalcitol, a vitamin D2 analog, regulates microglia-mediated neuroinflammation via decreased production of IL-1-β, inhibition of NF-κB and NLRP3 signaling, and caspase-1 overexpression . In examining the link between depression and cancer, numerous experimental studies have revealed that activation of the kynurenine pathway of tryptophan degradation due to inflammation plays an important role in the evolution and persistence of both diseases . The Hamilton group study showed that a history of depression, anxiety, and fear of tumor recurrence was associated with greater use of complementary treatment approaches . The supplements most commonly used by patients are selenium (Se), folic acid, and omega-3 fatty acids . Cancer patients often turn to antioxidants; among them, Se is particularly interesting, either from an inorganic source (sodium selenate) or the amino acid (selenomethionine) . However, it is questionable whether its action can be considered exclusively as an antioxidant because it can also act as an oxidant and exhibit an anticarcinogenic effect . Due to its antioxidant effect, Se is suitable as a supplement in depressive states and is an essential trace element for thyroxine metabolism. Thus, Se deficiency lowers antioxidant protection of the brain and may lead to brain damage—the turnover of dopamine and serotonin increases, while it decreases for noradrenaline and 5-hydroxy-3-indoleacetic acid compared to controls . The role of folic acid is reflected in the synthesis of serotonin, and its supplementation is advised for patients with depression . However, at high doses of folic acid, an adverse action may be observed because its role in metabolism controls the potential proliferative action for cancer cells . Other widely used supplements are omega-3 fatty acids, predominantly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). They influence optimal cell structure/function and affect synaptic neurotransmission . Therefore, they are recommended for complementary therapy in depression . Improvements are also expected from fatty acid supplementation in chemotherapy and radiotherapy, as they affect inflammation, apoptosis, eicosanoid synthesis, etc. . New therapeutic approaches may include drug-supporting/delivery systems as well as assorted supplements. Among the various supplements, zeolites are in the spotlight. There are a number of zeolite-associated positive effects reported in the literature: antioxidant and anticancer performance, ion exchange, and adsorption/encapsulation features, to name a few . These aluminosilicates can be of synthetic or natural origin, such as clinoptilolite, and are recognized for human application . Interestingly, synthetic zeolites can be designed to meet the specific demands of drug carrier systems and seem to be a far better choice, but are still awaiting general pharmaceutical recognition. Some therapeutic approaches may benefit from zeolite use, i.e., sustained drug-delivery systems, which are considered to be improved therapeutic pathways compared to regular ones . Over the past two decades, researchers have competed to find ideal carriers, exploring a possible synergistic effect between the selected support and the drug itself . There are several reasons for this—firstly, a specially designed carrier of nanometric dimensions must be considered to sustain BBB pass . To meet this requirement, animal testing is set forward with some interesting applications. For example, infrared-activated BBB permeability may be accomplished by utilizing zeolitic imidazolate-based nanocomposite for intracerebral quercetin delivery providing neuroprotective effects . Furthermore, the zeolite platform must encompass enough functional centers to efficiently adsorb/encapsulate drugs. Thus, zeolitic composites were proposed for synergetic tumor thermo-chemotherapy using doxorubicin drug delivery that sustains tumor reduction . In the field of mental disorders, zeolite testing is under-explored with the majority of studies employing only animal models. One way to treat induced bipolar disorder in rats with probiotic cultures, alone and in zeolite-supported formulations, was suggested by Alchujyan et al. . Interestingly, probiotics expressed a positive effect on arginase/nitric oxide synthase activities without significant benefits of zeolite carrier, as both formulations led to beneficial histopathological brain alterations and subsequent behavioral progress in rats. Several reports suggest that recovery of cancer patients can be promoted by zeolite supplementation . This hypothesis is based on zeolites’ excellent adsorption capacity for histamine which may be regarded as beneficial for pain relief . In vitro and in vivo experiments on zeolite frameworks safety are extensively studied, while others investigated double-blinded trials of oral clinoptilolite intake in cancer patients to treat peripheral neuropathy induced by chemotherapy . As reported by Vitale et al., the neuropathy extent was quite similar, occurring in 70.6% and 64.3% of patients in the placebo and zeolite supplementation groups, respectively . Bearing in mind the good adsorption properties of zeolites, their role in the removal of heavy metals is often mentioned in the context of the prevention of mental disorders. A prospective use of zeolite/ethylenediaminetetraacetic acid as a lead scavenger is reported , confirming the role of clinoptilolite in reducing neurotoxicity in mice. Another removal of lead addresses issues with autism spectrum disorder . Injection of zeolite particles is proposed with the possibility of stool excretion after metal adsorption, with no analysis of the detrimental effect zeolite nanoparticles could have on the hematological and gastrointestinal region. As a multifunctional material, Y zeolite is applied as an electrode support for the ruthenium ammine complex in the electrochemical detection of dopamine/serotonin . Extending this system toward zeolite’s possible interaction with L-dopa, as a dopamine precursor may be sound due to several hydrogen bonds that can be formed. However, this emerged as a premise for rising dopamine levels, which is challenging to test/confirm . Expectedly, these propositions are left with only hypothetical opinions.
Immune system alteration is the common denominator of depression and cancer. Additionally, alterations in the immune response seem to overlap in both pathological conditions. The psychiatric correlates are followed by immune disturbances, and we still wonder to what extent the resolution of inflammation in carcinoma might simultaneously contribute to the resolution of the associated depressive symptomatology. Recognition of acute mediators of inflammation is very important, and it is even more important to prevent the transition from acute to chronic inflammation through early anti-inflammatory interventions. The alarmins induce local (central) inflammation by TLR signaling and facilitating NF-κB transcriptional activity and NLRP3 inflammasome in neuronal and nonneuronal cells. Thus, pro-inflammatory cytokines produced in the periphery could activate inflammation in the brain and subsequently modulate the release and function of neurotransmitters, leading to the onset of depression. Previous clinical investigations have shown that the cytokines IL-1, IL-6, IFN-γ, and TNF-α play key roles in these processes. These same cytokines are among the major mediators of the anti-tumor immune response and the chronic inflammation that usually accompanies it. Hypersensitivity and chronification of inflammation suggest an exhausted and insufficient immune response. In conclusion, peripheral inflammation could trigger central immune-inflammatory pathways that lead to pain, fatigue, and depressive symptomatology in patients with cancer. Cancer treatment strategies, as well as conventional psychotropic drugs, could help balance the inflammatory milieu. The new equilibrium in both conditions may be achieved by variously targeted anti-inflammatory strategies. Anti-inflammatory drugs are well known, but new possible pathways and challenging add-on therapies have yet to be found.
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Cytological Samples: An Asset for the Diagnosis and Therapeutic Management of Patients with Lung Cancer | 98873e9a-130b-44b1-9558-aa645382e242 | 10001120 | Anatomy[mh] | Lung cancer has become the leading cause of cancer death for men and women . The management of this cancer has evolved over the last decade with the emergence of new therapies, such as tyrosine kinase inhibitors and immunotherapy. Therefore, patient samples must allow for both the diagnosis and molecular testing, as well as PD-L1 quantification. As patients are often diagnosed at an advanced stage , pathologists must use samples carefully and appropriately, as the diagnosis is no longer the only result needed for patient management. Cytological samples have a place in the management of patients with pulmonary mass, especially those with an advanced disease for whom surgery is not a therapeutic option. These patients with advanced diseases account for 45% of patients diagnosed with lung cancer . The last version of the World Health Organization (WHO) classification granted a section entirely dedicated to cytology in lung cancer, showing the importance of these samples in this context . In this article, we report the ability of cytological samples to perform the diagnosis of lung cancer and to obtain critical results, such as molecular profile and PD-L1 expression, which are essential for the therapeutic management.
2.1. Samples Collection This study included cytological samples in which suspicious cells were observed and where immunocytochemistry was performed to characterize these cells. The samples were collected between January 2021 and September 2022 in the Cell Biology Laboratory (Timone Hospital, Assistance Publique des Hôpitaux de Marseille, Marseille, France). The different types of these cytological samples were pleural, pericardial, and peritoneal effusions, bronchoalveolar lavage fluids, endobronchial ultrasound guided transbronchial needle aspiration (EBUS-TBNA) lymph nodes, EBUS-TBNA mediastinal or pulmonary mass, cerebrospinal fluid, and bone marrow aspiration. Samples were not initially fixed and were kept at 4 °C until slide preparation (smears or cytospin). Slides were stained with Papanicolaou and May-Grünwald–Giemsa stains. The conventional cytological diagnosis was performed by the Cell Biology Laboratory. PD-L1 testing was performed by the Anatomopathology Laboratory (Assistance Publique des Hôpitaux de Marseille, Marseille, France). The next generation sequencing (NGS) was performed by the Oncobiology Laboratory (Assistance Publique des Hôpitaux de Marseille, France). Samples included in this study were obtained from patients attending the Assistance Publique des Hôpitaux de Marseille for diagnosis and treatment. Results of the molecular testing and clinical data were retrospectively analyzed. This project was approved by the local ethics committee (PADS22-389). 2.2. Immunocytochemistry on Cytospins to Phenotype Tumor Cells Samples were prepared on cytospins as previously described in , following the manufacturer’s instructions. As a minimum, one wash was performed between each step. Slides were fixed with paraformaldehyde (PAF) 4% for 10 min, and then incubated with the peroxidase-blocking solution for 30 min. After being washed, slides were incubated with SensiTEK HRP kit (ScyTek Laboratories, Logan, UT, USA) for 10 min. Primary antibodies were incubated for 30 min (see for the list of primary antibodies). Then, the biotinylated secondary antibody was incubated for 15 min, followed by Streptavidin/HRP for 20 min and DAB Quanto chromogen (Diagomics, Blagnac, France) for 5 min. Nuclei were counterstained with Mayer’s hemalun solution. Slides were mounted with Aquatex ® (Merck Millipore, Darmstadt, Germany). Slides were observed under optical microscope (Leica, Wetzlar, Germany). Mouse isotype IgG and rabbit polyclonal antibodies were used as negative controls as part of best practice method. 2.3. Immunohistochemistry on Cytoblock for PD-L1 Expression Cytoblocks were prepared to perform PD-L1 testing. Cytological samples were fixed with formalin 4% for 6 h then centrifugated for 5 min at 670 g. The supernatant was discarded. The cytoblock TM kit (Epredia, Kalamazoo, MI, USA) was used to prepare cytoblocks following the manufacturer’s instructions. A slide stained with H&E was systematically performed before PD-L1 immunostaining to confirm the cytoblock quality and evaluate the adequate number of tumor cells. PD-L1 immunostaining (QR001, Quartett, Germany) was performed with the optiview DAB detection Kit on Benchmarck Ultra (Ventana, Roche, Bale, Switzerland). A positive control was systematically performed as part of best practice method. 2.4. Next Generation Sequencing NGS was performed from frozen cell pellets as previously described . In short, total nucleic acids were extracted with the Maxwell RSC Cell DNA Kit (Promega, Madison, WI, USA) and RNAs were extracted with the Maxwell RSC Simply RNA Blood Kit (Promega). The detection of mutations and fusions were performed by NGS on the Ion Torrent S5XL (ThermoFisher, Waltham, MA, USA) with a custom panel Oncomine Solid Tumor and Oncomine Solid Tumor+ (OST/OST+) and Oncomine Focus RNA assay kit (ThermoFisher, Waltham, MA, USA) (see for the fusion transcript panel and the mutation transcript panel). Ion Torrent Suite, Ion Reporter software (ThermoFisher, Waltham, MA, USA) and a pipeline developed in our laboratory were used for the interpretation of the results.
This study included cytological samples in which suspicious cells were observed and where immunocytochemistry was performed to characterize these cells. The samples were collected between January 2021 and September 2022 in the Cell Biology Laboratory (Timone Hospital, Assistance Publique des Hôpitaux de Marseille, Marseille, France). The different types of these cytological samples were pleural, pericardial, and peritoneal effusions, bronchoalveolar lavage fluids, endobronchial ultrasound guided transbronchial needle aspiration (EBUS-TBNA) lymph nodes, EBUS-TBNA mediastinal or pulmonary mass, cerebrospinal fluid, and bone marrow aspiration. Samples were not initially fixed and were kept at 4 °C until slide preparation (smears or cytospin). Slides were stained with Papanicolaou and May-Grünwald–Giemsa stains. The conventional cytological diagnosis was performed by the Cell Biology Laboratory. PD-L1 testing was performed by the Anatomopathology Laboratory (Assistance Publique des Hôpitaux de Marseille, Marseille, France). The next generation sequencing (NGS) was performed by the Oncobiology Laboratory (Assistance Publique des Hôpitaux de Marseille, France). Samples included in this study were obtained from patients attending the Assistance Publique des Hôpitaux de Marseille for diagnosis and treatment. Results of the molecular testing and clinical data were retrospectively analyzed. This project was approved by the local ethics committee (PADS22-389).
Samples were prepared on cytospins as previously described in , following the manufacturer’s instructions. As a minimum, one wash was performed between each step. Slides were fixed with paraformaldehyde (PAF) 4% for 10 min, and then incubated with the peroxidase-blocking solution for 30 min. After being washed, slides were incubated with SensiTEK HRP kit (ScyTek Laboratories, Logan, UT, USA) for 10 min. Primary antibodies were incubated for 30 min (see for the list of primary antibodies). Then, the biotinylated secondary antibody was incubated for 15 min, followed by Streptavidin/HRP for 20 min and DAB Quanto chromogen (Diagomics, Blagnac, France) for 5 min. Nuclei were counterstained with Mayer’s hemalun solution. Slides were mounted with Aquatex ® (Merck Millipore, Darmstadt, Germany). Slides were observed under optical microscope (Leica, Wetzlar, Germany). Mouse isotype IgG and rabbit polyclonal antibodies were used as negative controls as part of best practice method.
Cytoblocks were prepared to perform PD-L1 testing. Cytological samples were fixed with formalin 4% for 6 h then centrifugated for 5 min at 670 g. The supernatant was discarded. The cytoblock TM kit (Epredia, Kalamazoo, MI, USA) was used to prepare cytoblocks following the manufacturer’s instructions. A slide stained with H&E was systematically performed before PD-L1 immunostaining to confirm the cytoblock quality and evaluate the adequate number of tumor cells. PD-L1 immunostaining (QR001, Quartett, Germany) was performed with the optiview DAB detection Kit on Benchmarck Ultra (Ventana, Roche, Bale, Switzerland). A positive control was systematically performed as part of best practice method.
NGS was performed from frozen cell pellets as previously described . In short, total nucleic acids were extracted with the Maxwell RSC Cell DNA Kit (Promega, Madison, WI, USA) and RNAs were extracted with the Maxwell RSC Simply RNA Blood Kit (Promega). The detection of mutations and fusions were performed by NGS on the Ion Torrent S5XL (ThermoFisher, Waltham, MA, USA) with a custom panel Oncomine Solid Tumor and Oncomine Solid Tumor+ (OST/OST+) and Oncomine Focus RNA assay kit (ThermoFisher, Waltham, MA, USA) (see for the fusion transcript panel and the mutation transcript panel). Ion Torrent Suite, Ion Reporter software (ThermoFisher, Waltham, MA, USA) and a pipeline developed in our laboratory were used for the interpretation of the results.
3.1. General Results Between January 2021 and September 2022, 259 cytological samples containing cells suspected of malignancy were analyzed by immunocytochemistry to characterize the type and origin of cancer. Immunocytochemistry allowed for the characterization of the type of cancer in 248 cases (95.7%). In 59 samples (mostly pleural and peritoneal effusions), the immunocytochemistry confirmed the malignancy but with another origin than lung (for example, ovarian, breast, colorectal or pancreatic carcinoma, mesothelioma, neuroblastoma, lymphoma, or melanoma). Concerning the 189 samples with lung cancer, lung adenocarcinoma was diagnosed in 106 cases, followed by non-small cell lung cancer not-otherwise specified (NSCLC NOS) (44 cases), squamous cell carcinoma (20 cases) and neuroendocrine tumors (19 cases), including large cell neuroendocrine carcinoma (3 cases), small cell lung cancer (15 cases), and 1 case of carcinoid tumor (see and ). In 11 cases, samples contained cells that were suspected to be malignant, but the immunocytochemistry did not confirm the malignancy, either because the sample was too necrotic or because the samples contained a low number of tumor cells (<1% of total cells). Among the 189 samples diagnosed with lung cancer, 72 (38.1%) were pleural effusions, 71 (37.5%) were lymph nodes collected by EBUS-TBNA, 29 (15.3%) were mediastinal or pulmonary masses collected by EBUS-TBNA, 6 (3.2%) were bronchoalveolar lavage fluids (BAL), 6 (3.2%) were pericardial effusions, 2 (1.1%) were cerebrospinal fluids (CSF), 2 (1.1%) were peritoneal effusion, and one (0.5%) was bone marrow. Patients were mostly diagnosed at stage IV (75.1%) and were current or former smokers (73%) . 3.2. Molecular and PD-L1 Results PD-L1 was performed on cytoblocks from 115 cytological samples. No results could be reported in 29 cases (25.2%) because there were less than 50 tumor cells. Of the 86 samples that could be analyzed, PD-L1 expression was found negative (<1%) in 22 cases (25.6%), between 1 and 49% in 34 cases (39.5%) and ≥50% in 30 cases (34.9%). Examples of these are shown in . For 35 patients, the cytoblock was not performed because no sample remained after immunocytochemistry and NGS testing, or because the sample was too necrotic. For 20 patients, the cytoblock was available but PD-L1 was not performed on the cytoblock as it had already been done on a biopsy or on the resected tumor. Next generation sequencing (NGS) was performed on 140 out of 150 cases (93%) diagnosed with lung adenocarcinoma or NSCLC NOS. EGFR mutation was found in 20 cases (14.2%), of which 12 received a tyrosine kinase inhibitor. The other 8 patients were not at stage IV, or were treated with the best supportive care, or the treatment was not known. ALK fusions were found in 4 (2.8%) cases and ROS1 fusions in 2 (1.4%) cases. All these patients with ALK or ROS1 fusions were treated with appropriate tyrosine kinase inhibitors. A mutation in TP53 was found in 67 cases (47.8%), 46 of which had another associated mutation. KRAS mutation was found in 48 cases (34.3%), of which 29 had another associated mutation. Other mutations were found as HER2 , PIK3CA , STK11 , RET , BRAF , DDR2 , CTNNB1 , SMAD4 , PTEN and POLE . The absence of mutation or fusion was found in 19 cases (13.6%). 3.3. Improvement of Diagnosis and Therapeutic Management with Cytological Samples We analyzed the impact of cytology results on the management of 189 patients with lung cancer: For 124 (65.6%) cases, the result was conducive to the diagnosis and allowed for therapeutic management. Among them, 37 cases had a biopsy and a cytological sample during the same procedure: the same diagnosis was obtained on biopsy and cytology for 24 patients, while for 13 patients, the biopsy was free of tumor cells and the diagnosis was only performed on cytology. In 3 cases, a biopsy was recommended after cytology because they were necrotic or because there was not enough material to perform the NGS. For 40 (21.2%) patients already known to have lung cancer, a cytological sample was performed in the context of suspected lung cancer progression (demonstrated by imaging). The presence of tumor cells in the cytological sample confirmed the progression and led to a change of therapeutic line. For 19 (10.0%) cases, cytology results had no impact on the therapeutic management. In most of these cases, the metastatic site was already known (pleural, peritoneal, or pericardial) and the cytological analysis was performed because the effusion had to be drained. In other cases, the clinical status deteriorated rapidly, and the patient died within a few days. In 6 (3.2%) cases, no information was available. According to our data, the results obtained from cytological samples allow a therapeutic management for 87% of patients with lung cancer.
Between January 2021 and September 2022, 259 cytological samples containing cells suspected of malignancy were analyzed by immunocytochemistry to characterize the type and origin of cancer. Immunocytochemistry allowed for the characterization of the type of cancer in 248 cases (95.7%). In 59 samples (mostly pleural and peritoneal effusions), the immunocytochemistry confirmed the malignancy but with another origin than lung (for example, ovarian, breast, colorectal or pancreatic carcinoma, mesothelioma, neuroblastoma, lymphoma, or melanoma). Concerning the 189 samples with lung cancer, lung adenocarcinoma was diagnosed in 106 cases, followed by non-small cell lung cancer not-otherwise specified (NSCLC NOS) (44 cases), squamous cell carcinoma (20 cases) and neuroendocrine tumors (19 cases), including large cell neuroendocrine carcinoma (3 cases), small cell lung cancer (15 cases), and 1 case of carcinoid tumor (see and ). In 11 cases, samples contained cells that were suspected to be malignant, but the immunocytochemistry did not confirm the malignancy, either because the sample was too necrotic or because the samples contained a low number of tumor cells (<1% of total cells). Among the 189 samples diagnosed with lung cancer, 72 (38.1%) were pleural effusions, 71 (37.5%) were lymph nodes collected by EBUS-TBNA, 29 (15.3%) were mediastinal or pulmonary masses collected by EBUS-TBNA, 6 (3.2%) were bronchoalveolar lavage fluids (BAL), 6 (3.2%) were pericardial effusions, 2 (1.1%) were cerebrospinal fluids (CSF), 2 (1.1%) were peritoneal effusion, and one (0.5%) was bone marrow. Patients were mostly diagnosed at stage IV (75.1%) and were current or former smokers (73%) .
PD-L1 was performed on cytoblocks from 115 cytological samples. No results could be reported in 29 cases (25.2%) because there were less than 50 tumor cells. Of the 86 samples that could be analyzed, PD-L1 expression was found negative (<1%) in 22 cases (25.6%), between 1 and 49% in 34 cases (39.5%) and ≥50% in 30 cases (34.9%). Examples of these are shown in . For 35 patients, the cytoblock was not performed because no sample remained after immunocytochemistry and NGS testing, or because the sample was too necrotic. For 20 patients, the cytoblock was available but PD-L1 was not performed on the cytoblock as it had already been done on a biopsy or on the resected tumor. Next generation sequencing (NGS) was performed on 140 out of 150 cases (93%) diagnosed with lung adenocarcinoma or NSCLC NOS. EGFR mutation was found in 20 cases (14.2%), of which 12 received a tyrosine kinase inhibitor. The other 8 patients were not at stage IV, or were treated with the best supportive care, or the treatment was not known. ALK fusions were found in 4 (2.8%) cases and ROS1 fusions in 2 (1.4%) cases. All these patients with ALK or ROS1 fusions were treated with appropriate tyrosine kinase inhibitors. A mutation in TP53 was found in 67 cases (47.8%), 46 of which had another associated mutation. KRAS mutation was found in 48 cases (34.3%), of which 29 had another associated mutation. Other mutations were found as HER2 , PIK3CA , STK11 , RET , BRAF , DDR2 , CTNNB1 , SMAD4 , PTEN and POLE . The absence of mutation or fusion was found in 19 cases (13.6%).
We analyzed the impact of cytology results on the management of 189 patients with lung cancer: For 124 (65.6%) cases, the result was conducive to the diagnosis and allowed for therapeutic management. Among them, 37 cases had a biopsy and a cytological sample during the same procedure: the same diagnosis was obtained on biopsy and cytology for 24 patients, while for 13 patients, the biopsy was free of tumor cells and the diagnosis was only performed on cytology. In 3 cases, a biopsy was recommended after cytology because they were necrotic or because there was not enough material to perform the NGS. For 40 (21.2%) patients already known to have lung cancer, a cytological sample was performed in the context of suspected lung cancer progression (demonstrated by imaging). The presence of tumor cells in the cytological sample confirmed the progression and led to a change of therapeutic line. For 19 (10.0%) cases, cytology results had no impact on the therapeutic management. In most of these cases, the metastatic site was already known (pleural, peritoneal, or pericardial) and the cytological analysis was performed because the effusion had to be drained. In other cases, the clinical status deteriorated rapidly, and the patient died within a few days. In 6 (3.2%) cases, no information was available. According to our data, the results obtained from cytological samples allow a therapeutic management for 87% of patients with lung cancer.
In this study, we used cytospin specimens for staining and immunocytochemistry and cytoblocks for PD-L1 testing. The methods of cytological preparation each have their advantages and disadvantages. For example, cytoblocks can be compared to biopsies, while cytospin preparation provides good morphology that can easily be used for immunocytochemistry. Regardless of the method selected, rigorous quality controls are essential . Studies report that small biopsies and cytological samples can account for up to 70% of specimens for the diagnosis of lung cancer . In our study, immunocytochemistry on cytospin allowed to determine the type of cancer in 248 of the 259 cytological samples in which suspected tumor cells were observed. For the remaining 11 samples, the immunocytochemistry did not allow to characterize the cells either because of the quality (necrotic or too much altered cells), the lack of volume (e.g., cerebrospinal fluid), or the low number of suspected tumor cells (<1% of total cells). Despite this, the classification of tumor cells was successful in over 95% of cases. This rate shows the value of cytological samples for identifying tumor cells. Our results are consistent with other studies evaluating the ability of cytological samples to diagnose lung cancer . For example, Rekhtman et al. compared 192 pre-operative cytology specimens with histology and found a concordance of 96%. Proietti et al. assessed the efficacy of lung cancer subtyping in cytology and biopsy samples from 941 patients and found a concordance in 92.8% of cases. Arnold et al. conducted a prospective study to investigate the role of cytology in pleural effusions. They included 921 pleural effusions with 166 lung cancer and 100 lung adenocarcinomas. The sensitivity for the diagnosis of lung cancer was 56% and 82% for adenocarcinoma . Others studies found similar results for the detection and the characterization of tumor cells in pleural effusion . In the last decade, the emergence of immune checkpoint inhibitors and targeted therapies have modified the way in which cytological samples are managed. In addition to the diagnosis, the sample must allow for the assessment of PD-L1 expression and molecular testing. PD-L1 expression is a biomarker that predicts which patients are more likely to respond to immunotherapy. Immunotherapy can be prescribed in first line monotherapy for patients with advanced NSCLC and with ≥50% PD-L1 expression, and in second line therapy for metastatic NSCLC patients with ≥1% PD-L1 expression . In this context, evaluation of PD-L1 expression is essential on cytological samples . This test can be challenging; it needs an adequate protocol and quality controls . For example, macrophages and mesothelial cells must be properly recognized to avoid counting them in the percentage of PD-L1 expressing cells. Cytoblocks are the most commonly used material for analysis and provide concordant results compared to biopsies, even if smears can also be used . A recent multicenter study including 264 patients concluded that PD-L1 expression on cytological samples correctly predicts the efficacy of immunotherapy . In our study, PD-L1 expression was tested only on 115 cytoblocks. When PD-L1 status has already been determined for a patient on biopsy or surgical specimen, the analysis was not performed again on cytological sample. Molecular testing must be performed for patients with advanced NSCLC as several oncogenic drivers are targetable. The International Association for the Study of Lung Cancer (IASLC) recommends to test EGFR mutations and ALK and ROS1 fusions. HER2 , RET , MET , BRAF , and KRAS are not indicated as a routine stand-alone assay but may be included in a large molecular testing panel . In accordance, the European Society for Medical Oncology (ESMO) recommends the use of NGS that includes at least EGFR common mutations, ALK fusions, MET mutations, BRAF mutations, and ROS1 fusions . The absence of formalin in cytological samples facilitates molecular testing to be applied as NGS or polymerase chain reaction (PCR) . Molecular testing on cytological samples has the advantage of providing results even if the sample volume or the number of tumor cells is low . In our study, 93% of patients with lung adenocarcinoma or NSCLC had NGS results. Among them, only 13.5% did not show a molecular alteration in genes included in the tested panel. We have previously demonstrated the feasibility of detecting ALK and ROS1 fusions from cytological samples either by immunocytochemistry completed by fluorescence in situ hybridization (FISH) if positive, or by NGS, with high concordance of both techniques . ALK and ROS1 fusions are now routinely performed by immunocytochemistry on cytological samples as part of diagnosis results . Rekhtman et al. showed the feasibility of testing for EGFR and KRAS mutations in thoracic cytology . In our study, the NGS is performed on the frozen cell pellets. The supernatant from post-centrifuge liquid based cytology can also be used for NGS . The two main criteria that prevent all techniques (i.e immunocytochemistry, molecular testing and PD-L1) from being performed are low sample volume or the low representativeness of tumor cells. Regarding sample volume, pathologists should inform clinicians that the larger the volume sent to the laboratory, the more adequate the sample will be for diagnosis and additional testing. Particularly for effusions (pericardial, pleural, and peritoneal) where the puncture can evacuate up to several liters, the pathologist can receive only one or two milliliters. Adequacy of a standardized volume vary depending on the cellularity and the percentage of tumor cells. Currently, no standardized volume requirements exists, but several studies recommend 50 mL of fluid . Dalvi et al. recommend at least 20 mL but demonstrated that the tumor cell proportion is critical for assessing diagnosis and molecular analysis . The low number of tumor cells is a reason to perform a biopsy and obtain an accurate material for a new test. With a low number of tumor cells detected (1–5%), immunocytochemistry and NGS can potentially be interpreted . But PD-L1 interpretation requires at least 100 tumor cells, otherwise pathologists are unable to obtain a result.
Over the last decade, major therapeutic advances in the treatment of lung cancer, with the introduction of targeted therapies and immune checkpoint inhibitors, have forced pathologists to change their practice and use cytological samples differently. Diagnosis alone is no longer enough and the pathologist must keep a portion of the sample to perform PD-L1 analysis and molecular testing. Cytological samples are obtained by minimally invasive procedures and can provide enough material for the diagnosis and the therapeutic management in patients with lung cancer.
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A Novel Technique of Amniotic Membrane Preparation Mimicking Limbal Epithelial Crypts Enhances the Number of Progenitor Cells upon Expansion | c0b49748-feb6-4135-9fa1-15fdb55b159c | 10001367 | Anatomy[mh] | The homeostasis of the dynamic cellular organization in the cornea mainly depends on the regenerative efficiency of the stem cells in the surrounding limbus . Tissue-specific human limbal epithelial stem cells (hLESCs) residing in the limbal epithelial crypts of the palisades of Vogt continuously compensate for the loss of superficial human corneal epithelial cells (hCECs) . The insufficient compensation of diminished hCECs in the corneal epithelium due to the lack or malfunction of hLESCs leads to severe ocular surface disease or so-called limbal epithelial stem cell deficiency (LSCD) . The hLESCs play an essential role in epithelial differentiation, angiogenesis, and extracellular matrix (ECM) organization . Diverse therapeutic approaches have been used to treat both monocular and binocular LSCD. However, cultivated limbal epithelial stem cell transplantation (CLET) of expanded autologous limbal tissue seems to be the most common method for monocular LSCD . The CLET procedure is based on isolating the limbal biopsy from the contralateral eye and treatment with a proteolytic enzyme to digest the surrounding ECM, which helps the hLESCs get released and migrate from the niche. Furthermore, the digested limbal tissue or single isolated hLESCs are harvested ex vivo in a medium containing stem-cell-supporting growth factors and supplements, achieving cell expansion and graft tissue synthesis . Upon transplantation, hLESCs reside on the damaged corneal-limbal tissue, re-creating the limbal stem cell niche that allows epithelial regeneration. Following the existing standard protocols, the reported success rate of CLET varies. A favorable morphological outcome implying stable, intact, completely epithelized and avascular corneal surface is reported as 46.7% to 80.9%, whereas success as a functional outcome such as visual acuity varies from 60.5% to 78.7% . Successful transplantation is directly dependent on the graft tissue quality and the percentage of hLESCs/early progenitor cells in the graft. For successful transplantation, at least 3% of the cells in the expanded cell culture must express the p63 marker . Therefore, establishing a protocol that provides a high percentage of the hLESCs/early progenitor cells in the transplantation graft is of high importance. Human amniotic membrane (HAM) has proven to be a very efficient therapeutic tool in many ocular surface diseases, supporting wound healing and regeneration while suppressing inflammation , angiogenesis , and fibrosis , and it possesses anti-microbial features . It is used for corneal epithelial regeneration, conjunctival reconstruction, glaucoma interventions, and the treatment of corneal melting and perforations. Importantly, it is one of the most used carriers for the ex vivo expansion of hLESCs . HAM contains stem cell niche factors that support maintenance . Generally, such maintenance depends on the inhabitance of the stem cells in a specific niche that allows their anchoring and communication with supporting cells, the release of specific growth factors and cell cycle molecules, and the involvement of evolutionary conserved molecular pathways. Within the niche, the stem cells undergo symmetric or asymmetric division to transient amplifying cells (TACs) that leave the environment and become functionally mature corneal cells . It is known that the physical cues of the cellular environment guide stem cell fate . It is also suggested that biomechanical changes in the limbal stromal niche affect hLESCs fate . No less importantly, mechanical and environmental changes in the corneal tissue have implications for some corneal diseases and pathologies . We hereby present a novel suturing preparation technique that causes the three-dimensional (3D) radial folding of the HAM, mimicking crypt-like formations. The novel approach may allow the hLESCs, upon limbal biopsy expansion and cultivation ex vivo, to reside in the undulated crypts of HAM. This may potentially maintain the putative characteristics of the expanded hLESCs and thus ensure a higher quality of the expanded graft tissue compared to the conventional state-of-the-art method. Therefore, we aimed to compare the progenitor/differentiation state of the cells cultivated in the crypt-like HAMs vs. the cells cultivated on the flat-like HAMs.
The Regional Committee for Medical and Health Research Ethics in South-Eastern Norway (No 2017/418) approved tissue harvesting and laboratory procedures, and all tissue collections complied with the Guidelines of the Helsinki Declaration. Unless stated otherwise, all reagents were purchased from Merck (Darmstadt, Germany). 2.1. Human Amniotic Membrane (HAM) A Placenta was collected after a scheduled cesarian section from a full-term pregnancy. Informed consent and institutional board review approval had previously been obtained from the patient. According to the standard protocol, the placenta was immediately transported in a sterile container and further processed under sterile conditions . Proper washing with 0.9% NaCl (Fresenius Kabi AB, Uppsala, Sweden) or 0.9% NaCl containing 100 U/mL Penicillin, 100 μg/mL Streptomycin (P4333), and 2.5 μg/mL Amphotericin B (A2942) was repeatedly performed. The HAM was then separated from the chorion by blunt dissection, washed out from residual blood, and transferred onto a nitrocellulose filter carrier, pore size 0.45 μm (111306-47-CAN, Sartorius, Gottingen, Germany) with the epithelial side up, then divided into 3 × 3 cm and 5 × 5 cm pieces. HAM pieces were cryopreserved in 50% glycerol, 48.5% DMEM/F12 (31331028, Invitrogen, Carlsbad, CA, USA), 100 U/mL Penicillin, 100 μg/mL Streptomycin and 2.5 μg/mL Amphotericin B and stored at −80 °C. 2.2. HAM Preparation for hLESC Expansion and Cultivation Before use, HAMs were thawed, warmed to room temperature, and washed three times with a medium containing DMEM/F12, 100 U/mL Penicillin, and 100 μg/mL Streptomycin. Thereafter, the HAMs were placed on polyester membrane Netwell TM inserts (3479, Corning Inc., New York, NY, USA) 24 mm in diameter, with the epithelial side up by two different techniques : (1) HAMs 3 × 3 cm ( .1A) in size were peeled from the nitrocellulose filter paper. Further, HAMs were placed and stretched on the top of the polyester membrane and sutured by the eight individual sutures ( .1B) near the edge of the polyester membrane. The HAMs were then tightly stretched on the top of the membrane, making a flat surface ( .1C). Excess HAM tissue that remained at the edge of the polyester membrane was carefully removed with a disposable sterile scalpel. (2) The other approach was to use HAMs 5 × 5 cm in size ( .2A) placed on top of the membrane and sutured so that the HAMs were loosely attached. HAMs were sutured by individual sutures ( .2B). In addition, an individual suture was placed in the center of the HAM/polyester membrane to obtain the folding of the HAM and to keep it in close contact with the membrane ( .2C). The excessive HAM tissue at the edges was again removed accordingly. The sutured HAMs on the Netwell TM inserts were immersed in DMEM/F12 medium containing 100 U/mL Penicillin and 100 μg/mL Streptomycin and kept at 37 °C, 5% CO 2 , and 95% air overnight to obtain HAM free of any glycerol remains. 2.3. Limbal Biopsies and Human LESC Harvesting Following corneal transplantation, the remaining human corneal-scleral rings from three donors (n = 3) were divided into twelve limbal biopsies of equal size and thoroughly washed with DMEM/F12 medium containing 100 U/mL Penicillin and 100 μg/mL Streptomycin. The biopsies were treated with neutral protease and Dispase II (2.4 U/mL 4942078001, Roche Diagnostics, Mannheim, Germany) for 10 min at 37 °C. The dissociation process was blocked using Fetal Bovine Serum (FBS, F2442). The limbal biopsies were then placed centrally on the top of the HAMs, with the epithelial side down and submerged in a standardly used complex medium (COM). COM consisted of DMEM/F12, Penicillin (100 U/mL), Streptomycin (100 μg/mL), Amphotericin B (2.5 μg/mL), human epidermal growth factor (2 ng/mL, E9644), insulin (5 μg/mL), sodium selenite (5 ng/mL) and transferrin (5 μg/mL, l1884), cholera toxin A subunit from Vibrio cholerae (30 ng/mL, C8180), hydrocortisone (0.03 μg/mL, H0888), 5% FBS, and 0.5% dimethyl sulfoxide (DMSO, D2650). After 2 h of incubation, the attached limbal biopsies were completely covered by COM. Further, the limbal biopsies and outgrowing LESCs were harvested and incubated at 37 °C with 5% CO 2 , and 95% air for the following three weeks. The culture medium was changed every three days. 2.4. Immunohistochemistry (IHC) and Immunofluorescence Microscopy Limbal biopsies with hLESCs cultured on HAMs were cut from the Netwell TM inserts. Samples were fixed in 4% formalin overnight at 4 °C and then processed in dehydrated graded alcohol series of 70% (10–15 min), 80% (10–15 min), 96% (2 × 10 min), and 100% ethanol (2 × 10 min) before having xylene added (3 × 10 min) and being washed with melted paraffin (3 × 10 min) and then embedded in paraffin for immunohistochemistry (IHC). Paraffinized tissue was cut into 3–4 μm thick sections using an automated microtome (HM 355S, Thermo Fisher Scientific, Waltham, MA, USA) and attached to histological slides. Deparaffinization was performed in xylene (2 × 10 min), then rehydration was performed by sinking in 100%, 96%, and 70% ethanol, and then distilled water. Hematoxylin & Eosin (H&E) staining was primarily performed. Slides were immersed in Mayers hematoxylin plus solution (01825, Histolab, Askim, Sweden) for 10 min and then rinsed with distilled water (10 min) followed by eosin staining (10 min), and then they were rehydrated in upgraded alcohol series of 70%, 96%, 100% ethanol, and xylene. Slides were further mounted using Pertex (00840, Histolab) mounting medium. For IHC, heat-induced antigen retrieval was performed in a microwave for 5 min at 900W and 15 min at 600W in a citrate buffer (pH 6, C9999) or by PT module (LabVision, Fremont, CA, USA). Blocking of non-specific binding sites with 5% Bovine Serum Albumin (BSA, A9418) dissolved in Dulbecco’s Phosphate Buffered Saline (DPBS, 14190-144, Thermo Fisher Scientific) was conducted for 20 min. Further, slides were stained with primary antibodies diluted in 1% BSA for 1 h. Slides were stained using antibodies for the following progenitor markers: tumor protein p63 alpha (p63α, rabbit polyclonal, 1:200 dilution, 4892S, Cell Signaling, Beverly, MA, USA), SRY-Box Transcription Factor 9 (SOX9, 82630, Cell Signaling, rabbit monoclonal, 1:200), quiescence marker: CCAAT/enhancer-binding protein delta (CEBPD, rabbit polyclonal, 1:200 dilution, ab198320, Abcam, Cambridge, UK), and proliferation marker Ki-67 (rabbit monoclonal, 1:200, RM-9106-S, Thermo Scientific) and the following differentiation markers: cytokeratin 3/12 (KRT3/12, mouse monoclonal, 1:100 dilution, 08691431, MP biomedicals, Santa Ana, CA, USA) and connexin-43 (CX43, rabbit polyclonal, 1:300, C6219). Then, the slides were thoroughly washed three times for 5 min with PBS-tween buffer (28352, Thermo Fisher Scientific). Incubation was continued with the appropriate animal type of secondary antibody: Cy3 ® goat anti-rabbit IgG (rabbit monoclonal, 1:500 dilution, A10520, Abcam) for samples stained with p63α, CEBPD, SOX9, Ki67, and Alexa Fluor ® 488 donkey anti-mouse IgG (1:500 dilution, mouse monoclonal, 21202, Abcam) for samples stained with KRT3/12, and Alexa Fluor ® 488 donkey anti-rabbit IgG (1:500 dilution, rabbit monoclonal, A21206, Abcam) for antibody staining CX43. The secondary antibody was incubated for 45 min and washed three times for 5 min. Nuclear staining was performed using a 4′,6-daminidino-2-phenylindole (DAPI) mounting solution (P36931, Life technologies corporation, Carlsbad, CA, USA). Further, LabVision Autostainer 360 (Lab Vision Corporation, VT) was used for staining with antibodies against adherent junction molecules such as E-cadherin (CDH1, mouse monoclonal, 1:50 dilution, n1620, DakoCytomation, Santa Clara, CA, USA) and N-cadherin (CDH2, mouse monoclonal, 1:100 dilution, m3613, DakoCytomation). Visualization was done using the standard peroxidase technique (UltravisionOne HRP system, Thermo Fisher Scientific). Primary antibody binding to an expressed antigen was recognized by a secondary antibody conjugated with peroxidase-labeled polymer with diaminobenzidine (DAB). Each staining was performed at least three times, and each sample was tested in triplicate. Negative and positive controls were performed simultaneously for all antibodies. All antibodies used for IHC in this study are summarized in . Bright-field images of H&E and DAB-stained samples were taken by a ZEISS Axio Observer Z1 microscope (ZEISS, Oberkochen, Germany). Fluorescence was recorded by a ZEISS Axio Imager M1 fluorescence microscope (ZEISS). Three independent individuals used Image J software and counted nuclear antibody positivity (p63α, CEBPD, SOX9, and Ki67). 2.5. Statistical Analysis The technical replicates from the same donor and group of three donors of the hLESC harvested in two different conditions were averaged as a percentage mean ± standard error of the mean (SEM). Prism 8.3.0 (GraphPad, San Diego, CA, USA) was used for statistical analysis. The data were counted and analyzed by two different methods: in percentages representing the ratio of the number of cells positive for a specific marker and the total number of cells (DAPI positivity), or as the number of cells positive for a specific marker per mm 2 . Further, data were tested for normal distribution (Shapiro–Wilk test), and the difference was tested using an unpaired two-sample t-test. The significance level p ≤ 0.05 was counted as significant.
A Placenta was collected after a scheduled cesarian section from a full-term pregnancy. Informed consent and institutional board review approval had previously been obtained from the patient. According to the standard protocol, the placenta was immediately transported in a sterile container and further processed under sterile conditions . Proper washing with 0.9% NaCl (Fresenius Kabi AB, Uppsala, Sweden) or 0.9% NaCl containing 100 U/mL Penicillin, 100 μg/mL Streptomycin (P4333), and 2.5 μg/mL Amphotericin B (A2942) was repeatedly performed. The HAM was then separated from the chorion by blunt dissection, washed out from residual blood, and transferred onto a nitrocellulose filter carrier, pore size 0.45 μm (111306-47-CAN, Sartorius, Gottingen, Germany) with the epithelial side up, then divided into 3 × 3 cm and 5 × 5 cm pieces. HAM pieces were cryopreserved in 50% glycerol, 48.5% DMEM/F12 (31331028, Invitrogen, Carlsbad, CA, USA), 100 U/mL Penicillin, 100 μg/mL Streptomycin and 2.5 μg/mL Amphotericin B and stored at −80 °C.
Before use, HAMs were thawed, warmed to room temperature, and washed three times with a medium containing DMEM/F12, 100 U/mL Penicillin, and 100 μg/mL Streptomycin. Thereafter, the HAMs were placed on polyester membrane Netwell TM inserts (3479, Corning Inc., New York, NY, USA) 24 mm in diameter, with the epithelial side up by two different techniques : (1) HAMs 3 × 3 cm ( .1A) in size were peeled from the nitrocellulose filter paper. Further, HAMs were placed and stretched on the top of the polyester membrane and sutured by the eight individual sutures ( .1B) near the edge of the polyester membrane. The HAMs were then tightly stretched on the top of the membrane, making a flat surface ( .1C). Excess HAM tissue that remained at the edge of the polyester membrane was carefully removed with a disposable sterile scalpel. (2) The other approach was to use HAMs 5 × 5 cm in size ( .2A) placed on top of the membrane and sutured so that the HAMs were loosely attached. HAMs were sutured by individual sutures ( .2B). In addition, an individual suture was placed in the center of the HAM/polyester membrane to obtain the folding of the HAM and to keep it in close contact with the membrane ( .2C). The excessive HAM tissue at the edges was again removed accordingly. The sutured HAMs on the Netwell TM inserts were immersed in DMEM/F12 medium containing 100 U/mL Penicillin and 100 μg/mL Streptomycin and kept at 37 °C, 5% CO 2 , and 95% air overnight to obtain HAM free of any glycerol remains.
Following corneal transplantation, the remaining human corneal-scleral rings from three donors (n = 3) were divided into twelve limbal biopsies of equal size and thoroughly washed with DMEM/F12 medium containing 100 U/mL Penicillin and 100 μg/mL Streptomycin. The biopsies were treated with neutral protease and Dispase II (2.4 U/mL 4942078001, Roche Diagnostics, Mannheim, Germany) for 10 min at 37 °C. The dissociation process was blocked using Fetal Bovine Serum (FBS, F2442). The limbal biopsies were then placed centrally on the top of the HAMs, with the epithelial side down and submerged in a standardly used complex medium (COM). COM consisted of DMEM/F12, Penicillin (100 U/mL), Streptomycin (100 μg/mL), Amphotericin B (2.5 μg/mL), human epidermal growth factor (2 ng/mL, E9644), insulin (5 μg/mL), sodium selenite (5 ng/mL) and transferrin (5 μg/mL, l1884), cholera toxin A subunit from Vibrio cholerae (30 ng/mL, C8180), hydrocortisone (0.03 μg/mL, H0888), 5% FBS, and 0.5% dimethyl sulfoxide (DMSO, D2650). After 2 h of incubation, the attached limbal biopsies were completely covered by COM. Further, the limbal biopsies and outgrowing LESCs were harvested and incubated at 37 °C with 5% CO 2 , and 95% air for the following three weeks. The culture medium was changed every three days.
Limbal biopsies with hLESCs cultured on HAMs were cut from the Netwell TM inserts. Samples were fixed in 4% formalin overnight at 4 °C and then processed in dehydrated graded alcohol series of 70% (10–15 min), 80% (10–15 min), 96% (2 × 10 min), and 100% ethanol (2 × 10 min) before having xylene added (3 × 10 min) and being washed with melted paraffin (3 × 10 min) and then embedded in paraffin for immunohistochemistry (IHC). Paraffinized tissue was cut into 3–4 μm thick sections using an automated microtome (HM 355S, Thermo Fisher Scientific, Waltham, MA, USA) and attached to histological slides. Deparaffinization was performed in xylene (2 × 10 min), then rehydration was performed by sinking in 100%, 96%, and 70% ethanol, and then distilled water. Hematoxylin & Eosin (H&E) staining was primarily performed. Slides were immersed in Mayers hematoxylin plus solution (01825, Histolab, Askim, Sweden) for 10 min and then rinsed with distilled water (10 min) followed by eosin staining (10 min), and then they were rehydrated in upgraded alcohol series of 70%, 96%, 100% ethanol, and xylene. Slides were further mounted using Pertex (00840, Histolab) mounting medium. For IHC, heat-induced antigen retrieval was performed in a microwave for 5 min at 900W and 15 min at 600W in a citrate buffer (pH 6, C9999) or by PT module (LabVision, Fremont, CA, USA). Blocking of non-specific binding sites with 5% Bovine Serum Albumin (BSA, A9418) dissolved in Dulbecco’s Phosphate Buffered Saline (DPBS, 14190-144, Thermo Fisher Scientific) was conducted for 20 min. Further, slides were stained with primary antibodies diluted in 1% BSA for 1 h. Slides were stained using antibodies for the following progenitor markers: tumor protein p63 alpha (p63α, rabbit polyclonal, 1:200 dilution, 4892S, Cell Signaling, Beverly, MA, USA), SRY-Box Transcription Factor 9 (SOX9, 82630, Cell Signaling, rabbit monoclonal, 1:200), quiescence marker: CCAAT/enhancer-binding protein delta (CEBPD, rabbit polyclonal, 1:200 dilution, ab198320, Abcam, Cambridge, UK), and proliferation marker Ki-67 (rabbit monoclonal, 1:200, RM-9106-S, Thermo Scientific) and the following differentiation markers: cytokeratin 3/12 (KRT3/12, mouse monoclonal, 1:100 dilution, 08691431, MP biomedicals, Santa Ana, CA, USA) and connexin-43 (CX43, rabbit polyclonal, 1:300, C6219). Then, the slides were thoroughly washed three times for 5 min with PBS-tween buffer (28352, Thermo Fisher Scientific). Incubation was continued with the appropriate animal type of secondary antibody: Cy3 ® goat anti-rabbit IgG (rabbit monoclonal, 1:500 dilution, A10520, Abcam) for samples stained with p63α, CEBPD, SOX9, Ki67, and Alexa Fluor ® 488 donkey anti-mouse IgG (1:500 dilution, mouse monoclonal, 21202, Abcam) for samples stained with KRT3/12, and Alexa Fluor ® 488 donkey anti-rabbit IgG (1:500 dilution, rabbit monoclonal, A21206, Abcam) for antibody staining CX43. The secondary antibody was incubated for 45 min and washed three times for 5 min. Nuclear staining was performed using a 4′,6-daminidino-2-phenylindole (DAPI) mounting solution (P36931, Life technologies corporation, Carlsbad, CA, USA). Further, LabVision Autostainer 360 (Lab Vision Corporation, VT) was used for staining with antibodies against adherent junction molecules such as E-cadherin (CDH1, mouse monoclonal, 1:50 dilution, n1620, DakoCytomation, Santa Clara, CA, USA) and N-cadherin (CDH2, mouse monoclonal, 1:100 dilution, m3613, DakoCytomation). Visualization was done using the standard peroxidase technique (UltravisionOne HRP system, Thermo Fisher Scientific). Primary antibody binding to an expressed antigen was recognized by a secondary antibody conjugated with peroxidase-labeled polymer with diaminobenzidine (DAB). Each staining was performed at least three times, and each sample was tested in triplicate. Negative and positive controls were performed simultaneously for all antibodies. All antibodies used for IHC in this study are summarized in . Bright-field images of H&E and DAB-stained samples were taken by a ZEISS Axio Observer Z1 microscope (ZEISS, Oberkochen, Germany). Fluorescence was recorded by a ZEISS Axio Imager M1 fluorescence microscope (ZEISS). Three independent individuals used Image J software and counted nuclear antibody positivity (p63α, CEBPD, SOX9, and Ki67).
The technical replicates from the same donor and group of three donors of the hLESC harvested in two different conditions were averaged as a percentage mean ± standard error of the mean (SEM). Prism 8.3.0 (GraphPad, San Diego, CA, USA) was used for statistical analysis. The data were counted and analyzed by two different methods: in percentages representing the ratio of the number of cells positive for a specific marker and the total number of cells (DAPI positivity), or as the number of cells positive for a specific marker per mm 2 . Further, data were tested for normal distribution (Shapiro–Wilk test), and the difference was tested using an unpaired two-sample t-test. The significance level p ≤ 0.05 was counted as significant.
3.1. Epithelial and Basement Membrane (BM) Morphology in Corneal-limbal Tissue and Consequent Localization of hLESCs The distribution of hLESCs was examined in the different BM compartments of the human corneal-limbal tissue in situ and compared to the hLESCs cultured on conventionally flat and alternatively, HAM sutured in a radial pattern, mimicking limbal crypts ex vivo . The human corneal epithelium had 5–7 layers on the flat BM and an avascular Bowman’s layer ( A). The anterior limbus contained 7–10 epithelial layers on the irregular BM and vascularized stroma underneath ( B). The posterior limbal epithelium was attached to the undulated BM and limbal epithelial crypts that were placed deeper and were mainly surrounded by the limbal stroma ( C). The hLESCs were smaller in size, with a high nucleo-cytoplasmic (N-: C) ratio, and could be randomly detected in the basal epithelial layer of the anterior limbus ( B, black arrows). However, the hLESCs seemed to be more present and densely packed in the basal layer of the posterior limbus and limbal epithelial crypts ( C, black arrow). 3.2. Morphology of the hLESC Cultures Expanded on Conventional, Flat-sutured HAMs vs. hLESC Cultures Expanded on the Novel, Radially-sutured HAMs HAMs sutured by the novel radial suture technique comprised of flat and crypt-like areas. Furthermore, the crypts of the HAMs sutured by the novel radial suture technique consisted of (1) undulated HAM areas with the opened surface ( E) and (2) looped HAM areas that appeared to be almost closed ( F, black asterisk). The multi-layering of the epithelial cells was noted in ex vivo expanded hLESC cultures lying on the undulated and looped HAMs compared to cultures lying on the flat HAM ( E,F, black arrows vs. D). A higher presence of columnar-like epithelial cells was noted in the cultures harvested on the undulated ( E) and looped HAMs ( F) compared to cultures harvested on the flat HAM ( D). The polygonal and squamous cells were found in the middle and superficial layers of the hLESC cultures on the flat HAMs ( D). These polygonal and squamous cells appeared to be less present in the hLESC cultures in the crypt-like HAM compartments. To better understand the structural differences in cultivated tissue on flat and crypt-like HAMs, we aimed to compare the marker fingerprint of cultures growing on flat and looped-like HAMs, as these are two morphologically distinct settings. 3.3. Distribution of the Progenitor Markers In Situ Versus In Vitro Study Conditions The progenitor marker p63α was found in some of the cells of the basal and suprabasal layers of the cultures expanded on the flat and undulated HAMs ( .1A,B). However, the HAM loops contained a statistically higher number of p63α-positive hLESCs ( .1C) than cultures on the flat HAM, quantified as percentages: p63α vs. DAPI positivity ratio (flat vs. loop, 37.56 ± 3.34% vs. 62.53 ± 3.32%, p = 0.01, Figure 5A) or as a total number per mm 2 (377.8 ± 34.17 vs. 962.9 ± 167.2, p = 0.03, Figure 5B). Regarding the epithelium in the corneal-limbal tissue, p63α was not found in any of the cells of the corneal epithelium in situ ( .2A) but was identified in some cells of the basal and suprabasal layers of the anterior limbus ( .2B, arrow). The posterior limbal epithelium with undulated BM was enriched with p63α-positive cells in the basal and suprabasal layers ( .2C). Basal and suprabasal cells in the cultures expanded on flat ( .1D), undulated ( .1E), and looped ( .1F) HAMs expressed the SOX9 progenitor marker. However, the SOX9 progenitor marker positivity was significantly higher in the cultures expanded on crypt-like HAMs forming loops than in the cultures growing on flat HAMs, quantified as percentages (35.53 ± 0.96% vs. 43.23 ± 2.32%, p = 0.04, Figure 5A) or as a total number per mm 2 (442.3 ± 62.31 vs. 728.1 ± 65.97, p = 0.03, Figure 5B). In situ, the progenitor marker SOX9 was exclusive for the limbal basal epithelium ( .2E). In particular, the limbal epithelial crypts appeared to be enriched for this marker ( .2F). 3.4. Expression Profile of the Proliferation and Quiescence Markers in the Corneal-limbal Epithelial Tissue Versus In Vitro Study Conditions The expression distribution of the CEBPD marker was similar to p63α and present in the cultures expanded on flat ( .1A), undulated ( .1B), and looped HAMs ( .1C)—mainly in basal and suprabasal layers. In addition, CEBPD was found in the basal epithelial cells of both tissues in situ, the cornea ( .2D) and the anterior and posterior limbus ( .2B,C), accordingly. However, no statistical significance was noted in the number of CEBPD-positive cells expanded on HAM loops compared to cells expanded on flat HAMs, quantified as percentages (22.99 ± 2.96% vs. 30.49 ± 3.33 %, p = 0.17, A) or as a total number per mm 2 (243.00 ± 35.19 vs. 474.1 ± 138.5, p = 0.18, B). Many of the hLESCs expanded on a flat ( .1D, white arrow), undulated ( .1E), and looped HAM ( .1F) were found in the proliferation state. However, some sectors of the epithelial tissue on the flat HAM contained no Ki-67-positive cells ( .1D), while sections of the epithelial tissue on the undulated ( .1E) and/or looped HAM ( .1F) persistently maintained Ki-67-positive cells. Proliferation was significantly higher in cultures expanded on looped HAMs compared to the cultures on flat HAMs, quantified as percentages (8.43 ± 0.38 % vs. 22.38 ± 1.95 %, p = 0.002, A) or as a total number per mm 2 (100.7 ± 10.69 vs. 276.2 ± 33.34, p = 0.01, B). For comparison to the in situ state, proliferation marker Ki-67 was sporadically found in the suprabasal cells of the anterior ( .2E) and posterior ( .2F) limbal epithelium, whereas it was absent in the central corneal epithelium, and only sparsely present in some basal cells of the posterior cornea ( .2D). 3.5. Differentiation Marker profile in the Epithelium of the Corneal-limbal Tissue, and hLESC Cultures on the Flat and Crypt-like HAMs CX43 was uniformly distributed in all ex vivo expanded cells ( .1A–C), a finding similar to the CX43 pattern in the corneal epithelium in situ ( .2A). However, some basal cells in the anterior ( .2B) and posterior limbal epithelium and limbal epithelial crypts ( .2C) appeared to lack the CX43 marker. In the expanded hLESC cultures growing on flat HAMs, the differentiation marker KRT3/12 was present in the polygonal and squamous cells, mainly in the middle and top layers ( .1D). Less KRT3/12 presence could be noted in the cultures expanded on undulated ( .1E) and loop HAMs ( .1F), whereas this marker was almost absent in cells growing in small HAM loops. Regarding the corneal-limbal tissue, KRT3/12 was present in all corneal epithelial cells ( .2D). In the limbal epithelium, the majority of the cells were stained positive for the KRT3/12, whereas the cells in the lowest layers attached to the BM were devoid of this marker ( .2E,F). 3.6. Presentation of Cell Adhesion Molecules in the Corneal-Limbal Epithelium and Expanded hLESC Cultures on Flat and Crypt-like HAMs The transmembrane protein E-cadherin was present in most of the ex vivo expanded epithelial cells ( .1A–C). The same applied to the corneal-limbal tissue in situ ( .2A–C). In hLESC cultures, N-cadherin was present in a few cells of the basal layer on the flat HAMs ( .1D). On the other side, more cells in the basal layer of the crypt-like HAMs seemed to express N-cadherin since the surface of the basal layer of the cultivated tissue appeared enlarged in those crypts compared to the cultures on the flat HAMs ( .1E,F). N-cadherin was exclusive for the limbal basal epithelium in situ. Only a few basal cells in the epithelium of the anterior limbus expressed N-cadherin ( .2E). In contrast, almost all cells of the limbal basal epithelium in the posterior limbus expressed N-cadherin ( .2F).
The distribution of hLESCs was examined in the different BM compartments of the human corneal-limbal tissue in situ and compared to the hLESCs cultured on conventionally flat and alternatively, HAM sutured in a radial pattern, mimicking limbal crypts ex vivo . The human corneal epithelium had 5–7 layers on the flat BM and an avascular Bowman’s layer ( A). The anterior limbus contained 7–10 epithelial layers on the irregular BM and vascularized stroma underneath ( B). The posterior limbal epithelium was attached to the undulated BM and limbal epithelial crypts that were placed deeper and were mainly surrounded by the limbal stroma ( C). The hLESCs were smaller in size, with a high nucleo-cytoplasmic (N-: C) ratio, and could be randomly detected in the basal epithelial layer of the anterior limbus ( B, black arrows). However, the hLESCs seemed to be more present and densely packed in the basal layer of the posterior limbus and limbal epithelial crypts ( C, black arrow).
HAMs sutured by the novel radial suture technique comprised of flat and crypt-like areas. Furthermore, the crypts of the HAMs sutured by the novel radial suture technique consisted of (1) undulated HAM areas with the opened surface ( E) and (2) looped HAM areas that appeared to be almost closed ( F, black asterisk). The multi-layering of the epithelial cells was noted in ex vivo expanded hLESC cultures lying on the undulated and looped HAMs compared to cultures lying on the flat HAM ( E,F, black arrows vs. D). A higher presence of columnar-like epithelial cells was noted in the cultures harvested on the undulated ( E) and looped HAMs ( F) compared to cultures harvested on the flat HAM ( D). The polygonal and squamous cells were found in the middle and superficial layers of the hLESC cultures on the flat HAMs ( D). These polygonal and squamous cells appeared to be less present in the hLESC cultures in the crypt-like HAM compartments. To better understand the structural differences in cultivated tissue on flat and crypt-like HAMs, we aimed to compare the marker fingerprint of cultures growing on flat and looped-like HAMs, as these are two morphologically distinct settings.
The progenitor marker p63α was found in some of the cells of the basal and suprabasal layers of the cultures expanded on the flat and undulated HAMs ( .1A,B). However, the HAM loops contained a statistically higher number of p63α-positive hLESCs ( .1C) than cultures on the flat HAM, quantified as percentages: p63α vs. DAPI positivity ratio (flat vs. loop, 37.56 ± 3.34% vs. 62.53 ± 3.32%, p = 0.01, Figure 5A) or as a total number per mm 2 (377.8 ± 34.17 vs. 962.9 ± 167.2, p = 0.03, Figure 5B). Regarding the epithelium in the corneal-limbal tissue, p63α was not found in any of the cells of the corneal epithelium in situ ( .2A) but was identified in some cells of the basal and suprabasal layers of the anterior limbus ( .2B, arrow). The posterior limbal epithelium with undulated BM was enriched with p63α-positive cells in the basal and suprabasal layers ( .2C). Basal and suprabasal cells in the cultures expanded on flat ( .1D), undulated ( .1E), and looped ( .1F) HAMs expressed the SOX9 progenitor marker. However, the SOX9 progenitor marker positivity was significantly higher in the cultures expanded on crypt-like HAMs forming loops than in the cultures growing on flat HAMs, quantified as percentages (35.53 ± 0.96% vs. 43.23 ± 2.32%, p = 0.04, Figure 5A) or as a total number per mm 2 (442.3 ± 62.31 vs. 728.1 ± 65.97, p = 0.03, Figure 5B). In situ, the progenitor marker SOX9 was exclusive for the limbal basal epithelium ( .2E). In particular, the limbal epithelial crypts appeared to be enriched for this marker ( .2F).
The expression distribution of the CEBPD marker was similar to p63α and present in the cultures expanded on flat ( .1A), undulated ( .1B), and looped HAMs ( .1C)—mainly in basal and suprabasal layers. In addition, CEBPD was found in the basal epithelial cells of both tissues in situ, the cornea ( .2D) and the anterior and posterior limbus ( .2B,C), accordingly. However, no statistical significance was noted in the number of CEBPD-positive cells expanded on HAM loops compared to cells expanded on flat HAMs, quantified as percentages (22.99 ± 2.96% vs. 30.49 ± 3.33 %, p = 0.17, A) or as a total number per mm 2 (243.00 ± 35.19 vs. 474.1 ± 138.5, p = 0.18, B). Many of the hLESCs expanded on a flat ( .1D, white arrow), undulated ( .1E), and looped HAM ( .1F) were found in the proliferation state. However, some sectors of the epithelial tissue on the flat HAM contained no Ki-67-positive cells ( .1D), while sections of the epithelial tissue on the undulated ( .1E) and/or looped HAM ( .1F) persistently maintained Ki-67-positive cells. Proliferation was significantly higher in cultures expanded on looped HAMs compared to the cultures on flat HAMs, quantified as percentages (8.43 ± 0.38 % vs. 22.38 ± 1.95 %, p = 0.002, A) or as a total number per mm 2 (100.7 ± 10.69 vs. 276.2 ± 33.34, p = 0.01, B). For comparison to the in situ state, proliferation marker Ki-67 was sporadically found in the suprabasal cells of the anterior ( .2E) and posterior ( .2F) limbal epithelium, whereas it was absent in the central corneal epithelium, and only sparsely present in some basal cells of the posterior cornea ( .2D).
CX43 was uniformly distributed in all ex vivo expanded cells ( .1A–C), a finding similar to the CX43 pattern in the corneal epithelium in situ ( .2A). However, some basal cells in the anterior ( .2B) and posterior limbal epithelium and limbal epithelial crypts ( .2C) appeared to lack the CX43 marker. In the expanded hLESC cultures growing on flat HAMs, the differentiation marker KRT3/12 was present in the polygonal and squamous cells, mainly in the middle and top layers ( .1D). Less KRT3/12 presence could be noted in the cultures expanded on undulated ( .1E) and loop HAMs ( .1F), whereas this marker was almost absent in cells growing in small HAM loops. Regarding the corneal-limbal tissue, KRT3/12 was present in all corneal epithelial cells ( .2D). In the limbal epithelium, the majority of the cells were stained positive for the KRT3/12, whereas the cells in the lowest layers attached to the BM were devoid of this marker ( .2E,F).
The transmembrane protein E-cadherin was present in most of the ex vivo expanded epithelial cells ( .1A–C). The same applied to the corneal-limbal tissue in situ ( .2A–C). In hLESC cultures, N-cadherin was present in a few cells of the basal layer on the flat HAMs ( .1D). On the other side, more cells in the basal layer of the crypt-like HAMs seemed to express N-cadherin since the surface of the basal layer of the cultivated tissue appeared enlarged in those crypts compared to the cultures on the flat HAMs ( .1E,F). N-cadherin was exclusive for the limbal basal epithelium in situ. Only a few basal cells in the epithelium of the anterior limbus expressed N-cadherin ( .2E). In contrast, almost all cells of the limbal basal epithelium in the posterior limbus expressed N-cadherin ( .2F).
Different techniques of HAM suturing to the corneal surface have been used thus far. A HAM can be sutured as a graft (inlay) or as a patch (overlay). While used as a graft, a HAM is placed on the defect with the stromal face down and acts as a BM, allowing the epithelium to proliferate and regenerate over it. It can be used as a single or multilayered graft with a lamellar sac, filling, or roll-filling technique, mostly depending on the depth of the corneal defect. The patch technique is mostly used for epithelial defects without perforations. The epithelial side of the HAM is placed down towards the defect, and the HAM serves as a biological compressive bandage . Graft alone or as part of a sandwich technique, which is a combination of both graft and patch techniques, has been standardly used for HAMs carrying cultivated hLESCs and limbal explants . However, this is the first study to propose the manipulation of a HAM prior to the expansion of the hLESCs to ensure a better quality of the transplanted tissue as an adjuvant technique to the previously used HAM suturing techniques. Stem cell niches vary in size and functional organization in mammals . Stem cells can be found as individual structures under the BM of the skeletal muscle , or grouped as epithelial stem cells in the hair follicle bulges and neural stem cells in the forebrain subventricular zone in mammals . In this study, we provided a more optimal microenvironment for the expansion of hLESCs ex vivo by mimicking the BM folding in niches residing in the posterior limbus and limbal epithelial crypts. Generally, the maintenance of stem cells, including hLESCs, depends on a functional niche characteristic. These niches provide cell anchoring, mechanical protection, communication with underlying stroma and vasculature, the release of specific growth factors, cell cycle molecules, and the involvement of evolutionary conserved molecular pathways. Such 3D microenvironments allow the stem cells to hold the quiescence, maintain stemness, and undergo asymmetric or symmetric proliferation when needed . Our study supports earlier findings that most hLESC/early progenitor cells reside on the bottom of the limbal epithelial crypts, which are deep epithelial protrusions directly surrounded by a loose stromal matrix . When an epithelial stem cell niche is established along the stiff BM, it maintains its regular morphology. However, when the epithelial stem cell niche forms along a flexible and extensible BM, it may arrange in the form of finger-like protrusions, enabling a higher surface for stem cells to allocate, thus providing the protection and preservation of the putative stem cell characteristics . Stem cell progenies acquire differentiation properties by leaving the stem cell niche towards the more rigid and flat ground, such as the Bowman membrane, to eventually terminally differentiate and undergo apoptosis (26). A HAM is a desirable elastic and adaptable scaffold for creating 3D protrusions that can physically mimic limbal crypts ex vivo. It is a widely available natural semi-transparent and permeable membrane. Its mechanical and functional characteristics are desirable for the migration, adhesion, and growth of epithelial cells on the ocular surface. It possesses high elasticity, low stiffness, and high tensile strength properties , and it also resembles the cornea and conjunctiva in regards to the collagen arrangement . The stiffness should be similar between flat vs. crypt-like HAMs, as we used pieces from the same donor. Even though there might be some local differences in stiffness within the same HAM, we used nine pieces of both flat and crypt-like HAM, and all the pieces showed significant changes related to the suturing method. There was only one extra suture on the crypt-like vs. flat HAMs to enable the folding, so this should not have affected the overall stiffness of the crypt-like vs. flat HAMs. Functionally, HAM is immunotolerant and has low antigenicity, even though some immunomodulatory effects have been reported; it has an anti-fibrotic impact, mainly due to the TGF-β inhibition. It secretes a wide range of growth factors, such as EGF, bFGF, HGF, KGF and KGF receptors, TGFα, and TGFβ 1,2,3 isoforms, sharing some common features with stem cell niche composition . However, not all HAM properties seem beneficial for stem cell maintenance. An intact HAM promotes the epithelial differentiation of explanted limbal cultures. Therefore, removing the epithelium from the HAM upon preparation has been used by some authors to maintain progenitor properties, postpone differentiation, and thus, improve the quality of the explanted tissue . Also, not all of the cells expanded on a HAM have the features of hLESCs or early progenies. As previously shown, most of the hLESC/progenies are positioned in the basal epithelial cell layers—the ones attached to the HAM. In contrast, the cells in the upper/superficial layers exhibit more differentiation properties . With our novel suturing technique, we aimed to enlarge the surface area of the HAM, and hence enlarge the number of cells in the basal layer attached to the HAM, maintaining the more undifferentiated state. In addition, the expanded epithelial tissue appeared multilayered in the crypt-like HAMs, and it contained a higher number of columnar-like cells, likely indicating a higher proliferation rate. The novel suturing technique would also increase the supply of the stem-cell-supporting molecules secreted by the HAM. HAM is widely implemented in tissue engineering and regenerative medicine . However, its limited chemical and physical features, and the high cost of preserving it in a fresh condition, caused the urgency for new solutions . HAM has, so far, undergone additional adjustments to upgrade its properties for easier manipulation, duration, and utilization, and for higher resistance to microbes and broader applications, among others, in ocular surface reconstruction . For instance, AM can be used as a constituent of various composite scaffolds, in a form of extract, and as a hydrogel . Regarding the attempts of HAM modification for successful LESC transplantation, decellularized AM (dAM) conjugated with an electrospun polymer nanofiber mesh promoted LESC proliferation and adhesion in a rabbit model . An amniotic membrane in a form of extract (AME) and eye drops proved beneficial for the treatment of ocular surface disorders, injuries, and the in vivo cultivation of hLESCs . In addition, AME, as an animal-free product, was suggested as a suitable replacement for FBS upon LSC transplantation to avoid the risk of disease transmission and accumulation of bovine antigens . However, all the above-modified HAM methods require very complex processing or serve only as adjuvant therapy. Therefore, we present an easy-handling, widely available, and inexpensive method of HAM manipulation prior to hLESC expansion. As previously mentioned, the successful long-term restoration of the corneal epithelium after CLET requires more than 3% of the cells in the transplanted graft to be p63 positive . In our study, cells growing on either the flat or crypt-like HAMs were enriched with the p63α marker. However, we found a significantly larger number of cells positive for p63α in the looped regions of the crypt-like HAMs, compared to the cells growing on flat HAMs. Initially, the whole tumor protein p63 was perceived as a specific marker for hLESCs . Later studies discovered ΔNp63α to be more distinct for hLESCs and for early progenies residing in the limbal basal epithelium, while other p63 isoforms were detected in the suprabasal layers of the limbus and cornea, playing a role in corneal differentiation . Indeed, in our samples, p63α stained the particular cells in the basal limbal epithelium, while staining in the non-limbal cornea was absent. Significantly higher cell turnover was present in the cultures on the crypt-like HAMs compared to the cultures growing on flat HAMs, indicating the presence of cells with intense proliferation, such as early progenies/TACs. Compared to the in situ state, proliferation appeared to be much lower in corneal-limbal tissue, and it was noted in a few suprabasal cells of the anterior and posterior limbus and some cells in the posterior cornea as previously described . The CEBPD marker was not significantly more abundant in the cultures growing on crypt-like HAMs compared to those on flat-like HAMs. CEBPD is a quiescence marker that controls the cell cycle and inhibits the proliferation of hLESCs in ex vivo cultures . Since proliferation was significantly higher in ex vivo cultures growing on crypt-like HAMs compared to those growing on flat-like HAM, we expected these cells not to be positive for CEBPD; hLESCs do not co-express CEBPD with the Ki-67 marker in the limbus. In addition, CEBPD-positive hLESCs that co-express the ΔNp63α marker in the basal limbal epithelium in situ are the ones considered quiescent . However, it seems that the CEBPD marker is not specific for hLESCs in situ, since it is also found in the cells of the basal corneal epithelium in our samples. The transcriptional factor SOX9 plays diverse roles in the embryonal and adult development of mammals as well as in stem cell maintenance. This marker was upregulated in the cultures on crypt-like HAMs compared to those on flat-like HAMs. Its nuclear localization in TACs is essential for proliferation upon wound healing. However, SOX9 is particularly involved in the proliferation and differentiation steps of early progenies derived from hLESCs, but not in the terminal differentiation, which explains why SOX9 is absent in the cornea . This finding contributes to our conclusion that crypt-like HAMs contain numerous cells that are positive for SOX9 and are thus in a more undifferentiated state. We found no difference in the presence of the CX43 transmembrane protein between the cultures on crypt-like HAM and flat HAM. CX43 is a protein involved in the communication between mammalian cells through diverse mechanisms . Constitutionally, CX43 is present in the corneal epithelium and all suprabasal epithelial layers in the limbus, whereas it is absent in some cells of the basal limbal layer . A similar pattern of expression of CX43 applies to expanded cultures on the flat HAMs . The absence of cell interaction may be one of the mechanisms for maintaining stemness and the quiescence state . Thus, according to some authors, CX43 positivity in cells distinguishes the hLESCs from the TACs/early progenies in vivo. Since most of the cells in the basal and suprabasal layers of the cultivated epithelial tissue were CX43 positive, it seems that a very small cell fraction remains in a quiescent state ex vivo. Cytokeratin 3 (KRT3) and cytokeratin 12 (KRT12) are cornea-specific intermediate filaments, hallmarks of differentiated hCECs in the cornea and differentiated epithelial cells in the limbus . In cultured epithelial tissue, KRT3/12 has been found in the suprabasal and superficial layers, but not in the basal layer of cells cultured on flat HAM, a finding corresponding to our results . However, the KRT3/12 marker was reduced in the looped regions of the crypt-like HAMs. In some small looped regions, the respective marker was absent. The KRT3/12 marker is known to be absent from limbal basal layers—a finding that shows a more mature nature of the corneal basal cells compared to the limbal basal epithelial cells, due to different characteristics of the corresponding basal membranes . Also, KRT3/12 is absent from limbal epithelial crypts . Our study shows that unique cell fractions in the basal layers attached to the HAM are positive for the N-cadherin marker. It seems that the isolation of the cell cultures surrounded by a double HAM membrane increases cell positivity for this marker. N-cadherin is essential for maintaining the progenitor characteristics in cultured hLESCs . Differentiated corneal and limbal epithelial cells express E-cadherin, while N-cadherin is present in the hLESCs/progenitor cells in the limbal basal epithelium, a finding concomitant with ours. In particular, our basal epithelial layer in the posterior limbus appeared to be enriched with this marker. It is suggested that communication with the melanocytes is achieved via N-cadherin forming homotypic adhesions . A disadvantage of our technique may be that a larger area of HAM tissue is needed for transplantation and, evidently, a decreased transparency of the cultivated transplantation graft, in addition to any usual disadvantages of using HAM .
In conclusion, this novel HAM suturing technique increased the number of progenitor cells upon expansion and may thus increase the quality of the transplanted graft. We believe this technique can be a valuable, simple, and inexpensive tool to increase the success rate of corneal epithelial regeneration. However, the suturing technique needs to be tested in vivo to confirm its efficacy. Future clinical studies comparing conventional, flat suturing and the current suturing method are also required.
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Digital Health Literacy and Person-Centred Care: Co-Creation of a Massive Open Online Course for Women with Breast Cancer | b916f2db-4cef-467a-9228-d54798087089 | 10001393 | Patient-Centered Care[mh] | Breast cancer (BC) represents one of the most frequently diagnosed cancers in women worldwide . According to the most recent data from the European Cancer Information System (ECIS), there were approximately 355,460 new cases of BC diagnosed across Europe in 2020, with 34,088 of those cases occurring in Spain . However, thanks to early diagnosis and therapeutic advances, BC survival has increased in recent years , with a survival rate of around 85% . Increasing prevention and treatment for BC have lowered mortality, but the diagnosis and treatment continue to have a significant impact in many areas of patients’ lives (physical, emotional, cognitive and social) . The diagnosis of BC, which in most cases necessitates an effort to adjust and adapt to the new situation , is typically perceived as a traumatic event with a significant impact on the health-related quality of life of the women who suffer from it, making them more vulnerable to the potential consequences of using biased or low-quality health information. Person-centred care (PCC) is defined as the provision of care that considers a patient’s clinical needs, life circumstances, and personal values and preferences . A central component of PCC is to ensure quality communication between patients and healthcare professionals, with the aim of fostering the process of shared decision making (SDM) . SDM-based interventions, such as patient decision aids (PtDAs), have been shown to improve patients’ knowledge about available treatments and their benefits/risks, decisional conflict and other decisional process variables . There is a need to develop interventions to increase knowledge about PCC and digital health literacy (DHL) , particularly in chronic pathologies such as BC, where the impact of their diagnosis or treatment may increase the number of queries on the Internet and directly influence the understanding of health information . Health literacy (HL) integrates the skills and motivation to find, understand, evaluate and use health information. As a result, HL facilitates informed decision making and improves the ability to manage and address health disparities, giving patients more autonomy and empowerment to take responsibility for their own health, as well as the health of their families and communities. In turn, low HL impacts health outcomes and health-related costs, leading to inefficient healthcare utilization and delivery . DHL is an extension of HL that employs the same operational definition but in the context of information and communication technology resources. It involves both the provision of information and the degree to which information is understood. When these skills are lacking, technology solutions have the potential to either promote or hinder HL . Due to the complexity of health information, it is recommended that DHL interventions be based on a design of co-creation of resources, websites and health tools through collaborative work with patients, allowing them to improve the medical care they receive [ , , ]. Massive open online courses (MOOCs) are designed to engage a large number of participants learning remotely, offering the general population, clinical subpopulations or health professionals good quality knowledge on health issues through interactive and flexible technological resources, with little or no prior learning required . To date, most MOOCs have been developed for the education of medical students and health professionals , but they have also been directed at the general population or clinical subpopulations, showing positive effects in several areas such as healthy nutrition habits , self-management of diabetes or learning risk factors for dementia . As has been observed in some projects with other populations, the development of educational interventions with a MOOC based on a co-creation design, which combines several resources in different formats and adapts to different educational, cultural levels and needs of the users, could be a strategy to face the HL, self-care and empowerment challenges for women with BC. One example is the IC-Health European project ( https://cordis.europa.eu/project/id/727474/es accessed on 20 December 2022), whose results have shown good acceptance of co-created teaching resources aimed at improving the DHL of people with chronic diseases and the general population [ , , ]. In recent years, the framework of participatory action research has been used for the development of eHealth. It is an approach that involves collaboration to develop a process through the construction of knowledge and social change in a community following a cyclical approach and involving stakeholders as co-investigators in the process . As occurs in other participatory processes, the co-design of health interventions contributes to improving the services offered, to the extent that they are adjusted to the needs and priorities of its participants while incorporating their own skills [ , , ]. In general, digital interventions, such as MOOCs, have the potential to improve the quality of life and outcomes for women with BC by providing access to information from anywhere at any time, thereby increasing accessibility and flexibility, as well as support to complement traditional medical treatments. Therefore, the aim of this study is to co-create a MOOC of PCC and DHL for and with women with BC.
2.1. Design The MOOC was co-created using a modified experience-based design approach . The co-creation process was divided into three sequential phases: (a) exploratory phase, (b) development phase and (c) evaluation phase (see ). 2.2. Participants and Recruitment Adult women (≥18 years) in any of their cancer stages and BC survivors (regardless of DHL level and knowledge about PCC), their families/carers and any healthcare professionals involved in the management of BC (oncologists, gynaecologists, nurses, psycho-oncologists, etc.) were invited to participate voluntarily in the MOOC co-creation process. A theoretical sample optimized the maximum variability of sociodemographic and clinical profiles (age, educational level, time since diagnosis and active treatment) of women with BC. The recruitment was carried out via snowball sampling through healthcare professionals and expert patients (BC survivors) between May and June 2020. Participants signed an informed consent declaration. 2.3. Procedure The co-creation process was carried out in three online sessions of 120 min each (via the Zoom platform due to the COVID-19 pandemic and delivered by members of the research team) between June 2020 and March 2021 and was supported by a Moodle platform. The first session (exploratory phase) was held in June 2020 and consisted of (i) a brief presentation of the participants; (ii) identifying the different diagnosis, treatment and long-term follow-up paths for BC represented through a patient journey map (PJM)—a scheme that aims to reflect the care pathway followed by a person —based on their experiences, emotions, feelings and thoughts; (iii) exploring their empowerment and information needs in each phase of the disease; (iv) and exploring patients’ information needs and experiences on patient empowerment and SDM. Health professionals did not participate in the development of the PJM; they offered advice and their experiences on the most frequent concerns found in clinical practice with these patients, according to the phase of the disease. In the second session (development phase), held in July 2020, the participants reviewed the PJM and designed the structure and proposed the contents of the MOOC (self-care, myths related to BC, strategies to improve DHL, etc.) based on the empowerment and information needs identified in the first session and their previous experiences managing BC information online. At the end of this session, participants were encouraged to continue the process of co-creation online between July and December 2020 through a Moodle platform where the participants were registered and which they accessed with an individual username and password (assessment phase). The research team developed and shared some content proposals weekly for the different units of the MOOC, and participants were asked to provide feedback and/or new content proposals (see ). Initially, the content of the units was presented in infographic format (see ) and was mainly related to PCC, self-care and DHL applied to BC. Once all the suggestions for improvement provided by the participants on the content were compiled, a graphic designer developed videos and edited the infographics to provide them with interactivity and visually improve their appearance. Updated contents were shared again with the participants in March 2021. Through questionnaires on the Moodle platform (see ), they could give feedback on the definitive contents of the MOOC (see ). A third session (evaluation phase) was held in March 2021 to offer final feedback about the content and interface of the MOOC (acceptability pilot) and to evaluate the experience in the co-creation of the MOOC by means of specific questionnaires (see ). Four gift cards were raffled off as a token of appreciation during this last meeting. 2.4. Measures 2.4.1. Experience in the Co-Creation Process A 13-item questionnaire was specifically developed to explore patients’ and healthcare professionals’ experience in the co-creation process. The first 6 items were measured using a 5-point Likert scale (from “strongly disagree” to “strongly agree”), addressing satisfaction with communication, objective adequacy, usefulness of patient involvement in the co-creation process, importance of co-creation to design relevant content for patients, self-perception of increased knowledge and feeling of being part of the team project. The following 4 items were also assessed on a 5-point Likert scale (from “insufficient” to “excellent”) and were related to participants’ opinions on the quality and clarity of the co-creation sessions, the methodology employed, the interactions between participants and the researchers’ implication. The last 3 items were open-ended questions about what participants liked the most and the least about the MOOC co-creation process, which aspects they found most useful and which aspects could be improved in the co-creation process (see ). 2.4.2. Acceptability Pilot of the MOOC The MOOC’s acceptability was evaluated using a specific scale created in the context of the project following the technology acceptance model’s (TAM) methodology and based on previous related studies . This scale assessed factors such as ease of navigation, clarity of objectives and language, appropriateness of learning activities and quizzes, and other characteristics of the MOOC. The acceptability questionnaire, answered by both patients and healthcare professionals, included 18 items: the first 15 were rated on a 5-point Likert scale, and the last 3 items were open-ended questions about strengths and weaknesses, improvement suggestions and the main points learned throughout the MOOC (see ). 2.5. Analysis The PJM and MOOC content were progressively developed in conjunction with participants. A draft was created with the information obtained from the online co-creation sessions. The different sections of the PJM summarize the experiences of participants with BC or survivors. The research group reviewed the contributions of the participants and proposed a draft version based on a PCC framework. Subsequently, this version of PJM and MOOC content was reviewed by all participants through an iterative process until consensus was reached. For the experience in the co-creation process and the acceptability pilot of the MOOC measures, means and standard deviations (SD) were calculated for all items assessed, and we also analysed the response distribution for each item.
The MOOC was co-created using a modified experience-based design approach . The co-creation process was divided into three sequential phases: (a) exploratory phase, (b) development phase and (c) evaluation phase (see ).
Adult women (≥18 years) in any of their cancer stages and BC survivors (regardless of DHL level and knowledge about PCC), their families/carers and any healthcare professionals involved in the management of BC (oncologists, gynaecologists, nurses, psycho-oncologists, etc.) were invited to participate voluntarily in the MOOC co-creation process. A theoretical sample optimized the maximum variability of sociodemographic and clinical profiles (age, educational level, time since diagnosis and active treatment) of women with BC. The recruitment was carried out via snowball sampling through healthcare professionals and expert patients (BC survivors) between May and June 2020. Participants signed an informed consent declaration.
The co-creation process was carried out in three online sessions of 120 min each (via the Zoom platform due to the COVID-19 pandemic and delivered by members of the research team) between June 2020 and March 2021 and was supported by a Moodle platform. The first session (exploratory phase) was held in June 2020 and consisted of (i) a brief presentation of the participants; (ii) identifying the different diagnosis, treatment and long-term follow-up paths for BC represented through a patient journey map (PJM)—a scheme that aims to reflect the care pathway followed by a person —based on their experiences, emotions, feelings and thoughts; (iii) exploring their empowerment and information needs in each phase of the disease; (iv) and exploring patients’ information needs and experiences on patient empowerment and SDM. Health professionals did not participate in the development of the PJM; they offered advice and their experiences on the most frequent concerns found in clinical practice with these patients, according to the phase of the disease. In the second session (development phase), held in July 2020, the participants reviewed the PJM and designed the structure and proposed the contents of the MOOC (self-care, myths related to BC, strategies to improve DHL, etc.) based on the empowerment and information needs identified in the first session and their previous experiences managing BC information online. At the end of this session, participants were encouraged to continue the process of co-creation online between July and December 2020 through a Moodle platform where the participants were registered and which they accessed with an individual username and password (assessment phase). The research team developed and shared some content proposals weekly for the different units of the MOOC, and participants were asked to provide feedback and/or new content proposals (see ). Initially, the content of the units was presented in infographic format (see ) and was mainly related to PCC, self-care and DHL applied to BC. Once all the suggestions for improvement provided by the participants on the content were compiled, a graphic designer developed videos and edited the infographics to provide them with interactivity and visually improve their appearance. Updated contents were shared again with the participants in March 2021. Through questionnaires on the Moodle platform (see ), they could give feedback on the definitive contents of the MOOC (see ). A third session (evaluation phase) was held in March 2021 to offer final feedback about the content and interface of the MOOC (acceptability pilot) and to evaluate the experience in the co-creation of the MOOC by means of specific questionnaires (see ). Four gift cards were raffled off as a token of appreciation during this last meeting.
2.4.1. Experience in the Co-Creation Process A 13-item questionnaire was specifically developed to explore patients’ and healthcare professionals’ experience in the co-creation process. The first 6 items were measured using a 5-point Likert scale (from “strongly disagree” to “strongly agree”), addressing satisfaction with communication, objective adequacy, usefulness of patient involvement in the co-creation process, importance of co-creation to design relevant content for patients, self-perception of increased knowledge and feeling of being part of the team project. The following 4 items were also assessed on a 5-point Likert scale (from “insufficient” to “excellent”) and were related to participants’ opinions on the quality and clarity of the co-creation sessions, the methodology employed, the interactions between participants and the researchers’ implication. The last 3 items were open-ended questions about what participants liked the most and the least about the MOOC co-creation process, which aspects they found most useful and which aspects could be improved in the co-creation process (see ). 2.4.2. Acceptability Pilot of the MOOC The MOOC’s acceptability was evaluated using a specific scale created in the context of the project following the technology acceptance model’s (TAM) methodology and based on previous related studies . This scale assessed factors such as ease of navigation, clarity of objectives and language, appropriateness of learning activities and quizzes, and other characteristics of the MOOC. The acceptability questionnaire, answered by both patients and healthcare professionals, included 18 items: the first 15 were rated on a 5-point Likert scale, and the last 3 items were open-ended questions about strengths and weaknesses, improvement suggestions and the main points learned throughout the MOOC (see ).
A 13-item questionnaire was specifically developed to explore patients’ and healthcare professionals’ experience in the co-creation process. The first 6 items were measured using a 5-point Likert scale (from “strongly disagree” to “strongly agree”), addressing satisfaction with communication, objective adequacy, usefulness of patient involvement in the co-creation process, importance of co-creation to design relevant content for patients, self-perception of increased knowledge and feeling of being part of the team project. The following 4 items were also assessed on a 5-point Likert scale (from “insufficient” to “excellent”) and were related to participants’ opinions on the quality and clarity of the co-creation sessions, the methodology employed, the interactions between participants and the researchers’ implication. The last 3 items were open-ended questions about what participants liked the most and the least about the MOOC co-creation process, which aspects they found most useful and which aspects could be improved in the co-creation process (see ).
The MOOC’s acceptability was evaluated using a specific scale created in the context of the project following the technology acceptance model’s (TAM) methodology and based on previous related studies . This scale assessed factors such as ease of navigation, clarity of objectives and language, appropriateness of learning activities and quizzes, and other characteristics of the MOOC. The acceptability questionnaire, answered by both patients and healthcare professionals, included 18 items: the first 15 were rated on a 5-point Likert scale, and the last 3 items were open-ended questions about strengths and weaknesses, improvement suggestions and the main points learned throughout the MOOC (see ).
The PJM and MOOC content were progressively developed in conjunction with participants. A draft was created with the information obtained from the online co-creation sessions. The different sections of the PJM summarize the experiences of participants with BC or survivors. The research group reviewed the contributions of the participants and proposed a draft version based on a PCC framework. Subsequently, this version of PJM and MOOC content was reviewed by all participants through an iterative process until consensus was reached. For the experience in the co-creation process and the acceptability pilot of the MOOC measures, means and standard deviations (SD) were calculated for all items assessed, and we also analysed the response distribution for each item.
Twenty-eight participants from Tenerife and Gran Canaria (Canary Islands, Spain) were contacted between May and June 2020, of whom 19 participated in the co-creation process: 17 patients ( ) and two healthcare professionals (nurses from gynaecology and breast pathology units; mean age 40 (1.41) years and with more than 10 years of professional experience). 3.1. Patient Journey Map Points of contact, experience with healthcare received, emotions, feelings and thoughts, diagnostic and therapeutic treatments, and perception about own participation in shared decision making for the three stages of the trajectory of care of BC (early detection and diagnosis, treatment and long-term follow-up) were collected on the co-designed PJM ( ). 3.1.1. Early Detection and Diagnosis Stage Most of the participants received their diagnosis during routine controls (specialized care) or as a result of the presence of symptoms (primary care), and the main emotions that emerged during this time were shock, anxiety, uncertainty and worry about the future. The main diagnostic techniques that the participants underwent were physical examination (palpation), imaging tests (mammography and ultrasound) and biopsy. The experiences collected about the healthcare received in this stage were related to the perception of professionalism, friendliness, a predisposition to resolve doubts and the transmission of calm and encouragement from the healthcare professionals who attended to them. However, the participants expressed that there were other drawbacks in the medical care received at this time related to the challenges of early detection and the complexity of some administrative processes (e.g., medical appointments). Some participants expressed that they would have liked more advice from medical staff. Other participants expressed that they felt involved in the decision-making process in this phase, and this helped them accept the disease and have trust in the therapeutic approach to be used. 3.1.2. Treatment Stage Participants identified the involvement of other healthcare professionals (e.g., oncology, gynaecology, surgery and rehabilitation, among others). While uncertainty remained the predominant emotion in this stage, other emotions started to emerge as well, including concern for appearance and shock by the physical changes that were occurring as a result of the therapeutic techniques used in this stage (e.g., chemotherapy, radiotherapy, surgery, etc.). In general, the participants experienced empathetic care and a certain psychological accompaniment by the healthcare professionals who assisted them. The participants felt more involved in the decision-making process in the gynaecology units than in the oncology units. They all concurred that the experience of informed participation in their treatment process was positive. 3.1.3. Long-Term Follow-Up Stage The main experience was less follow-up by healthcare professionals, giving rise to feelings of helplessness or loneliness and uncertainty about self-care. Other concerns, such as going back to work or looking for a new job more adapted to their health needs, were shared among the participants. The treatments at this stage focused on breast reconstruction surgery and medication. All the participants said they had received limited information on self-care, medical care to follow from this stage and possible new treatments required by healthcare professionals. However, they commented that at this stage they felt empowered to choose the aspects of their health in which they wanted to be involved, leading them to request personalized attention and to ask questions in order to be more involved in the decision-making process. 3.1.4. Recommendations of the Participants for Other Women with BC or Survivors Additionally, and at their own initiative, the participants in the co-creation sessions provided a series of recommendations or tips for other women diagnosed with BC and suggested their inclusion in the MOOC as another resource. These tips were about family, social, work and empowerment areas and specifically for each of the stages worked ( ). 3.2. Empowerment and Information Needs shows the empowerment and information needs identified in each phase. The main empowerment needs identified were related to strategies for emotional management and guidelines for self-care throughout the process from diagnosis until long-term follow-up. The main information needs were related to the lack of understanding of the meanings of biomarkers, parameters and acronyms found in reports, as well as medical jargon, treatment options and the likelihood of cancer recurrence. The need to have guidelines for accessing information and support resources available online, including association websites and online experiences of other women with BC, was highlighted. 3.3. MOOC Content Development Between July and October 2020, a weekly activity was published on the Moodle platform to carry out the process of co-design of the MOOC content. shows the themes of these activities. Finally, the MOOC was composed of five units: (i) BC (definition, types and stages, diagnostic process, treatments, myths, etc.), (ii) PCC (definition, implementation strategies, tips for preparing consultations with the healthcare professional, etc.); (iii) DHL (definition, guidelines to improve each skill, etc.), (iv) self-care (management of physical side effects, emotional management, etc.) and (v) experiences and advice from patients in different areas (healthcare, family, social and work area) and moments of the disease (diagnosis, treatment and long-term follow-up). 3.4. Experience in the Co-Creation Process Data was available for seventeen participants (89.47%) ( ). All of them strongly agreed or agreed that the general objectives of the project were adequate (item 2) and that the participation of women who have or have had BC is useful for the development of a MOOC on this content (item 3). More than 88% of the participants strongly agreed or agreed that being part of the MOOC co-creation process made the content more relevant to them (item 4) and rated the quality of the activities carried out in the co-creation process (item 7) and the methodology applied (item 8) as very good or excellent. Regarding open questions, participants appreciated the way their experiences were incorporated into the MOOC and how they felt part of something meaningful, sharing experiences with other women in similar situations (item 11). In order to fully engage in the co-creation process, participants expressed that they would have liked to attend a face-to-face session. Additionally, some participants found it challenging to devote more time to the MOOC due to personal issues (item 12). See to consult illustrative quotes from participants’ responses to open questions. 3.5. Acceptability Pilot of the MOOC Data was available for seven participants (36.84%) ( ). Combining the “totally agree” and “agree” categories, most of the participants positively evaluated the acceptability of the MOOC in terms of language, content, relevance, proposed activities and suitability of the MOOC objectives. Regarding open questions, most participants emphasized the usefulness of the MOOC’s content (especially related to SDM) and the way it is presented (through infographics and other audio-visual materials) as strengths. Nevertheless, one participant pointed out some navigation difficulties, while another emphasized the lengthy process (item 16). When it was possible, improvements suggested by participants were implemented, such as adding an initial summary of the MOOC’s content (item 17). All the contents were mentioned as important topics learned after completing the MOOC (item 18). See to consult illustrative quotes from participants’ responses to open questions.
Points of contact, experience with healthcare received, emotions, feelings and thoughts, diagnostic and therapeutic treatments, and perception about own participation in shared decision making for the three stages of the trajectory of care of BC (early detection and diagnosis, treatment and long-term follow-up) were collected on the co-designed PJM ( ). 3.1.1. Early Detection and Diagnosis Stage Most of the participants received their diagnosis during routine controls (specialized care) or as a result of the presence of symptoms (primary care), and the main emotions that emerged during this time were shock, anxiety, uncertainty and worry about the future. The main diagnostic techniques that the participants underwent were physical examination (palpation), imaging tests (mammography and ultrasound) and biopsy. The experiences collected about the healthcare received in this stage were related to the perception of professionalism, friendliness, a predisposition to resolve doubts and the transmission of calm and encouragement from the healthcare professionals who attended to them. However, the participants expressed that there were other drawbacks in the medical care received at this time related to the challenges of early detection and the complexity of some administrative processes (e.g., medical appointments). Some participants expressed that they would have liked more advice from medical staff. Other participants expressed that they felt involved in the decision-making process in this phase, and this helped them accept the disease and have trust in the therapeutic approach to be used. 3.1.2. Treatment Stage Participants identified the involvement of other healthcare professionals (e.g., oncology, gynaecology, surgery and rehabilitation, among others). While uncertainty remained the predominant emotion in this stage, other emotions started to emerge as well, including concern for appearance and shock by the physical changes that were occurring as a result of the therapeutic techniques used in this stage (e.g., chemotherapy, radiotherapy, surgery, etc.). In general, the participants experienced empathetic care and a certain psychological accompaniment by the healthcare professionals who assisted them. The participants felt more involved in the decision-making process in the gynaecology units than in the oncology units. They all concurred that the experience of informed participation in their treatment process was positive. 3.1.3. Long-Term Follow-Up Stage The main experience was less follow-up by healthcare professionals, giving rise to feelings of helplessness or loneliness and uncertainty about self-care. Other concerns, such as going back to work or looking for a new job more adapted to their health needs, were shared among the participants. The treatments at this stage focused on breast reconstruction surgery and medication. All the participants said they had received limited information on self-care, medical care to follow from this stage and possible new treatments required by healthcare professionals. However, they commented that at this stage they felt empowered to choose the aspects of their health in which they wanted to be involved, leading them to request personalized attention and to ask questions in order to be more involved in the decision-making process. 3.1.4. Recommendations of the Participants for Other Women with BC or Survivors Additionally, and at their own initiative, the participants in the co-creation sessions provided a series of recommendations or tips for other women diagnosed with BC and suggested their inclusion in the MOOC as another resource. These tips were about family, social, work and empowerment areas and specifically for each of the stages worked ( ).
Most of the participants received their diagnosis during routine controls (specialized care) or as a result of the presence of symptoms (primary care), and the main emotions that emerged during this time were shock, anxiety, uncertainty and worry about the future. The main diagnostic techniques that the participants underwent were physical examination (palpation), imaging tests (mammography and ultrasound) and biopsy. The experiences collected about the healthcare received in this stage were related to the perception of professionalism, friendliness, a predisposition to resolve doubts and the transmission of calm and encouragement from the healthcare professionals who attended to them. However, the participants expressed that there were other drawbacks in the medical care received at this time related to the challenges of early detection and the complexity of some administrative processes (e.g., medical appointments). Some participants expressed that they would have liked more advice from medical staff. Other participants expressed that they felt involved in the decision-making process in this phase, and this helped them accept the disease and have trust in the therapeutic approach to be used.
Participants identified the involvement of other healthcare professionals (e.g., oncology, gynaecology, surgery and rehabilitation, among others). While uncertainty remained the predominant emotion in this stage, other emotions started to emerge as well, including concern for appearance and shock by the physical changes that were occurring as a result of the therapeutic techniques used in this stage (e.g., chemotherapy, radiotherapy, surgery, etc.). In general, the participants experienced empathetic care and a certain psychological accompaniment by the healthcare professionals who assisted them. The participants felt more involved in the decision-making process in the gynaecology units than in the oncology units. They all concurred that the experience of informed participation in their treatment process was positive.
The main experience was less follow-up by healthcare professionals, giving rise to feelings of helplessness or loneliness and uncertainty about self-care. Other concerns, such as going back to work or looking for a new job more adapted to their health needs, were shared among the participants. The treatments at this stage focused on breast reconstruction surgery and medication. All the participants said they had received limited information on self-care, medical care to follow from this stage and possible new treatments required by healthcare professionals. However, they commented that at this stage they felt empowered to choose the aspects of their health in which they wanted to be involved, leading them to request personalized attention and to ask questions in order to be more involved in the decision-making process.
Additionally, and at their own initiative, the participants in the co-creation sessions provided a series of recommendations or tips for other women diagnosed with BC and suggested their inclusion in the MOOC as another resource. These tips were about family, social, work and empowerment areas and specifically for each of the stages worked ( ).
shows the empowerment and information needs identified in each phase. The main empowerment needs identified were related to strategies for emotional management and guidelines for self-care throughout the process from diagnosis until long-term follow-up. The main information needs were related to the lack of understanding of the meanings of biomarkers, parameters and acronyms found in reports, as well as medical jargon, treatment options and the likelihood of cancer recurrence. The need to have guidelines for accessing information and support resources available online, including association websites and online experiences of other women with BC, was highlighted.
Between July and October 2020, a weekly activity was published on the Moodle platform to carry out the process of co-design of the MOOC content. shows the themes of these activities. Finally, the MOOC was composed of five units: (i) BC (definition, types and stages, diagnostic process, treatments, myths, etc.), (ii) PCC (definition, implementation strategies, tips for preparing consultations with the healthcare professional, etc.); (iii) DHL (definition, guidelines to improve each skill, etc.), (iv) self-care (management of physical side effects, emotional management, etc.) and (v) experiences and advice from patients in different areas (healthcare, family, social and work area) and moments of the disease (diagnosis, treatment and long-term follow-up).
Data was available for seventeen participants (89.47%) ( ). All of them strongly agreed or agreed that the general objectives of the project were adequate (item 2) and that the participation of women who have or have had BC is useful for the development of a MOOC on this content (item 3). More than 88% of the participants strongly agreed or agreed that being part of the MOOC co-creation process made the content more relevant to them (item 4) and rated the quality of the activities carried out in the co-creation process (item 7) and the methodology applied (item 8) as very good or excellent. Regarding open questions, participants appreciated the way their experiences were incorporated into the MOOC and how they felt part of something meaningful, sharing experiences with other women in similar situations (item 11). In order to fully engage in the co-creation process, participants expressed that they would have liked to attend a face-to-face session. Additionally, some participants found it challenging to devote more time to the MOOC due to personal issues (item 12). See to consult illustrative quotes from participants’ responses to open questions.
Data was available for seven participants (36.84%) ( ). Combining the “totally agree” and “agree” categories, most of the participants positively evaluated the acceptability of the MOOC in terms of language, content, relevance, proposed activities and suitability of the MOOC objectives. Regarding open questions, most participants emphasized the usefulness of the MOOC’s content (especially related to SDM) and the way it is presented (through infographics and other audio-visual materials) as strengths. Nevertheless, one participant pointed out some navigation difficulties, while another emphasized the lengthy process (item 16). When it was possible, improvements suggested by participants were implemented, such as adding an initial summary of the MOOC’s content (item 17). All the contents were mentioned as important topics learned after completing the MOOC (item 18). See to consult illustrative quotes from participants’ responses to open questions.
This study presents the development of a MOOC aimed to improve the DHL of women with BC. We used a co-creation approach involving 17 patients and survivors and two nurses. In order to inform the content of the MOOC, we explored participants’ perceptions of the extent they were involved in the decision-making process, as well as their feelings, emotions and information needs throughout the therapeutic process. Most participants indicated that the MOOC co-creation experience was positive and made them feel involved in the project, and they positively valued the final product. Similar results were obtained by our team with other MOOCs developed for pregnant and lactating women and people with type-2 diabetes , including larger samples than the one used in this study. In these two studies, participants’ self-perceived DHL significantly improved after completing the MOOC development compared to baseline. Future work is warranted to evaluate the effectiveness of this MOOC at improving BC patients’ actual DHL (not only self-perceived), objective knowledge of the disease and treatments, and their involvement in treatment decisions. Women in this study pointed out information needs concerning different stages of the cancer, from diagnosis to long-term follow-up, as shown in previous studies . Increasingly, these patients want to be involved in the decisions related to their health, and some studies have focused on involving the patient experience to improve the healthcare they receive . As a result of an exchange of information and values between patients and healthcare providers, SDM engages patients as partners in their own care and optimizes the decision-making process . To support SDM and the use of PtDA in the practice, it is important that patients also have a certain level of HL to increase patient empowerment and allow them to adopt a more participatory role in their healthcare . Online interventions that provide information and support to women with BC appear to cushion the uncertainty they experience at different stages of the disease, and MOOCs can be an effective educational resource for meeting these unmet needs and promoting both DHL and SDM processes . The PJM considered the evolving requirements for empowerment during the stages of diagnosis, treatment and long-term follow-up. Knowledge of the patients’ experiences, through a PJM, facilitates the identification of key moments in which to provide more precise information . As we have seen in the results of this study, depending on the individual experiences of each woman, the care received during various BC periods could be perceived as more or less satisfactory. Based on our results, women with BC positively valued the experience of participating in the co-creation process of the MOOC, which made the content more relevant to them. This result aligns with previous evidence suggesting that a user-centred design process involves the participation of groups of users throughout the entire development cycle, during which they describe the context in which the generated resources will be used, their needs as users, and take part in user tests . These are all contributions for designing and building health information technology through iterations . This intervention represents an opportunity to reach a larger population that, due to health, availability and/or travel circumstances, may find it impossible to attend another type of face-to-face training on this subject. Technology provides great options for enhancing patient care; however, disparities in access and DHL continue to negatively impact vulnerable populations because of potential barriers in the digital sphere for those with low HL . This problem can be especially aggravated as more information is provided online and healthcare professionals must be involved in the development of these skills in their patients with BC, but they also require support and a strategy at the institutional level. Therefore, healthcare organizations must prioritize achieving accessibility for all patients when designing eHealth services . In this regard, the integration of educational materials designed by a representative sample of the target population to which they are addressed makes this proposal an opportunity to contribute to obtaining relevant health results for both affected patients and their healthcare professionals and, ultimately, decision-makers with financial capacity. From a managerial perspective, healthcare organizations should reframe their strategies, procedures and approaches, embracing a patient-centred perspective to become health literate . From a policy perspective, it suggests that individual HL and organizational HL should be handled as two complementary tools to empower people and to engage them in self-care and health policy making . The main strength of this project is having involved the intended audience in the creation of MOOCs, which enhances the significance of the material covered and how it is delivered. This is important because they have valuable insights and perspectives on the subject matter and can provide feedback on the relevance and effectiveness of the content and its delivery. This can lead to the creation of more engaging and effective MOOCs that better meet the needs and expectations of the intended audience. Nonetheless, there are several limitations to the study. Initially, it had been proposed that the co-creation process be based on face-to-face sessions with the participants followed by some online sessions through the Moodle platform. However, due to the COVID-19 pandemic, face-to-face sessions were replaced by online sessions carried out through the Zoom platform. This fact made the co-creation process last a few weeks longer than expected by adapting the work rhythms to the availability and web resources of the participants. However, the online sessions had several advantages: participants did not have to travel, the meetings were easier to organize and fewer financial resources were needed to support the development of the sessions. Another limitation is that, although all professionals related to BC were invited to participate, only two nurses did so. Perhaps the participation of other professionals involved in the process (e.g., gynaecologists and oncologists), as well as family members and/or caregivers, could have been beneficial for the generation of more useful resources. Even though women of all educational levels participated, the majority had higher education, so there was not much variability in this regard and lower educational levels may have been under-represented. In addition, there is a need for independent evaluation of acceptability to confirm the results obtained. Likewise, it is necessary to carry out an evaluation of the effectiveness of the MOOC with an independent sample that allows us to know if there really is an improvement in the levels of DHL and a change in knowledge in all the areas that are included in the different modules of the MOOC (BC, PCC, DHL, etc.).
The work carried out in this project is an example of how the development of educational interventions in MOOC format directed and designed by women with BC, with resources in different formats adapted to different educational/cultural levels and needs of the users, seems to be a viable strategy to generate higher-quality and useful resources for this population. The co-creation methodology and this type of resource aim to address the literacy and empowerment challenges of women with BC.
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Comparison of Frailty Assessment Tools for Older Thai Individuals at the Out-Patient Clinic of the Family Medicine Department | f54bceae-cb01-428e-bb96-f1a3598691db | 10001464 | Family Medicine[mh] | Frailty is defined as a reduction in the ability to cope with everyday or acute stressors, particularly among older adults . Frailty results in an increased vulnerability brought about by age-associated declines in physiological reserves and functioning across multiple organ systems . The consequences of this condition heighten an individual’s susceptibility to increased dependency and vulnerability, as well as to an increased risk of death . The health care system is affected by increases in health care needs, admissions to hospital, and admissions to long-term care. However, frailty is a dynamic process which can emerge from pre-frail or robust statuses . Validated assessment tools and appropriate interventions are important to reduce morbidity and mortality. A systematic review and meta-analysis of a survey of the models used to evaluate frailty among ≥ 50-year-olds in 62 countries found that 12% of prevalence used physical frailty models and 24% used deficit accumulation models. The prevalences of the consideration of pre-frailty were 46% and 49% for the physical frailty models and the deficit accumulation models, respectively . In terms of geographical location, using physical frailty models, the highest prevalence of physical frailty was found in Africa (22%) and the lowest prevalence was in Europe (8%), while the pre-frailty prevalence was highest in the Americas (50%) and lowest in Europe (42%). However, using deficit accumulation models, the prevalence of frailty was found to be highest in Oceania (31%) and lowest in Europe (22%), while pre-frailty prevalence was highest in Oceania (51%) and lowest in Europe and Asia (49%). The population-level frailty prevalence among community-dwelling adults varied by age, gender, and frailty classification . Several studies have reported that frailty is related to a variety of negative health outcomes and diseases. In 2013, cognitive frailty was described as a group of heterogeneous clinical symptoms based on the presence of both physical frailty and cognitive impairment, excluding consistent Alzheimer’s disease or other dementias. The prevalence of cognitive frailty among community-dwelling older adults was reported to be 9% in a systematic review and meta-analysis . Similarly, the prevalences of frailty and pre-frailty were found to be 20.1% and 49.1%, respectively, in a systematic review and meta-analysis study of community-dwelling older adults with diabetes. Older adults with diabetes were more susceptible to being frail than those without diabetes . Additional factors were found to have an influence on frailty; for example, fruit and vegetable consumption was associated with a lower risk of frailty . There are many measurement tools available which can provide frailty scores when used to screen for or assess the degree of frailty; however, no single score metric is considered the gold standard . It has been recommended that geriatricians in the Asia-Pacific region use a validated measurement tool to identify frailty . There are three major approaches used, i.e., the physical frailty phenotype model of Fried et al. and its rapid screening tool, FRAIL; the deficit accumulation model of Rockwood and Mitnitski, which captures multimorbidity; and mixed physical and psychosocial models, such as the Tilburg Frailty Indicator and the Edmonton Frailty Scale . Another approach by Aguayo GA et al. consists of the use of four models, including a phenotype of the frailty model, a multidimensional model, an accumulation of deficits model, and a disability model. The most commonly used method in the literature is the physical frailty phenotype . The phenotype diagnosis is based on three of the following five criteria: weight loss, exhaustion, physical inactivity, slow walking speed, and weak grip strength . The present study reviews five phenotypic criteria that have been measured in different ways across various studies which could potentially affect the estimates of the prevalence of frailty and the predictive ability of the aforementioned phenotype, potentially leading to different classifications and results . Kutner and Zhang commented on the replacement of the performance-based measures (i.e., grip strength and walking speed) in the original frailty phenotype definition with self-reported items. In Thailand, a study by Boribun N. et. al. found that the prevalence of frailty in Thai community-dwelling older adults was 24.6%, based on the Frail Non-Disabled (FiND) questionnaire. A 2020 study by Sukkriang and Punsawad , which used various frailty assessment tools, found that the prevalence of frailty of older individuals in Thai communities was 11.7%, using Fried’s Frailty Phenotype (Cardiovascular Heart Study) criteria, and studied the validity of various frailty assessment tools. The Clinical Frailty Scale (CFS) used in the same study had a sensitivity of 56% and a specificity of 98.41%; the simple FRAIL questionnaire had a sensitivity of 88% and a specificity of 85.71%; the PRISMA-7 questionnaire sensitivity was 76%; and the specificity was 86.24%. The Timed Up and Go (TUG) test had a sensitivity of 72% and a specificity of 82.54%. The Gerontopole frailty screening tool (GFST) sensitivity was 88% and the specificity was 83.56%. The study by Sriwong et al. (2022) developed a Thai version of the Simple Frailty Questionnaire (T-FRAIL) and modified it to improve its diagnostic properties in the preoperative setting. Their study found that the incidence of frailty diagnosed using the Thai Frailty Index was 40.0%. The identification of frailty using a score of two points or more provided the best Youden index, at 63.1, with a sensitivity of 77.5% (95% CI 69.0–84.6) and a specificity of 85.6% (95% CI 79.6–90.3). There is currently a need for simple, valid, accurate, and reliable methods and tools for detecting frailty which are appropriate for the Thai population. Our team works in an academic hospital and has developed evidence data in our clinic in the hospital. Therefore, the present study was conducted in this clinic. This study compared selected frailty assessment tools, including Fried’s Frailty phenotype (FFP), which is the most commonly used assessment tool used for reference; the Frailty Assessment Tool of the Thai Ministry of Public Health (FATMPH), which is recommended in the Thai check-up manual but lacks published validation; and the FiND questionnaire, which is used in communities but, as yet, there is no evidence of its use at the Out-Patient Department (OPD) of Maharaj Nakorn Chiang Mai Hospital (a university-level hospital).
2.1. Samples This cross-sectional study included 251 older patients (age 60 years or older) who came to the OPD of the Family Medicine Department, Maharaj Nakorn Chiang Mai Hospital, Faculty of Medicine, Chiang Mai University, during the period of December 2016–March 2017. The patients signed a consent form declaring their agreement to participate in this research. This study was approved by the Research Ethics Committee of the Faculty of Medicine of Chiang Mai University (no. 380/2016). The inclusion criteria for participants were: (1) Thais 60 years or older and who had been seen at the OPD for more than 1 year, (2) the ability to communicate orally in Thai and read the Thai language, (3) the ability to walk by themselves or with walking aids. The exclusion criteria were: (1) being bed ridden, (2) being handicapped in both hands, (3) currently having a serious illness, and (4) having impaired cognition. The sample size was calculated to be 230 using the following formula: n = Z2α/2 × Se(1 − Se)/d2 × Prev where n = sample size, Se = sensitivity (0.9), Prev = prevalence (0.15) , d = precision of the estimate (1.0), and alpha = 0.1. 2.2. Frailty Assessment Tools 2.2.1. Fried’s Frailty Phenotype The five criteria of Fried’s Frailty Phenotype (FFP) assessment were used as the reference assessment tool in this study, following Fried et al. , with slight modification. These criteria were: (1) Weight loss. My weight has decreased at least 4.5 kg in the past year or I have had an unintentional weight loss of at least 5% of my previous year’s body weight (no = 0, yes = 1). (2) Exhaustion. Self-reported results of the Center for Epidemiologic Studies Depression scale (CES–D). Two statements were provided: (2.1) I felt that everything I did was an effort and (2.2) I could not get going. The question is then asked, “How often in the last week did you feel this way?” The alternative answers are: 0 = rarely or none of the time (<1 day), 1 = some or a little of the time (1–2 days), 2 = a moderate amount of the time (3–4 days), or 3 = most of the time. Answers of “2” or “3” to either of these questions were categorized as frail by the exhaustion criterion (no = 0, yes = 1). (3) Slowness. My walking speed is 20% below baseline (adjusted for gender and height) (no = 0, yes = 1). (4) Weakness. Grip strength is 20% below baseline (adjust for gender and body mass index) (no = 0, yes = 1). (5) Low activity was evaluated with the following question: How often do you engage in activities that require a low or moderate amount of energy such as gardening, cleaning the car, or walking? (more than once a week = 1, once a week = 2, one to three times a month = 3 and hardly ever or never = 4) . A combined FFP score of 0 was considered a “non-frail” phenotype; a score of 1 or 2 was considered a “pre-frail” phenotype; and a score of 3 or more was considered a “frail” phenotype. 2.2.2. Frailty Assessment Tool of the Thai Ministry of Public Health The Frailty Assessment Tool of the Thai Ministry of Public Health (FATMPH) is a modification of Fried’s Frailty Phenotype, and is included in the Elderly Screening/Assessment Manual (2015) . The assessment tool has 5 criteria: four questions are self-reports and one is based on measurement by medical staff: (1) In the past year, has your weight has decreased by more than 4.5 kg? (no = 0, yes = 1) (2) Do you feel tired all the time? (no = 0, yes = 1) (3) Are you unable to walk alone and need someone for support? (no = 0, yes = 1) (4) The participants walked in a straight line for a distance of 4.5 m. Time was measured from when they started walking (Time < 7 s = 0, time ≥ 7 s or could not walk = 1). (5) The participant had an obvious weakness in their hands, arms, and legs (no = 0, yes = 1). A FATMPH score of 0 was considered a phenotype of “non-frail”; a score of 1 or 2 was considered a phenotype of “pre-frail”; and a score of 3 or more was consider a phenotype of “frail”. 2.2.3. Frail Non-Disabled (FiND) Questionnaire The Frail Non-Disabled (FiND) questionnaire is designed to differentiate between frailty and disability. FiND was used for community-dwelling older Thai adults by Boribun N et. al. . The content validity index (CVI) was 0.8 and Cronbach’s alpha was 0.89 . The FiND questionnaire consists of 5 questions: Do you have any difficulty walking 400 m? (no or some difficulty = 0, much difficulty or unable = 1) Do you have any difficulty climbing up a flight of stairs? (no or some difficulty = 0, much difficulty or unable = 1) During the last year, have you involuntarily lost more than 4.5 kg? (no = 0, yes = 1) How often in the last week did you feel that everything you did was an effort or that you could not get going? (2 times or less = 0, 3 or more times = 1) What is your level of physical activity? (at least 2–4 h per week = 0, mainly sedentary = 1) A combined score of A + B + C + D + E = 0 was considered as “non-frail”; A + B = 0 and C + D + E ≥ 1 was considered as “frail”; and A + B ≥ 1 was considered as “disabled”. 2.3. Data Collection Data were collected using questionnaires and assessed using various tools. The general characteristics recorded included age, sex, religion, education, income, source of payment of medical expenses, history of family disease, present weight, weight one year ago, height, and body mass index. All participants were assessed using the Thai-language version of FATMPH, FFP, and FiND. The inter-rater reliability was 1.0 between researchers and assistants. 2.4. Statistical Analysis The data were analyzed using Stata 12.0 and are presented as frequency, percentage, mean, and standard deviation (SD). The frailty assessment tools were analyzed for their sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV); Cohen’s kappa was used to measure the reliability of these assessment tools. 2.5. Evaluation Consequence All frail participants who were involved in any of the study of the assessment tools were advised to undergo comprehensive geriatric assessment. The appropriate interventions were then provided to these individuals.
This cross-sectional study included 251 older patients (age 60 years or older) who came to the OPD of the Family Medicine Department, Maharaj Nakorn Chiang Mai Hospital, Faculty of Medicine, Chiang Mai University, during the period of December 2016–March 2017. The patients signed a consent form declaring their agreement to participate in this research. This study was approved by the Research Ethics Committee of the Faculty of Medicine of Chiang Mai University (no. 380/2016). The inclusion criteria for participants were: (1) Thais 60 years or older and who had been seen at the OPD for more than 1 year, (2) the ability to communicate orally in Thai and read the Thai language, (3) the ability to walk by themselves or with walking aids. The exclusion criteria were: (1) being bed ridden, (2) being handicapped in both hands, (3) currently having a serious illness, and (4) having impaired cognition. The sample size was calculated to be 230 using the following formula: n = Z2α/2 × Se(1 − Se)/d2 × Prev where n = sample size, Se = sensitivity (0.9), Prev = prevalence (0.15) , d = precision of the estimate (1.0), and alpha = 0.1.
2.2.1. Fried’s Frailty Phenotype The five criteria of Fried’s Frailty Phenotype (FFP) assessment were used as the reference assessment tool in this study, following Fried et al. , with slight modification. These criteria were: (1) Weight loss. My weight has decreased at least 4.5 kg in the past year or I have had an unintentional weight loss of at least 5% of my previous year’s body weight (no = 0, yes = 1). (2) Exhaustion. Self-reported results of the Center for Epidemiologic Studies Depression scale (CES–D). Two statements were provided: (2.1) I felt that everything I did was an effort and (2.2) I could not get going. The question is then asked, “How often in the last week did you feel this way?” The alternative answers are: 0 = rarely or none of the time (<1 day), 1 = some or a little of the time (1–2 days), 2 = a moderate amount of the time (3–4 days), or 3 = most of the time. Answers of “2” or “3” to either of these questions were categorized as frail by the exhaustion criterion (no = 0, yes = 1). (3) Slowness. My walking speed is 20% below baseline (adjusted for gender and height) (no = 0, yes = 1). (4) Weakness. Grip strength is 20% below baseline (adjust for gender and body mass index) (no = 0, yes = 1). (5) Low activity was evaluated with the following question: How often do you engage in activities that require a low or moderate amount of energy such as gardening, cleaning the car, or walking? (more than once a week = 1, once a week = 2, one to three times a month = 3 and hardly ever or never = 4) . A combined FFP score of 0 was considered a “non-frail” phenotype; a score of 1 or 2 was considered a “pre-frail” phenotype; and a score of 3 or more was considered a “frail” phenotype. 2.2.2. Frailty Assessment Tool of the Thai Ministry of Public Health The Frailty Assessment Tool of the Thai Ministry of Public Health (FATMPH) is a modification of Fried’s Frailty Phenotype, and is included in the Elderly Screening/Assessment Manual (2015) . The assessment tool has 5 criteria: four questions are self-reports and one is based on measurement by medical staff: (1) In the past year, has your weight has decreased by more than 4.5 kg? (no = 0, yes = 1) (2) Do you feel tired all the time? (no = 0, yes = 1) (3) Are you unable to walk alone and need someone for support? (no = 0, yes = 1) (4) The participants walked in a straight line for a distance of 4.5 m. Time was measured from when they started walking (Time < 7 s = 0, time ≥ 7 s or could not walk = 1). (5) The participant had an obvious weakness in their hands, arms, and legs (no = 0, yes = 1). A FATMPH score of 0 was considered a phenotype of “non-frail”; a score of 1 or 2 was considered a phenotype of “pre-frail”; and a score of 3 or more was consider a phenotype of “frail”. 2.2.3. Frail Non-Disabled (FiND) Questionnaire The Frail Non-Disabled (FiND) questionnaire is designed to differentiate between frailty and disability. FiND was used for community-dwelling older Thai adults by Boribun N et. al. . The content validity index (CVI) was 0.8 and Cronbach’s alpha was 0.89 . The FiND questionnaire consists of 5 questions: Do you have any difficulty walking 400 m? (no or some difficulty = 0, much difficulty or unable = 1) Do you have any difficulty climbing up a flight of stairs? (no or some difficulty = 0, much difficulty or unable = 1) During the last year, have you involuntarily lost more than 4.5 kg? (no = 0, yes = 1) How often in the last week did you feel that everything you did was an effort or that you could not get going? (2 times or less = 0, 3 or more times = 1) What is your level of physical activity? (at least 2–4 h per week = 0, mainly sedentary = 1) A combined score of A + B + C + D + E = 0 was considered as “non-frail”; A + B = 0 and C + D + E ≥ 1 was considered as “frail”; and A + B ≥ 1 was considered as “disabled”.
The five criteria of Fried’s Frailty Phenotype (FFP) assessment were used as the reference assessment tool in this study, following Fried et al. , with slight modification. These criteria were: (1) Weight loss. My weight has decreased at least 4.5 kg in the past year or I have had an unintentional weight loss of at least 5% of my previous year’s body weight (no = 0, yes = 1). (2) Exhaustion. Self-reported results of the Center for Epidemiologic Studies Depression scale (CES–D). Two statements were provided: (2.1) I felt that everything I did was an effort and (2.2) I could not get going. The question is then asked, “How often in the last week did you feel this way?” The alternative answers are: 0 = rarely or none of the time (<1 day), 1 = some or a little of the time (1–2 days), 2 = a moderate amount of the time (3–4 days), or 3 = most of the time. Answers of “2” or “3” to either of these questions were categorized as frail by the exhaustion criterion (no = 0, yes = 1). (3) Slowness. My walking speed is 20% below baseline (adjusted for gender and height) (no = 0, yes = 1). (4) Weakness. Grip strength is 20% below baseline (adjust for gender and body mass index) (no = 0, yes = 1). (5) Low activity was evaluated with the following question: How often do you engage in activities that require a low or moderate amount of energy such as gardening, cleaning the car, or walking? (more than once a week = 1, once a week = 2, one to three times a month = 3 and hardly ever or never = 4) . A combined FFP score of 0 was considered a “non-frail” phenotype; a score of 1 or 2 was considered a “pre-frail” phenotype; and a score of 3 or more was considered a “frail” phenotype.
The Frailty Assessment Tool of the Thai Ministry of Public Health (FATMPH) is a modification of Fried’s Frailty Phenotype, and is included in the Elderly Screening/Assessment Manual (2015) . The assessment tool has 5 criteria: four questions are self-reports and one is based on measurement by medical staff: (1) In the past year, has your weight has decreased by more than 4.5 kg? (no = 0, yes = 1) (2) Do you feel tired all the time? (no = 0, yes = 1) (3) Are you unable to walk alone and need someone for support? (no = 0, yes = 1) (4) The participants walked in a straight line for a distance of 4.5 m. Time was measured from when they started walking (Time < 7 s = 0, time ≥ 7 s or could not walk = 1). (5) The participant had an obvious weakness in their hands, arms, and legs (no = 0, yes = 1). A FATMPH score of 0 was considered a phenotype of “non-frail”; a score of 1 or 2 was considered a phenotype of “pre-frail”; and a score of 3 or more was consider a phenotype of “frail”.
The Frail Non-Disabled (FiND) questionnaire is designed to differentiate between frailty and disability. FiND was used for community-dwelling older Thai adults by Boribun N et. al. . The content validity index (CVI) was 0.8 and Cronbach’s alpha was 0.89 . The FiND questionnaire consists of 5 questions: Do you have any difficulty walking 400 m? (no or some difficulty = 0, much difficulty or unable = 1) Do you have any difficulty climbing up a flight of stairs? (no or some difficulty = 0, much difficulty or unable = 1) During the last year, have you involuntarily lost more than 4.5 kg? (no = 0, yes = 1) How often in the last week did you feel that everything you did was an effort or that you could not get going? (2 times or less = 0, 3 or more times = 1) What is your level of physical activity? (at least 2–4 h per week = 0, mainly sedentary = 1) A combined score of A + B + C + D + E = 0 was considered as “non-frail”; A + B = 0 and C + D + E ≥ 1 was considered as “frail”; and A + B ≥ 1 was considered as “disabled”.
Data were collected using questionnaires and assessed using various tools. The general characteristics recorded included age, sex, religion, education, income, source of payment of medical expenses, history of family disease, present weight, weight one year ago, height, and body mass index. All participants were assessed using the Thai-language version of FATMPH, FFP, and FiND. The inter-rater reliability was 1.0 between researchers and assistants.
The data were analyzed using Stata 12.0 and are presented as frequency, percentage, mean, and standard deviation (SD). The frailty assessment tools were analyzed for their sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV); Cohen’s kappa was used to measure the reliability of these assessment tools.
All frail participants who were involved in any of the study of the assessment tools were advised to undergo comprehensive geriatric assessment. The appropriate interventions were then provided to these individuals.
The demographic characteristics of the 251 older participants from the OPD are shown in . Most were female and ranged in age from 60 to 69. The majority of the participants were married or living with a partner, had lower than a high school education, and were Buddhist. Half the participants were government officials. Most participants had an income of more than 10,000 baht per month. Their major source of income was from pensions, which provided an adequate income. The health status of the participants is shown in . Several medical conditions were identified among the participants. The most prevalent was hypertension, followed (in declining order of incidence) by dyslipidemia, diabetes mellitus, hyperuricemia, glaucoma or cataracts, chronic kidney disease, benign prostatic hypertrophy, coronary artery disease, cerebrovascular disease, and malignancy, followed by others. In this study, frailty status was evaluated using frailty assessment tools including FFP, FATMPH, and FiND. The frail and non-frail phenotypes were defined based on the combined results of all the assessment tools. The study found that the overall prevalence of frailty was 8.37% based on FFP, most of whom were female (90.47%). The frailty phenotype prevalence determined using FATMPH was 17.53% (female = 65.91%); using FiND, the frailty phenotype prevalence determined was 3.98% (female = 80.00%) ( and ). The sensitivity, specificity, positive predictive value, and negative predictive value of the FATMPH and FiND tools were analyzed and compared with the standard FFP tool. As shown in , FATMHP had a sensitivity of 57.14%, a specificity of 86.09%, a positive predictive value (PPV) of 27.27%, and a negative predictive value (NPV) of 95.65%. FiND had a sensitivity of 19.05%, a specificity of 97.39%, a PPV of 40.00% and an NPV of 92.94%. The comparison of FATMPH and FiND with FFP found the Cohen kappa statistics to be 0.298 for FATMPH and 0.147 for FiND.
Fried’s Frailty Phenotype (FFP) is a well-known and regularly utilized tool for identifying frailty in older individuals . In Thailand, FATMPH was developed as a frailty assessment tool based on FFP. Even though the Fried criteria were not initially intended to be used as a self-reported questionnaire, researchers now usually employ modified questionnaires based on this frailty phenotype . The Frail Non-Disabled (FiND) questionnaire, a self-administered frailty screening instrument designed to differentiate frailty from disability, was developed as a screening tool . We focused on the comparison of both FATMPH and FiND with FFP, which is currently used to assess older patients at the OPD of the Family Medicine Department of the Maharaj Nakorn Chiang Mai Hospital Faculty of Medicine. Most of the participants had a chronic disease (92.43%), most frequently hypertension (65.75%). The prevalence of frailty in this study was 8.37% using FFP, which is lower than the prevalence of frailty among community-based elderly people (9.9%) . Differences in frailty prevalence were due at least in part to differences in the assessment tools used, as well as the different geographical locations covered in this study. Frailty prevalence increased with age and was higher for females than males . The relatively low prevalence of frailty in the study may be due to the fact that most of the participants were in the younger group of the elderly participants (60–69 years, 65.34%). A screening test is defined as a medical test or procedure performed on members (subjects) of a defined asymptomatic population or population subgroup to assess the likelihood of their members having a particular disease or condition . A screening test has only two possible outcomes: positive, suggesting that the subject has the disease or condition; or negative, suggesting that the subject does not have the disease or condition . In prior research, a Korean version of the FRAIL scale (K-FRAIL) was found to be consistent with the multidimensional frailty index and to be a concise tool for screening for frailty in a clinical setting in Korea . In Thailand, many frailty assessment tools have been established for use both for community-dwelling individuals and in hospitals . There have, however, been few studies in Thailand that have included a comparison and validation of the frailty assessment tools used for older Thai adults in order to evaluate their diagnostic efficacy. A previous comparative study of the Thai version of the Simple Frailty Questionnaire (T-FRAIL) and the Thai Frailty Index (TFI) found that T-FRAIL was valid and reliable for frailty detection in elderly patients at a surgery out-patient clinic . Another study of community-dwelling elderly compared several screening tests, including CFS, the simple FRAIL questionnaire, the PRISMA-7 questionnaire, the TUG, and the GFST with Fried’s Frailty Phenotype method. That study found the simple FRAIL questionnaire and the GFST were the most appropriate tests for screening frailty due to their high sensitivity . The present study is the first study to compare the use of FATMPH and FiND with FFP regarding patients in an OPD for older Thai adults. The comparison of FATMPH and FiND found that the sensitivity of FATMPH (57.14%) was higher than that of FiND (19.05%), but that the specificity of FATMPH (86.09%) was lower than that of FiND (97.39%). FATMPH and FiND were both had a lower sensitivity than CFS (56%), the simple FRAIL questionnaire (88%), the PRISMA-7 questionnaire (76%), the TUG (72%), and the GFST (88%), as reported in the study by Sukkriang and Punsawad , as well as the modified T-Frails, including T-Frail M1 (83.3%) and T-Frail M2 (85.8%), as reported in the study by Sriwong . However, the categorizations of FiND (non-frail, frail, and disabled) are different from that of both FATMPH and FFP (non-frail, pre-frail, and frail), which could affect the sensitivity of the tests and which might be a reason that FiND had the lowest sensitivity in the present study. FATMPH had a higher sensitivity than FiND because it was modified from FFP, but its sensitivity as a screening tool remains poor. In addition, FATMPH and FiND both had high specificity, similar to other tools used in previous studies . Most of the screening tools had a specificity of higher than 85%: CSF, at 98.41%, as found in a previous study ; and FiND, at 97.39%, as found in the present study. The sensitivity of both FATMPH and FiND were lower than 85%, suggesting that neither is an adequate screening tool , while the high specificities of both CSF and FiND suggest they are appropriate for confirming the absence of the condition. FiND is a self-assessment questionnaire suitable for use for individuals in communities, as well as in primary care, whereas FFP is appropriate in primary care and acute care for both individuals in communities and in clinical settings, although the assessment time of FFP is longer than that of FiND . The final judgement of whether or not these methods are appropriate will depend on the context. If the score is used as part of a sequence of screening steps, sensitivity is likely to be more important than specificity, while if the score is used to guide treatment initiation, specificity is equally important . The reliability of FATMPH and FiND were compared with FFP and evaluated using Cohen’s kappa statistic. The kappa values of FATMPH and FiND were 0.289 (95% CI = 0.132–0.445) and 0.147 (95% CI = 0.004–0.241), respectively. The levels of agreement of these values were fair (0.21 ≤ K ≤ 0.40) and slight (0.00 ≤ K ≤ 0.20) , respectively. Additionally, in a research context, this measure depends on the prevalence of the condition (with a very low prevalence, κ will be very low, even with high agreement between the raters) . FATMPH’s kappa agreement level was higher than FiND because FATMPH was modified from FFP. Aguayo GA et al. , in a study of the agreement between 35 published frailty scores in the general population, found a very wide range of agreement (Cohen’s kappa = 0.10–0.83). The frailty phenotype properties were impacted by the modified frailty phenotype criteria . The prevalence of frailty was 31.2% for modified self-reported walking, 33.6% for modified self-reported strength, and 31.4% for modified self-reported walking and strength . The agreement with the primary phenotype was 0.651 for modified self-reported walking, 0.913 for modified self-reported strength, and 0.441 for modified self-report walking and strength . FATMPH had a lower agreement (0.268) than that of the Modified Frailty Phenotype. We think that the physical inactivity criteria of FATMPH, i.e., the “Can you walk by yourself or do you need someone help you? (no = 0, yes = 1)” should be re-evaluated, as it appears to be very similar to the walk speed criteria (4.5 m walk time; <7 s = 0, ≥7 s or cannot walk = 1). FFP has two measurements (grip strength and walking speed), but FATMPH uses only walking speed and includes fewer detailed questions. Frailty scores show marked heterogeneity because they are based on different concepts of frailty and research results based on different frailty scores cannot be compared or pooled . A limitation of our study is that it was not representative of all community-dwelling older Thais because the participants were all older patients at the OPD of an academic hospital (Maharaj Nakorn Chiang Mai Hospital) and most were urban residents receiving regular government welfare payments. Further study of validated frailty assessment tools such as multicenter studies, as well as other assessment tools, are necessary to ensure their suitability for the Thai population context.
Our academic hospital-based study using the Thai-language version of the Frailty Assessment Tool of the Thai Ministry of Public Health (FATMPH) and the FiND questionnaire found that both have only a fair to slight agreement with Fried’s Frailty Phenotype (FFP). Additionally, their predictive power is low and, thus, insufficient for frailty detection in a clinical setting. Further multicenter study of these and other assessment tools is needed to improve frailty screening in older Thai populations.
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