ONEST (Observers Needed to Evaluate Subjective Tests) Analysis of Stromal Tumour-Infiltrating Lymphocytes (sTILs) in Breast Cancer and Its Limitations.

Tumour-infiltrating lymphocytes (TILs) reflect antitumour immunity. Their evaluation of histopathology specimens is influenced by several factors and is subject to issues of reproducibility. ONEST (Observers Needed to Evaluate Subjective Tests) helps in determining the number of observers that would be sufficient for the reliable estimation of inter-observer agreement of TIL categorisation. This has not been explored previously in relation to TILs. ONEST analyses, using an open-source software developed by the first author, were performed on TIL quantification in breast cancers taken from two previous studies. These were one reproducibility study involving 49 breast cancers, 23 in the first circulation and 14 pathologists in the second circulation, and one study involving 100 cases and 9 pathologists. In addition to the estimates of the number of observers required, other factors influencing the results of ONEST were examined. The analyses reveal that between six and nine observers (range 2-11) are most commonly needed to give a robust estimate of reproducibility. In addition, the number and experience of observers, the distribution of values around or away from the extremes, and outliers in the classification also influence the results. Due to the simplicity and the potentially relevant information it may give, we propose ONEST to be a part of new reproducibility analyses.

ONEST (Observers Needed to Evaluate Subjective Tests) suggests four or more observers for a reliable assessment of the consistency of histological grading of invasive breast carcinoma: A reproducibility study with a retrospective view on previous studies.

Histological grade is one of the most important prognosticators of breast cancer which is available for nearly all cases. It also makes part of several multivariable analysis derived combined prognostic profiles despite concerns about its reproducibility. The aims included a reproducibility study of grading in the light of a recently described statistical approach, ONEST (Observers Needed to Evaluate Subjective Tests) and review earlier reproducibility studies in the light of the ONEST analysis. Nine pathologists reviewed 50 core needle biopsies and 50 slides from different excision specimens and recorded the scores for gland (tubule) formation, nuclear pleomorphism and mitotic activity as well as histological grade. Overall percent agreement, Fleiss kappa and the intraclass correlation coefficient (ICC) were used for the analysis of reproducibility. ONEST data and curves were generated from 100 random permutations of the participants. ONEST suggested a minimum of 4 observers for the reliable evaluation of reproducibility for both the scored components and grade in either type of specimen. Our results suggested moderate or moderate to good reproducibility of grading (kappa values of 0.51 for excisions, and 0.54 for biopsies and ICCs of 0.70 and 0.69, respectively) with gland formation being the most and nuclear pleomorphism the worst consistently evaluated feature. In studies with sufficient participants (at least 4) and non-pairwise comparisons in the analysis, the reproducibility of histological grading is fair to moderate, whereas studies with fewer participants or pairwise kappa analysis suggest moderate to almost prefect agreement of the results. ONEST is a valuable complementation of reproducibility analyses.

Inter-observer agreement for the histological diagnosis of invasive lobular breast carcinoma.

Invasive lobular breast carcinoma (ILC) is the second most common breast carcinoma (BC) subtype and is mainly driven by loss of E-cadherin expression. Correct classification of BC as ILC is important for patient treatment. This study assessed the degree of agreement among pathologists for the diagnosis of ILC. Two sets of hormone receptor (HR)-positive/HER2-negative BCs were independently reviewed by participating pathologists. In set A (61 cases), participants were provided with hematoxylin/eosin (HE)-stained sections. In set B (62 cases), participants were provided with HE-stained sections and E-cadherin immunohistochemistry (IHC). Tumor characteristics were balanced. Participants classified specimens as non-lobular BC versus mixed BC versus ILC. Pairwise inter-observer agreement and agreement with a pre-defined reference diagnosis were determined with Cohen's kappa statistics. Subtype calls were correlated with molecular features, including CDH1/E-cadherin mutation status. Thirty-five pathologists completed both sets, providing 4,305 subtype calls. Pairwise inter-observer agreement was moderate in set A (median κ = 0.58, interquartile range [IQR]: 0.48-0.66) and substantial in set B (median κ = 0.75, IQR: 0.56-0.86, p < 0.001). Agreement with the reference diagnosis was substantial in set A (median κ = 0.67, IQR: 0.57-0.75) and almost perfect in set B (median κ = 0.86, IQR: 0.73-0.93, p < 0.001). The median frequency of CDH1/E-cadherin mutations in specimens classified as ILC was 65% in set A (IQR: 56-72%) and 73% in set B (IQR: 65-75%, p < 0.001). Cases with variable subtype calls included E-cadherin-positive ILCs harboring CDH1 missense mutations, and E-cadherin-negative ILCs with tubular elements and focal P-cadherin expression. ILCs with trabecular growth pattern were often misclassified as non-lobular BC in set A but not in set B. In conclusion, subtyping of BC as ILC achieves almost perfect agreement with a pre-defined reference standard, if assessment is supported by E-cadherin IHC. CDH1 missense mutations associated with preserved E-cadherin protein expression, E- to P-cadherin switching in ILC with tubular elements, and trabecular ILC were identified as potential sources of discordant classification.

Comparison of the antiremodeling effects of losartan and mirabegron in a rat model of uremic cardiomyopathy.

Uremic cardiomyopathy is characterized by diastolic dysfunction (DD), left ventricular hypertrophy (LVH), and fibrosis. Angiotensin-II plays a major role in the development of uremic cardiomyopathy via nitro-oxidative and inflammatory mechanisms. In heart failure, the beta-3 adrenergic receptor (ß3-AR) is up-regulated and coupled to endothelial nitric oxide synthase (eNOS)-mediated pathways, exerting antiremodeling effects. We aimed to compare the antiremodeling effects of the angiotensin-II receptor blocker losartan and the ß3-AR agonist mirabegron in uremic cardiomyopathy. Chronic kidney disease (CKD) was induced by 5/6th nephrectomy in male Wistar rats. Five weeks later, rats were randomized into four groups: (1) sham-operated, (2) CKD, (3) losartan-treated (10 mg/kg/day) CKD, and (4) mirabegron-treated (10 mg/kg/day) CKD groups. At week 13, echocardiographic, histologic, laboratory, qRT-PCR, and Western blot measurements proved the development of uremic cardiomyopathy with DD, LVH, fibrosis, inflammation, and reduced eNOS levels, which were significantly ameliorated by losartan. However, mirabegron showed a tendency to decrease DD and fibrosis; but eNOS expression remained reduced. In uremic cardiomyopathy, ß3-AR, sarcoplasmic reticulum ATPase (SERCA), and phospholamban levels did not change irrespective of treatments. Mirabegron reduced the angiotensin-II receptor 1 expression in uremic cardiomyopathy that might explain its mild antiremodeling effects despite the unchanged expression of the ß3-AR.

The additional value of ONEST (Observers Needed to Evaluate Subjective Tests) in assessing reproducibility of oestrogen receptor, progesterone receptor, and Ki67 classification in breast cancer.

The reproducibility of assessing potential biomarkers is crucial for their implementation. ONEST (Observers Needed to Evaluate Subjective Tests) has been recently introduced as a new additive evaluation method for the assessment of reliability, by demonstrating how the number of observers impact on interobserver agreement. Oestrogen receptor (ER), progesterone receptor (PR), and Ki67 proliferation marker immunohistochemical stainings were assessed on 50 core needle biopsy and 50 excision samples from breast cancers by 9 pathologists according to daily practice. ER and PR statuses based on the percentages of stained nuclei were the most consistently assessed parameters (intraclass correlation coefficients, ICC 0.918-0.996), whereas Ki67 with 5 different theoretical or St Gallen Consensus Conference-proposed cut-off values demonstrated moderate to good reproducibility (ICC: 0.625-0.760). ONEST highlighted that consistent tests like ER and PR assessment needed only 2 or 3 observers for optimal evaluation of reproducibility, and the width between plots of the best and worst overall percent agreement values for 100 randomly selected permutations of observers was narrow. In contrast, with less consistently evaluated tests of Ki67 categorization, ONEST suggested at least 5 observers required for more trustful assessment of reliability, and the bandwidth of the best and worst plots was wider (up to 34% difference between two observers). ONEST has additional value to traditional calculations of the interobserver agreement by not only highlighting the number of observers needed to trustfully evaluate reproducibility but also by highlighting the rate of agreement with an increasing number of observers and disagreement between the better and worse ratings.

The new TNM-based staging of breast cancer.

This review describes the changes that have been implemented in the Tumor-Node-Metastasis (TNM)-based staging of breast cancers by the new, 8th editions of the relevant Union for International Cancer Control (UICC) and American Joint Committee on Cancer (AJCC) publications. After giving a background for TNM being the common language of cancer staging and related activities like cancer treatment and registration, it summarizes not only the changes but reviews some highlights important for pathologists, and lists and comments on the differences between the publications and diagnostic practices based on them. A section is dedicated to the prognostic stages of breast carcinomas introduced in the AJCC Cancer Staging Manual, but not mentioned in the UICC TNM classification of malignant tumors. A few issues that are not appropriately covered by TNM according to the authors' view (e.g., multifocal tumors, larger lymph node metastases identified by molecular methods, the heterogeneous prognosis of M1-defined stage IV disease) close the review with the final thoughts raising the vision of a potential loss of the common staging language.

[Evaluation of anatomic and prognostic stages of breast cancer according to the 8th edition of the TNM staging system - Retrospective analysis based on data from deceased patients once diagnosed with breast cancer]. / Emlorákok TNM-8 szerinti anatómiai és prognosztikai stádiumainak retrospektív vizsgálata elhunyt, valaha emlorákos betegek adatai alapján.

INTRODUCTION: The 8th edition of the Tumor-Node-Metastasis (TNM) based staging of breast cancer introduces a prognostic stage influenced by biomarkers along the traditional T, N and M categories. AIM: To retrospectively assess stage influencing prognostic variables; and the anatomic and prognostic stages on the basis of the overall survival (OS) of a cohort of deceased patients once diagnosed with breast cancer. METHOD: We included patients with known causes of death certified at the Bács-Kiskun County Teaching Hospital and having a history of breast cancer diagnosed on a resection specimen at the same institution. Prognostic factors were obtained from the histopathological reports. Statistics included one-way ANOVA, Dunn's post hoc test and Kaplan-Meier curve analyses. RESULTS: The 303 patients grouped as breast cancer related death (n = 168) or unrelated (n = 135) showed significant differences in most stage defining prognostic factors and the anatomic and prognostic stages. Significant differences in 5-year OS were observed between pT and pN categories, histological grades and estrogen receptor statuses. Except for stages I and II, significant differences were found between both different anatomic and prognostic stages (p<0.001). Stage IV is by definition uniform, but we identified survival differences between biomarker based subgroups: triple negative carcinomas had worse OS than estrogen receptor positive and HER2 negative carcinomas. CONCLUSIONS: Our analysis based on real survival data suggests that the prognostic stages separate patients according to OS similarly to the anatomic stages. The results validate the prognostic stages, but also suggest that separating stage IV disease according to biomarkers makes sense. Orv Hetil. 2017; 158(35): 1373-1381.

High-dose Radiation Induced Heart Damage in a Rat Model.

BACKGROUND/AIM: Radiation-induced heart disease (RIHD) is a concern during radiotherapy. For its comprehensive study, an in vivo selective heart irradiation model was developed. MATERIALS AND METHODS: Sprague-Dawley rats were irradiated with 50 Gy and functional imaging, biochemical (circulating growth differentiation factor-15 (GDF-15), transforming growth factor-beta (TGF-beta) and morphological (picrosirius red staining of the heart) objectives were tested. RESULTS: Signs and symptoms of RIHD occurred >12 weeks after irradiation with tachypnea, systolic and diastolic dysfunction, cardiac hypertrophy and body development retardation. Plasma GDF-15 was increased 3, 12 and 26, while plasma TGF-beta was increased 12 weeks after irradiation. At autopsy, extensive pleural fluid was found in the irradiated animals. Interstitial fibrosis could be reliably detected and quantified in irradiated hearts after a follow-up time of 19 weeks. CONCLUSION: The studied parameters could be used in future experiments for testing protective agents for prevention of radiation heart injury.