Demonstrating the interference of tissue processing in the evaluation of tissue biomarkers: The case of PD-L1.

The immunohistochemical (IHC) expression of PD-L1 in cancer models is used as a predictive biomarker of response to immunotherapy. We aimed to evaluate the impact of the usage of 3 different tissue processors in the IHC expression of PD-L1 antibody clones: 22C3 and SP142. Three different topographies of samples (n = 73) were selected at the macroscopy room: 39 uterine leiomyomas, 17 placentas and 17 palatine tonsils. Three fragments were collected from each sample and were inked with a specific color that represented their separate processing in a different tissue processor (A, B or C). During embedding, the 3 fragments with distinct processing were ensemble in the same cassette for sectioning of 3 slides/each: hematoxylin-eosin, 22C3 PDL1 IHC staining and SP142 PD-L1 IHC staining, that were blindly observed by 2 pathologists under digital environment. All but one set of 3 fragments were considered adequate for observation even in the presence of artifacts associated with processing issues that were recorded as high as 50.7 % for processor C. The occurrence of background non-specific staining and the presence of false positive results appear to be unrelated with the PD-L1 clone or the type of tissue processing. 22C3 PD-L1 was more frequently considered adequate for evaluation than SP142 PD-L1 that, in 29.2 % of WSIs (after tissue processor C) were considered not adequate for observation due to lack of the typical pattern of expression. Similarly, the intensity of PD-L1 staining was significantly decreased in fragments processed by C (both PD-L1 clones) in tonsil and placenta specimens, and by A (both clones) in comparison with those processed by B. This study demonstrates the need to standardize the tissue processing in pathology to cope with the growing needs of precision medicine quantifications and the production of high-quality material necessary for computational pathology usage.

p40/PD-L1 and TTF1/PD-L1 Immunohistochemical Double Staining Contributes to Intraindividual Variability Associated With PD-L1 Staining Interpretation in Lung Cancer Samples.

The important developments achieved in recent years with a consequent paradigm shift in the treatment of non-small cell lung cancer (NSCLC), including the latest immune checkpoint inhibitors, have led to an increasing need to optimize the scarce material usually available in the diagnosis of these tumors. In this sense, this study intends to evaluate the performance of double immunohistochemistry (IHC) in comparison to simple IHC for programmed death-ligand 1 (PD-L1) evaluation with 22C3 clone for selection to therapy with pembrolizumab. For that, 38 histologic samples of NSCLC small biopsies sent to our laboratory were selected. Double IHC were performed with the doublets TTF1/PD-L1 and p40/PD-L1, after all the usual diagnostic routine and molecular study was performed. The slides were interpreted by 2 independent pathologists and the results obtained were compared with each other and with the results obtained at diagnosis. A perfect agreement was observed when comparing the immunoexpression of TTF1 and p40 in double IHC in relation to single IHC. Although the agreement was substantial in the analysis of the positive/negative PD-L1 IHC (81.6% to 92.1%; κ=0.610 to 0.829) and in the analysis of the 50% cut-off (86.8% to 89.5%; κ=0.704 to 0.759), it fell short of the expected and desirable agreement for a biomarker such as PD-L1, since this result will have a major role in the institution of a treatment. In conclusion, this small series does not allow us to recommend this methodology for the evaluation of the PD-L1 biomarker in double staining IHC with the 22C3 clone for therapy selection.

The spectrum of HER2 expression in breast cancer: linking immunohistochemistry quantification with in situ hybridization assay.

We aimed to document the pathological characteristics of breast cancer (BC) cases with different scores of HER2 by immunohistochemistry (IHC), as well as to establish a relationship between HER2 expression and HER2 amplification by in situ hybridization (ISH). A cohort of 258 primary BC cases was evaluated for HER2 gene amplification with bright-field ISH. All HER2-negative and HER2-positive cases by IHC were concordant with the ISH classification. BC cases with score of 0 had lower average of HER2 copy number compared to cases with score of 1 + . HER2-equivocal cases by IHC had intermediate pathological characteristics between HER2-negative and HER2-positive cases. About 12% of HER2-equivocal cases were classified as ISH-positive. HER2-equivocal cases with HER2 gene amplification had proliferation index, HER2/CEP17 ratio, and average of HER2 copy number between HER2-equivocal cases without HER2 gene amplification and HER2-positive cases by IHC. Additionally, HER2-equivocal cases with HER2 amplification had score of 2 + in at least 50% of the total tumor area, with a proportion of ISH-positive cases increasing with the amount of score of 2 + present in the tumor. The quantification of score of 2 + in the tumor predicted the ISH classification with an AUC of 0.902. A logistic regression model using the same HER2 quantification and the nuclear score was able to increase the abovementioned prediction to an AUC of 0.929. As such, we were able to link HER2 quantification by IHC and morphological analysis with HER2 amplification by ISH.

EWSR1 rearrangement in papillary thyroid microcarcinoma is related to classic morphology and the presence of small-cell phenotype.

The EWSR1 rearrangements with unknown genes were detected in a high percentage of classic variants of papillary thyroid carcinoma. The small-cell carcinoma of the thyroid with Ewing family tumor elements (CEFTE) typically presents with EWSR1-FLI1 rearrangement suggesting the possible role of EWSR-FLI1 translocation in the loss of thyroid differentiation and acquisition of a small-cell phenotype. In order to determine the frequency and association of EWSR1 rearrangements, particularly the EWSR1-FLI1 fusion with clinicopathological features of papillary thyroid microcarcinoma (m-PTC) and the presence of small cells, we analyzed a series of 99 m-PTCs using the fluorescence in situ hybridization method.  Ninety cases (90.9%) of m-PTC were positive for small cells. This group of m-PTC has shown more often invasive growth, lymphatics invasion, and moderate/extended intratumoral fibrosis. Three cases out of 99 were inconclusive for EWSR1 rearrangement. Eighty-nine (92.7%) and twenty-seven (28.1%) out of 96 m-PTC cases were positive for EWSR1 rearrangement and EWSR1-FLI1 fusion, respectively. m-PTC with classical architectural pattern presented more frequently with EWSR1 rearrangement relative to m-PTC with other patterns (p = 0.005). Other clinicopathological features were not related to the presence of EWSR1 rearrangement or EWSR1-FLI1 fusion. The percentage of small cells present significantly correlated with the percentage of cells positive for EWSR1-FLI1 fusion (p = 0.05) and EWSR1 rearrangement (p <0.001). EWSR1-FLI1 fusion is not rare in m-PTC and it is associated with the acquisition of small-cell phenotype. The EWSR1 gene rearrangement is a frequent event in m-PTC and is related to the classical pattern of m-PTC.

Digital Pathology Workflow Implementation at IPATIMUP.

The advantages of the digital methodology are well known. In this paper, we provide a detailed description of the process for the digital transformation of the pathology laboratory at IPATIMUP, the major modifications that operate throughout the processing pipeline, and the advantages of its implementation. The model of digital workflow implementation at IPATIMUP demonstrates that careful planning and adoption of simple measures related to time, space, and sample management can be adopted by any pathology laboratory to achieve higher quality and easy digital transformation.

Expression of PD-L1 in medullary thyroid carcinoma-a new therapeutic target?

PD-L1 expression in MTC is hot topic since, if it is demonstrated that PD-L1 is highly expressed in this cancer, thus immunotherapy against checkpoint inhibitors could become an important therapeutic tool in MTC treatment. To answer this question, we evaluated PD-L1 expression in MCT tumour tissues, using an anti-PD-L1 22C3 antibody and found a high expression in 6 of the 8 patients (75%). Similarly, two other recent studies reported a higher PD-L1 expression. According to our results, MTC cells present a significative PD-L1 expression, raising the hypothesis that immunotherapy, such as pembrolizumab, could have a role on MCT treatment. The authors believe this is a fundamental question and may impact the future of MTC treatment.

Translation and Cross-Cultural Adaptation of Quality of Life Scale in Patients with Onychomycosis.

(1) Background: Onychomycosis (OM) is a fungal nail infection, considered a risk factor for diabetic foot ulcers. It is associated with changes in quality of life, in terms of pain, self-confidence and self-esteem. The aim was to translate and adapt the OM quality of life questionnaire "OnyCOE-tTM-Quality Of Life Questionnaire Onychomycosis (Nail Fungal Condition)". (2) Methods: This study followed the guidelines proposed by Beaton et al. (2000), where two English to Portuguese translations were performed and, after an expert consensus, a common version was obtained. This was followed by two back-translations. The expert committee achieved semantic equivalence, idioms and concepts. The pre-test was applied to 49 people. The final version and processed data were sent to the authors. (3) Results: We adapted terms semantically, modified statements syntactically, altering items from interrogative to affirmative. (4) Conclusions: The translated version of the "Quality of life-Onychomycosis" questionnaire suggested that it can be used for further studies to test validity and reliability in this population.

HER2 in situ hybridization test in breast cancer: quantifying margins of error and genetic heterogeneity.

The aim of the present study was to evaluate the effect of counting increasing number of invasive cancer cells in the result of the HER2 in situ hybridization (ISH) test in breast cancer as well as to compare two different approaches of measuring genomic heterogeneity (single cell and population based). A cohort of 100 consecutive breast cancer cases (primary and metastatic) were evaluated for HER2 gene amplification with bright-field ISH. The evaluation of the samples included scoring 20 nuclei, in five different areas, measuring the margins of error for each case. Genomic heterogeneity (GH) was defined by the 2018 ASCO/CAP guideline as a discrete population of tumor cells with HER2 amplification. We also evaluated GH as single tumor cells with HER2 amplification. The stabilization of the coefficient of variation of HER2/CEP17 ratio requires about 60 invasive cancer cells. The average margin of error of HER2/CEP17 ratio and of HER2 copy number was 0.40 and 0.53, respectively, when counting 20 cells, decreasing to 0.20 and 0.26 when counting 100 cells. Population GH was observed in 1% of the cases, while single cell GH was observed in 27% of the cases, reaching its maximum value in cases near the thresholds of positivity. Therefore, margins of error in HER2 ISH test are high, and the minimal cell number recommended in current guidelines should be raised to at least 60 cells. Population GH is a rare event and single cell GH is maximal in cases near the thresholds.

[Application of the 2015 Beers Criteria Operationalized for Portugal in Institutionalized Elderly: A Cross-Sectional Study]. / Aplicação dos Critérios de Beers de 2015 Operacionalizados para Portugal em Pessoas Idosas Institucionalizadas: Um Estudo Transversal.

INTRODUCTION: The consumption of potentially inappropriate medicines is high among institutionalized elderly, predisposing to potential drug interactions, adverse drug events, risk of iatrogenic cascade, increased morbidity and mortality and health costs. Medication review is a promising strategy for therapeutic optimization, although scarcely documented in Portugal. The aim of this study was to characterize, using explicit criteria, the existence of potentially inappropriate medicines, among institutionalized elderly, and to calculate the eventual cost savings, with their discontinuation. MATERIAL AND METHODS: Descriptive and cross-sectional study conducted in three residential homes for the elderly, from different geographic regions, based on a random sample of 33 health records. In order to characterize the existence of potentially inappropriate medicines, we used the 2015 Beers criteria, revised by the American Geriatrics Society and in the Portuguese version. RESULTS: On average, 11 drugs are prescribed to elderly residents of three residential structures for the elderly. All health records contain potentially inappropriate medicines (mean 4.8 ± 2.0 per resident), with anxiolytics (17.7%), antidepressants (17.7%) and antipsychotics (15.8%) being the most prevalent. Its reduction would result in an average monthly savings of €9.6 per resident. DISCUSSION: The consumption of potentially inappropriate medicines is higher than the literature describes, and the cost of medicines is high. The involvement of nurses in the process of drug management and reconciliation, in coordination with the physician, could be an effective strategy. This is the first study using the latest Portuguese version of the Beers criteria, which makes the comparability of the results difficult. CONCLUSION: The consumption of potentially inappropriate medicines is high, which suggest the need for adoption of improvement measures.

Introdução: O consumo de medicamentos potencialmente inapropriados é elevado entre idosos institucionalizados, predispondo à ocorrência de potenciais interações medicamentosas, eventos adversos a medicação, risco de cascata iatrogénica, aumento da morbimortalidade e dos custos em saúde. A revisão da medicação é uma estratégia promissora com vista à otimização terapêutica, ainda que pouco documentada em Portugal. Este estudo pretende caraterizar, utilizando critérios explícitos, a existência de medicamentos potencialmente inapropriados, entre idosos institucionalizados, e calcular a eventual poupança, em medicamentos, com a sua supressão.Material e Métodos: Estudo descritivo e transversal, realizado em três estruturas residenciais para pessoas idosas, de regiões geográficas distintas, a partir de uma amostra aleatória de 33 processos clínicos. Para a caracterização da existência de medicamentos potencialmente inapropriados, utilizaram-se os Critérios de Beers de 2015, revistos pela American Geriatrics Society e na versão operacionalizada para Portugal.Resultados: Em média, 11 fármacos são prescritos aos idosos residentes das estruturas residenciais para pessoas idosas. Todos os processos contêm medicamentos potencialmente inapropriados (média de 4,8 ± 2,0 por residente), sendo os ansiolíticos (17,7%), antidepressivos (17,7%) e antipsicóticos (15,8%) os mais prevalentes. A sua redução resultaria numa poupança média mensal de €9,6, por residente.Discussão: O consumo de medicamentos potencialmente inapropriados é superior ao que a bibliografia descreve e o custo com os medicamentos é elevado. O envolvimento dos enfermeiros no processo de gestão e reconciliação medicamentosa, em articulação com o médico, poderá ser uma estratégia eficaz. O estudo é pioneiro na utilização da última versão portuguesa dos critérios de Beers, o que dificulta a comparabilidade dos resultados.Conclusão: O consumo de medicamentos potencialmente inapropriados é elevado, o que sugere a necessidade de adoção de medidas de melhoria.

Digital Versus Optical Diagnosis of Follicular Patterned Thyroid Lesions.

OBJECTIVES: To study the concordance between pathologists in the diagnosis of follicular patterned thyroid lesions using both digital and conventional optical settings. MATERIAL AND METHODS: Five pathologists reviewed 50 hematoxylin and eosin-stained slides of follicular patterned thyroid lesions using both digital (the D-Sight 2.0 scanner and navigator viewer) and conventional optical instruments with washout interval time. RESULTS: The mean concordance rate with the ground truth (GT) was similar between conventional optical and digital observation (83.2 and 85.2%, respectively). The most frequent reason for diagnostic discordance with GT on both systems was the evaluation of nuclear features (69.1% for conventional optical observation and 59.4% for digital observation). The intraobserver diagnostic concordance mean was 86.8%. Time for digital observation (mean time per case = 2.9 ± 0.8 min) was higher than that for conventional optical observation (mean time per case = 2.0 ± 0.7 min). Interobserver correlation of measurements was higher in the digital observation than the conventional optical observation. CONCLUSION: Conventional optical and digital observation settings showed a comparable accuracy for the diagnosis of follicular patterned thyroid nodules, as well as substantial intraobserver agreement and a significant improvement in the reproducibility of the measurements that support the use of digital diagnosis in thyroid pathology. The origins underlying the variability of the diagnosis were the same in both conventional optical microscopy and digital pathology systems.

What to expect from the 2018 ASCO/CAP HER2 guideline in the reflex in situ hybridization test of immunohistochemically equivocal 2+ cases?

To evaluate the effect of the 2018 ASCO/CAP guideline in the identification of HER2-positive breast carcinomas (BC) in reflex in situ hybridization (ISH) test. A total of 592 primary invasive BC cases from before and after the publication of the updated ASCO/CAP guideline were evaluated for HER2 amplification by silver ISH according to the 2013 and 2018 guidelines. Cases were mostly (95%) HER2 equivocal by immunohistochemistry (IHC), not centrally reviewed. Other reasons for referring cases were IHC confirmation, IHC discordancy (either between needle-core-biopsy (NCB) and surgical excision specimen (SES) or between different laboratories) and IHC result unexpected for histopathologic features. Cases evaluated with the 2013 guideline (1st cohort) were 14.6% HER2-positive, decreasing significantly after the reclassification with the 2018 guideline due to the exclusion of group 2 cases without HER2 protein overexpression. Cases studied after the implementation of the 2018 guideline (2nd cohort) were 8.7% HER2-positive, a frequency that was not significantly different from the reclassification of the 1st cohort with the 2018 guideline. All cases referred for IHC confirmation had the expected ISH result. Cases with IHC discordancy between NCB and SES were ISH concordant. Only one out of 14 cases with an IHC score 3+ and classified as histological grade 1 or with a Ki67 below 10% was classified as ISH HER2-positive. The 2018 ASCO/CAP guideline resulted in a decrease of HER2-positive cases in reflex ISH test, selecting less patients for anti-HER2-targeted therapy.

O bem-estar espiritual dos professores de medicina e de enfermagem / The spiritual well-being of the faculty of medicine and nursing

Objetivo: avaliar o bem-estar espiritual dos professores de Medicina e de Enfermagem na Universidade de Marília. Método: estudo descritivo, exploratório, de abordagem quantitativa. A população foi constituída por uma amostra de 49 professores e a escolha foi aleatória. Utilizou-se a "Escala de Avaliação Espiritual". A coleta de dados foi realizada em fevereiro e março de 2012, analisados pelo Programa SPSS 20.0. O projeto de pesquisa foi aprovado pelo Comitê de Ética em Pesquisa, CAAE n. 25000.113733/2010-14. Resultados: os professores dos cursos de Medicina e Enfermagem apresentaram escores positivos em todos os termos/conceitos comedidos: Fé Pessoal 90%, Prática Religiosa 74% e Paz Espiritual 88%. Conclusão: em sua maioria, os professores desta Universidade têm fé, prática religiosa e apresentam paz espiritual.(AU)