Determination of well-being-related markers in nails by liquid chromatography tandem mass spectrometry.

Well-being is a multifactorial positive state that is highly influenced by some endogenous molecules that control happiness and euphoric feelings. These molecules, e.g., neurotransmitters, hormones and their derivatives, play a crucial role in metabolism and may be referred to as "well-being-related markers". The deregulation of well-being-related markers can lead to organism malfunctions and life-threatening states. In this research, we aimed to evaluate the potential of nails for the chronic production of several well-being-related markers. For this purpose, we developed an LCMS /MS-based method for the determination of 10 well-being-related markers, including melatonin, serotonin, cortisol, kynurenine and several precursors and metabolites. The method was optimized regarding different analytical steps: required sample amount, extraction time, number of required extractions, preconcentration, injection volume and MS conditions. Method validation was performed by two different approaches: (i) using surrogate nail matrix and (ii) using authentic nail samples by standard additions. The method was found to be linear in the expected endogenous range and sensitive enough to determine the low endogenous concentration levels in nails. Accuracy and precision were appropriate in both validation approaches. As proof of concept, the method was used (i) to correlate fingernail and toenail levels for all metabolites in 22 volunteers, (ii) to establish the endogenous concentration range of all metabolites in females (n = 50) and males (n = 34) and (iii) to correlate the metabolite levels with age. For some metabolites, the calculated ranges have been reported for the first time. In summary, the present strategy to evaluate well-being-related markers in nails may be a useful tool for the evaluation of the production of these important compounds with high potential for a wide range of clinical purposes.

Psychosocial needs of pediatric cancer patients and their caregivers at end of treatment: Why psychosocial screening remains important.

OBJECTIVE: The pediatric cancer Psychosocial Standards of Care calls for psychosocial screening across the cancer trajectory. The current study aims to describe pediatric cancer family needs at the end of treatment (EOT) and summarize feedback on a clinical EOT screening and education program. METHODS: During a clinic visit, families attended an education session regarding general EOT considerations and caregivers and youth aged 11+ years completed questionnaires. Scores were coded for clinical significance based on cutoff scores per questionnaire, and clinical significance frequencies were calculated. Caregivers provided qualitative feedback on the EOT program via an open-ended prompt. RESULTS: Screening was completed by 151 families. Ninety-four patients (67.1%) endorsed risk by self- or proxy-report in at least one domain. Across all patient age groups, a symptom of neurocognitive functioning was the most frequently endorsed risk, including executive functioning, sustained focused, and thinking slower than others. For caregivers, 106 (74.1%) endorsed risk in at least one domain, with concerns for ability to manage their child's medical condition as the most frequent endorsement. Families were agreeable to an EOT program with many caregivers advocating for receiving this program earlier. CONCLUSIONS: Both patients and caregivers experienced clinically significant needs that require intervention at EOT. While patients are experiencing neurocognitive effects and distress, their caregivers are balancing management of their own distress with management of their child's needs during a transition to decreased support from the medical team. The findings affirm the need for systematic screening at EOT and anticipatory guidance for off treatment expectations.

Laboratory interpretative comments and guidance: clinical and operative outcomes on moderate to severe hyponatraemia patient management.

AIMS: Hyponatraemia is the most common body fluid disorders but often goes unnoticed. Our laboratory incorporated a standardised procedure to help clinicians detect moderate/severe hyponatraemia. The study aims were to evaluate the outcomes on patient care and clinicians' satisfaction. METHODS: The study, observational and retrospective, included 1839 cases, adult and paediatric patients, with sodium concentration <130 mmol/L. The procedure consisted of interpretative comments in the emergency and core laboratories report and the point-of-care testing blood gas network report. We evaluated hyponatraemia length in two equal periods: before and after the implementation. We conducted a survey addressed to the staff of the clinical settings involved to know their satisfaction. RESULTS: The median hyponatraemia length decreased significantly from 4.95 hours (2.08-16.57) in the first period to 2.17 hours (1.06-5.39) in the second period. The lack of hyponatraemia patients follow-up was significantly less after the procedure implementation. The survey was answered by 92 (60 senior specialists and 32 residents) out of 110 clinicians surveyed. Ninety of them (98%) answered positively. CONCLUSIONS: We have demonstrated the reduction in the time for diagnosing and management by physicians, the higher uniformity in the time required to solve hyponatraemia episodes following our laboratory procedure and the clinicians' satisfaction.

Subungual melanoma with blue naevus-like morphological features: a clinicopathological retrospective analysis of nine cases.

Melanocytic lesions in the nail apparatus are often challenging. Both subungual melanomas (SUM) and blue naevus of the nail are very rare. Occasionally, melanomas may mimic blue naevus histologically. Benign and malignant blue melanocytic lesions are commonly associated with G protein mutations, a distinct abnormality not associated with conventional subungual melanomas. We describe the clinical, histological and immunohistochemical features of nine cases of SUM with blue naevus-like morphological features. Mutations in exon 4 and 5 of GNAQ and GNA11 were investigated in two cases, which showed no mutations. RNA-seq of one case revealed unknown mutations along with mutations in ATM, METK and ARID1A. Our study delineates a variant of SUM that mimics blue naevus. Awareness of this pitfall is important when evaluating heavily pigmented lesions around the nail in order to avoid misdiagnosis. Appropriate sampling of subungual lesions and clinicopathological correlation are paramount to reach the correct diagnosis.

Clinical, histopathological and prognostic features of primary cutaneous acral CD8+ T-cell lymphoma and other dermal CD8+ cutaneous lymphoproliferations: results of an EORTC Cutaneous Lymphoma Group workshop.

BACKGROUND: The differential diagnosis of atypical dermal nonepidermotropic CD8+ lymphocytic infiltrates includes a heterogeneous spectrum of lymphoproliferations with overlapping histological and phenotypic features, but divergent clinical manifestations and prognoses. As these neoplasms are rare, more data on their clinicopathological presentation and course are needed. OBJECTIVES: To assess the clinical, histological and immunophenotypic features; outcomes of; and differences between dermal CD8+ lymphoproliferations. METHODS: Retrospective analysis of a series of 46 patients and biopsies by the international EORTC Cutaneous Lymphoma Group. RESULTS: The dermal CD8+ lymphoproliferations (n = 46) could be assigned to one of three groups: (i) cutaneous acral CD8+ T-cell lymphoma (n = 31), characterized mostly by a solitary nodule arising at acral sites, a monotonous dermal infiltrate of small-to-medium-sized CD8+ lymphocytes with a characteristic dot-like pattern of CD68, a low proliferation rate and an excellent prognosis; (ii) primary cutaneous CD8+ peripheral T-cell lymphoma, unspecified/NOS (n = 11), presenting with one or multiple rapidly evolving tumours, mostly medium-sized pleomorphic CD8+ tumour cells with expression of several cytotoxic markers, and high proliferative activity; and (iii) cutaneous CD8+ lymphoproliferations (n = 4), associated with congenital immunodeficiency syndromes in two patients with persisting localized or disseminated violaceous to brownish plaques on the extremities, a histiocyte-rich infiltrate of mostly small CD8+ lymphocytes with subtle atypia and a protracted course; and papular CD8+ eruptions in two patients with acquired immunosuppression. CONCLUSIONS: A constellation of distinct clinical, histopathological and phenotypic features allows discrimination and assignment of dermal CD8+ infiltrates into distinct disease entities. Primary cutaneous acral CD8+ lymphoma, assigned a provisional category in current lymphoma classifications, is a distinct and reproducible entity. A correct diagnosis is essential to avoid unnecessarily aggressive treatment for indolent CD8+ lymphoproliferations and to identify cases with underlying immuno-deficiency or potential for dismal outcome.

Determination of up to twenty carboxylic acid containing compounds in clinically relevant matrices by o-benzylhydroxylamine derivatization and liquid chromatography-tandem mass spectrometry.

Carboxylic acid containing compounds (R-COOH) are involved in a large number of biological processes and they are relevant for several pathological processes such as neurodegeneration or cancer. Comprehensive methodologies for the quantitative determination of R-COOH in biological samples are required. In this study we have developed a LC-MS/MS method for the quantification of 20 endogenous R-COOH belonging to different pathways such as kynurenine metabolism, serotoninergic pathway, glycolysis, tricarboxylic acid cycle, dopaminergic pathway, short chain fatty acids and glycine metabolism. The approach included derivatization with o-benzylhydroxylamine (reaction time 1 h), liquid-liquid extraction with ethyl acetate and LC-MS/MS detection (run time 10 min). The method was optimized and validated in 5 different matrices (urine, plasma, saliva, brain and liver) following two different approaches: (i) using surrogate matrices and (ii) using actual human samples by standard additions. A suitable linearity was obtained in the endogenous range of the analytes. Adequate intra and inter-assay accuracies (80-120%) and intra- and inter-assay precisions (<20%) were achieved for almost all analytes in all studied matrices. The method was applied in several scenarios to confirm (i) human urinary changes produced in glycolysis after exercise, (ii) metabolic changes produced in rat brain and plasma by methamphetamine administration and (iii) metabolic alterations in human plasma caused by vitamin B6 deficiency. Additionally, the application of the method allowed for establishing previously unreported alterations in R-COOH metabolites under these conditions. Due to the comprehensive analyte and matrix coverage and the wide applicability of the developed methodology, it can be considered as a suitable tool for the study of R-COOH status in health and disease by targeted metabolomics.

Nail Melatonin Content: A Suitable Non-Invasive Marker of Melatonin Production.

Melatonin plays multiple physiological roles in the human body. Evaluation of melatonin production by the determination of urinary 6-sulfatoxymelatonin in 24-h samples has important drawbacks which hinder the successful evaluation of melatonin production in large cohorts. Here, we evaluated the potential of nail analysis for estimating melatonin production. Firstly, mass spectrometry methodology for the determination of melatonin in nails was optimized and successfully validated. The method was found to be linear in the range 6.5-830 fg/mg with intraday and interday accuracy in the range 100-104 %, precision below 15 % and a LOD of 3.5 fg/mg. Secondly, nail melatonin concentrations from 84 volunteers (age 5-96) were determined. The expected correlation between melatonin and age was obtained (correlation coefficient -0.615; p < 0.001). Additionally, we showed that fingernails are preferable to toenails to determine nail melatonin content. Finally, fingernails collected for 180 days after melatonin administration (two volunteers, 1.9 mg/night during 5 days) were analyzed. Nail melatonin concentrations immediately rose after administration and went back to pre-administration values after ≈100 days in both volunteers. Our results suggest that melatonin determination in nails is a suitable non-invasive tool for the estimation of global melatonin production. Due to the easy collection and storage of nails, the long-term information obtained and the multiple functions of melatonin, nail melatonin content might complement dim light melatonin onset, which is commonly measured from plasma/saliva samples, paving the way for melatonin research.

Acceptability and feasibility of survivorship care plans and an accompanying mobile health intervention for adolescent and young adult survivors of childhood cancer.

BACKGROUND: Self-management interventions for adolescent and young adult (AYA) survivors of childhood cancer are needed. The present study reports on the acceptability and feasibility of delivering survivorship care plans (SCPs) and an accompanying app to AYA. PROCEDURE: AYA (n = 224) ages 15-29 who completed treatment for cancer were randomized and received a digital SCP only or an SCP plus a mobile app intended to enhance self-management. For 16 weeks, the app delivered one to two daily messages complementing information in their SCP and tailored based on age, treatment, and health goal. Data are presented on feasibility, self-reported acceptability (including satisfaction and perceived benefits) and its relationship to app engagement (for those in app group), and feedback from qualitative interviews conducted with 10 AYA. RESULTS: The SCP and app proved feasible as evidenced by high recruitment and retention, access to technology, time analysis, moderate app engagement, and minimal technical issues. However, 12% reported never reading the SCP and 8% never used the app. The app and SCP were acceptable to AYA, and SCP acceptability ratings did not differ between groups. For those with the app, acceptability was positively related to message engagement. AYA recommended enhanced individualization and design features of the SCP and app. CONCLUSIONS: Results support the use of tailored SCPs and mobile health interventions for most AYA, as well as the need for further refinement and research. Delivery of SCPs and digital interventions are acceptable and feasible to AYA survivors, and may help promote health-related knowledge and survivorship self-management.

Study of Chemotherapy-Induced Cognitive Impairment in Women with Breast Cancer.

BACKGROUND: Oncology patients experience a large number of symptoms and, those referring to cognitive performance has an ever-increasing importance in clinical practice, due to the increase in survival rates and interest in the patient's quality of life. The studies reviewed showed that chemotherapy-related cognitive impairment might occur in 15 and 50% of oncology patients. The main objective of this research was to study the impact of chemotherapy on the cognitive function of patients with locoregional breast cancer. METHOD: Analytical, prospective, longitudinal study using three measures, unifactorial intrasubject design, non-probability, and random selection sampling. The sample comprised women newly diagnosed with locoregional breast cancer in stages I, II, IIIA who received chemotherapy at the University Hospital of Salamanca (Complejo Asistencial Universitario de Salamanca), randomly selected for three years. Semi-structured interviews were conducted, and anxiety and depression (Hospital Anxiety and Depression scale, HAD); quality of life (QLQ-BR23 scale) and the following cognitive variables were assessed-processing speed, attention, memory, and executive functions (subtests of the Wechsler Intelligence Scale and the Trail Making Test). RESULTS: The final sample size included 151 participants; 23 were excluded. A decline in cognitive performance was observed in patients, which did not completely recover two months after chemotherapy was completed. Additionally, worse cognitive performance was observed in patients with anxious or depressive symptoms. There was a negative impact on the quality of life. CONCLUSION: Chemotherapy had an impact on the cognitive performance of oncology patients in most cognitive domains studied.

Systematic Review: Monoclonal Antibody-Induced Subacute Cutaneous Lupus Erythematosus.

BACKGROUND: Subacute cutaneous lupus erythematosus (SCLE) lacks consensus diagnostic criteria and the pathogenesis is poorly understood. There are increasing reports of SCLE induced by monoclonal antibodies (mAbs), but there are limited data on the aetiology, clinical characteristics and natural course of this disease. METHODS: We devised a set of diagnostic criteria for SCLE in collaboration with a multinational, multispecialty panel. This systematic review employed a two-layered search strategy of five databases for cases of mAb-induced SCLE (PROSPERO registered protocol CRD42019116521). To explore the relationship between relative mAb use and the number of SCLE cases reported, the estimated number of mAb users was modelled from 2013 to 2018 global commercial data and estimated annual therapy costs. RESULTS: From 40 papers, we identified 52 cases of mAb-induced SCLE, occurring in a cohort that was 73% female and with a median age of 61 years. Fifty percent of cases were induced by anti-tumour necrosis factor (TNF)-ɑ agents. A median of three drug doses preceded SCLE onset and the lesions lasted a median of 7 weeks after drug cessation. Oral and topical corticosteroids were most frequently used. Of the licensed mAbs, adalimumab, denosumab, rituximab, etanercept and infliximab were calculated to have the highest relative number of yearly users based on global sales data. Comparing the number of mAb-induced SCLE cases with estimated yearly users, the checkpoint inhibitors pembrolizumab and nivolumab showed strikingly high rates of SCLE relative to their global use, but ipilimumab did not. CONCLUSION: We present the first systematic review characterising mAb-induced SCLE with respect to triggers, clinical signs, laboratory findings, prognosis and treatment approaches. We identify elevated rates associated with the use of checkpoint inhibitors and anti-TNFɑ agents.

Occupational airborne contact urticaria, anaphylaxis and asthma in farmers and agronomists due to Bruchus pisorum.

BACKGROUND: Occupational contact urticaria due to inhalation of legume pest allergens has rarely been described. OBJECTIVE: To determine the cause of an outbreak of airborne contact urticaria, asthma, and anaphylaxis in farmers and agronomists studying the disinsectization of dried peas. Peas were treated with aluminum phosphide (EPA). METHODS: Extracts prepared from Bruchus lentis and Bruchus pisorum bodies and from healthy legumes, EPA-treated legumes, and infested legumes, were used for in vivo tests (skin prick testing, oral, and specific bronchial challenge for the diagnosis of asthma and anaphylaxis) in affected patients and in five control patients with severe legume hypersensitivity. Open application, patch testing with the most common legume insecticides, molecular analysis, and IgE immunoblotting were undertaken. RESULTS: We found positive responses (prick, provocation, immune detection) to parasitic pea extracts and B. pisorum. A 25 kDa band was detected in the western blot of all patients who worked with infested EPA-treated peas. Bands from B. pisorum extract were detected in all patients. No responses to pea allergens were found in any patient, unlike in legume allergy controls. Oral provocation tests were negative. CONCLUSION: B. pisorum is a cause of contact urticaria and may cause occupational hives, anaphylaxis, and asthma. The allergen may enter by inhalation or puncture of setae released by B. pisorum.

Cutaneous malignant glomus tumours: applicability of currently established malignancy criteria for tumours occurring in the skin.

Glomus tumours (GTs) have traditionally been classified into benign GTs, GTs with uncertain malignant potential and malignant GTs, based on a combination of criteria such as size of the tumour, degree of nuclear atypia and the level of mitotic activity. Several of the proposed grading criteria are difficult, or even impossible to apply for GTs occurring in the skin. The aim of the study was to analyse the applicability of the currently established GT malignancy criteria for tumours occurring in the skin and to establish their prognostic significance. A total of 25 benign cutaneous GTs, 11 new cutaneous malignant GTs and 36 cutaneous malignant GTs previously published in the literature were studied. We analysed the following clinicopathological features and correlated them with disease outcome: age, sex, site, size, depth of invasion, degree of nuclear atypia, mitotic activity, growth pattern, vascular invasion, spindle-cell morphology and tumoural necrosis. Of all the clinicopathological parameters analysed, only tumoural necrosis was found by univariate analysis (p = 0.001) to be associated with adverse biological behaviour, and none by multivariate analysis. Multivariate statistical analysis failed to detect any clinicopathological features predictive of the disease outcome (e.g., local recurrence, development of metastatic spread and/or death of disease) in cutaneous malignant GTs. Furthermore, the currently established malignancy criteria for cutaneous GTs can be difficult to apply, mainly due to their smaller size. Likewise, counting mitotic activity per 50 high power fields can often not be accomplished in GTs occurring at superficial locations. Complete excision of these tumours coupled with long-term follow-up is the mainstay of treatment for cutaneous malignant GTs. The results of our study also suggest that cutaneous malignant GTs follow a more indolent clinical course than their deep soft tissue counterparts.

Squamoid Eccrine Ductal Carcinoma: A Clinicopathologic Study of 30 Cases.

Squamoid eccrine ductal carcinoma is a poorly documented skin adnexal carcinoma showing squamous and duct differentiation. It is regarded to be of low-grade malignant potential, but limited follow-up information is available. To study their clinical behavior and histologic features, 30 squamoid eccrine ductal carcinomas were identified from departmental and referral files. Hematoxylin and eosin-stained sections were reviewed, and immunohistochemistry for carcinoembryonic antigen and epithelial membrane antigen was examined to confirm duct differentiation. Clinical follow-up was obtained from patient records and referring pathologists. The tumors presented as nodules or plaques (median size, 1.0 cm; range, 0.5 to 2.5 cm) with a predilection for the head and neck (77%). The patients were elderly (median age, 79.5 y; range, 10 to 96 y) with a male predominance. Histologically, these poorly demarcated tumors were characterized by an infiltrative growth pattern within the dermis and additional invasion of subcutis in 70%. Median tumor thickness was 4.3 mm (range, 1.5 to 18 mm). Superficially, the tumors resembled well-differentiated squamous cell carcinoma. In the deeper reaches, they were organized in cords and strands showing duct differentiation in a desmoplastic stroma. Cytologic atypia was moderate to severe. Ulceration (47%), necrosis (23%), and perineural and lymphovascular infiltration (27% and 6%, respectively) were additional features. Follow-up data (median, 29 mo; range, 7 to 99), available for 24 patients (80%), revealed a local recurrence rate of 25%. Three patients had lymph node metastasis, and 1 patient died of metastatic disease. Our study outlines the histologic characteristics of squamoid eccrine carcinoma and emphasizes its clinical behavior with risk for local recurrence and potential for more aggressive behavior with metastasis and rare disease-related mortality.

Providing Children and Adolescents Opportunities for Social Interaction as a Standard of Care in Pediatric Oncology.

Experiences with peers constitute an important aspect of socialization, and children and adolescents with cancer may experience reduced social interaction due to treatment. A literature review was conducted to investigate the evidence to support a standard of care evaluating these experiences. Sixty-four articles were reviewed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) criteria. Moderate quality of evidence suggest that social interaction can be beneficial to increase knowledge, decrease isolation, and improve adjustment and constitute an important, unmet need. The evidence supports a strong recommendation for youth with cancer to be provided opportunities for social interaction following a careful assessment of their unique characteristics and preferences.

Component-resolved diagnostics in vernal conjunctivitis.

BACKGROUND: Conventional diagnostic tests in allergy are insufficient to clarify the origin of vernal conjunctivitis (VC). OBJECTIVES: To evaluate IgE-mediated hypersensitivity by component-resolved diagnosis (CRD) in tears and serum from patients with VC and to evaluate how to treat patients with identified triggering allergens by specific immunotherapy. METHODS: Patients were divided into 3 groups: (1) patients with VC (25 patients), (2) patients allergic to grass pollen with seasonal allergic conjunctivitis (AC) (50 patients), and (3) healthy blood donors (50 patients). If triggering allergens were detected, specific conventional immunotherapy was administered for 1 year. RESULTS: Twenty-five patients with VC were evaluated. The identified triggering allergens were n Lol p 1 (11 patients), n Cyn d 1 (8 patients), group 4 and 6 grasses (6 patients), and group 5 grasses (5 patients). Prick test and pollen IgE test results were positive in one patient. Clinical improvement was observed in 13 of the 25 patients with VC after 1 year of specific immunotherapy. CONCLUSION: CRD seems to be a more sensitive diagnostic tool compared with prick test and IgE detection. Specific CRD-led immunotherapy may achieve clinical improvements in patients with VC.

Evaluation of follicular T-helper cells in primary cutaneous CD4+ small/medium pleomorphic T-cell lymphoma and dermatitis.

BACKGROUND: CD4+ small/medium-sized pleomorphic T-cell lymphoma (SMPTCL) is a controversial primary cutaneous lymphoma, in which the candidate neoplastic cells express a follicular T-helper phenotype. We describe 16 cases of SMPTCL and compare expression of PD-1, CXCL-13 and ICOS in these tumors with 40 dermatitis cases. METHODS: Histopathologic examination and immunocytochemistry were performed for 16 tumors and 40 assorted dermatitis cases. RESULTS: All but one patient presented with solitary lesions. Each biopsy revealed a dense nodular non-epitheliotropic infiltrate of atypical T-cells. Neoplastic cells were CD3+/CD4+/CD8(-)/CD30(-). Cutaneous recurrence occurred in one patient over a median follow up of 8 months (range 5-36). All tumors widely expressed PD-1 and ICOS to a lesser extent. CXCL-13 stained much fewer cells. Of the dermatitis cases, PD-1 (most numerous) and ICOS labeled lymphoid cells in all cases, albeit fewer than in the tumors, and CXCL-13 was negative in 32. A rosette pattern of PD-1 expression was identified in all the SMPTCL cases but not in dermatitis. CONCLUSIONS: There remains uncertainty about the appropriate nosological status of SMPTCL, which some authors consider to be a pseudolymphoma. However, this study suggests a significant difference in the prevalence and pattern of follicular T-helper cell markers between this tumor and lymphoid proliferations known to be reactive.

An ulcerated nodule on the nose.

A 75-year-old retired nurse, originally from Barbados, presented to her general practitioner (GP) with a scaling ulcerated nodule on the left side of her nose. She was taking medication for type 2 diabetes, hypertension and glaucoma, but was otherwise well with no systemic symptoms. Her GP diagnosed a patch of eczema; however, a trial of topical steroids was not effective and she was referred to dermatology. A skin biopsy confirmed the clinical suspicion that this patient had a nodular basal cell carcinoma (BCC). BCCs account for 75% of all skin cancers; they very rarely metastasise, but can spread to invade local structures. Our patient has type VI skin. Skin cancer is rare in patients with skin type VI; however, in this group, morbidity and mortality are disproportionately high in relation to cancer incidence.

Merkel cell carcinoma with divergent differentiation: histopathological and immunohistochemical study of 15 cases with PCR analysis for Merkel cell polyomavirus.

AIMS: To report on 15 cases of Merkel cell carcinoma (MCC) with divergent differentiation, to characterize its clinicopathological spectrum and its relationship with Merkel cell polyomavirus (MCV). METHODS AND RESULTS: Fifteen patients with a mean age of 81 years were included. Follow-up was available for 13 cases (range 12 days to 6 years; median 6 months). Recurrence, metastasis and mortality rates were 15.4%, 53.8% and 61.5%, respectively. All tumours showed the typical histological and immunohistochemical features of MCC, with at least one additional divergent component. Eight cases had a single aberrant component (squamous in six cases, follicular in one case, and porocarcinoma in one case), six cases had two aberrant components (squamous and sarcomatous in three cases, glandular and squamous in two cases, and sarcomatous and neuroblastic in one case), and one case had three aberrant components (glandular, squamous, and sarcomatous). All cases had dysplastic changes in the overlying epithelium, and four of 15 showed epidermotropism. PCR analysis for Merkel cell polyomavirus (MCV) gave negative results in all 12 cases tested. CONCLUSIONS: Merkel cell carcinoma with divergent differentiation is a highly aggressive tumour that might be difficult to recognize, owing to its wide histological variability. Negativity for MCV suggests that the virus is not implicated in the development of this subtype of MCC.