Tumor Microenvironment in Male Breast Carcinoma with Emphasis on Tumor Infiltrating Lymphocytes and PD-L1 Expression.

Male breast cancer (MBC) is rare and usually presents as a locally advanced disease. Stromal tumor-infiltrating lymphocytes (sTILs) are associated with a better response to neoadjuvant chemotherapy and improved prognosis in all molecular subtypes of female breast cancer, but their role in MBC is less clear. We studied sTILs and the expression of programmed cell death ligand 1 (PD-L1) and pan-TRK in MBC. We retrospectively studied 113 cases of MBC surgically treated between 1988 and 2015. The tumors were evaluated for histological type and grade, stage, intrinsic subtype and sTILs. We performed immunohistochemistry for PD-L1 (clone SP142) and pan-TRK (clone EPR17341) on tissue microarrays. Pan-TRK positive cases were further analyzed by next-generation sequencing. The median age was 69 years (range 60−77). Invasive carcinoma of no special type was found in 94.7% of cases, of which 53.1% were grade 2. Estrogen receptor was positive in 92% of the tumors, progesterone receptor in 85.8%, androgen receptor in 70.8%; 4.4% were human epidermal growth factor receptor 2 (HER2)-positive, and 55.8% HER2-low. 40.7% of tumors were luminal A and 51.3% luminal B, 4.4% HER2-enriched and 3.5% triple negative carcinoma. sTILs density was <50% in 96.4% of the tumors, >50% in 3.6% of the tumors. PD-L1 immune cell score >1% was found in 7.1% of the tumors (all of luminal subtype). A weak focal cytoplasmic pan-TRK staining was present in 8.8% but without NTRK fusion. Neither sTILs nor PD-L1 had statistically significant outcomes. Our findings suggest that a subset of MBC patients harbors an immunological environment characterized by increased sTILs with PD-L1 expression. These patients may potentially benefit from immune checkpoint inhibitor therapy. Frequent HER2-low may offer novel anti-HER2 treatment options.

Sensitive and robust liquid biopsy-based detection of PIK3CA mutations in hormone-receptor-positive metastatic breast cancer patients.

BACKGROUND: The benefit of alpelisib in hormone-receptor-positive (HR+) metastatic breast cancer patients provided clinical evidence for the increasing importance of PIK3CA testing. We performed a comparison of liquid biopsy and tissue-based detection of PIK3CA mutations. MATERIALS AND METHODS: PIK3CA hotspot mutation analysis using a high-resolution SiMSen-Seq assay was performed in plasma from 93/99 eligible patients with HR+/HER2- breast cancer. Additionally, mFAST-SeqS was used to estimate the tumour fractions in plasma samples. In 72/93 patients, matched tissue was available and analysed using a customised Ion Torrent panel. RESULTS: PIK3CA mutations were detected in 48.6% of tissue samples and 47.3% of plasma samples, with identical PIK3CA mutation detected in 24/72 (33.3%) patients both in tissue and plasma. In 10 (13.9%) patients, mutations were only found in plasma, and in 6 (8.3%) patients, PIK3CA mutations found in tissue were not detectable in ctDNA. In 49/93 plasma samples without detectable PIK3CA mutations, 22 (44.9%) samples had elevated tumour fractions, implying true negative results. CONCLUSION: SiMSen-Seq-based detection of PIK3CA mutations in plasma shows advantageous concordance with the tissue analyses. A combination with an untargeted approach for detecting ctDNA fractions may confirm a negative PIK3CA result and enhance the performance of the SiMSen-Seq test.

Biosafety Requirements for Autopsies of Patients with COVID-19: Example of a BSL-3 Autopsy Facility Designed for Highly Pathogenic Agents.

Information obtained from autopsies of patients infected with high-risk pathogens is an important pillar in managing a proper response to pandemics, particular in the early phase. This is due to the fact that autopsy allows efficient evaluation of comorbidities for risk assessment, as well as identification of key pathophysiological and molecular mechanisms in organs driving the severity of disease which might be important targets for therapeutic interventions. In the case of patients who have died of infection with unknown pathogens, isolation and culture of pathogens from the affected organs is another important opportunity for a proper response to (re)emerging infectious diseases. However, the situation of COVID-19 demonstrated that there were concerns about performing autopsies because of biosafety risks. In this review we compare requirements for biosafety level 3 (BSL-3) laboratories from the European Commission and the World Health Organization and summarize specific recommendations for postmortem analysis of COVID-19-deceased patients from the Centers for Disease Control and Prevention. Furthermore, we describe in detail a BSL-3 facility with enhanced protection of personnel and an environment that has been designed for performing autopsies, biobanking of collected tissue specimens, and culture of pathogens in cases of high-risk pathogen infections and report on the experience obtained in operating this facility in the context of COVID-19.

Prognostic value of histopathological DCIS features in a large-scale international interrater reliability study.

PURPOSE: For optimal management of ductal carcinoma in situ (DCIS), reproducible histopathological assessment is essential to distinguish low-risk from high-risk DCIS. Therefore, we analyzed interrater reliability of histopathological DCIS features and assessed their associations with subsequent ipsilateral invasive breast cancer (iIBC) risk. METHODS: Using a case-cohort design, reliability was assessed in a population-based, nationwide cohort of 2767 women with screen-detected DCIS diagnosed between 1993 and 2004, treated by breast-conserving surgery with/without radiotherapy (BCS ± RT) using Krippendorff's alpha (KA) and Gwet's AC2 (GAC2). Thirty-eight raters scored histopathological DCIS features including grade (2-tiered and 3-tiered), growth pattern, mitotic activity, periductal fibrosis, and lymphocytic infiltrate in 342 women. Using majority opinion-based scores for each feature, their association with subsequent iIBC risk was assessed using Cox regression. RESULTS: Interrater reliability of grade using various classifications was fair to moderate, and only substantial for grade 1 versus 2 + 3 when using GAC2 (0.78). Reliability for growth pattern (KA 0.44, GAC2 0.78), calcifications (KA 0.49, GAC2 0.70) and necrosis (KA 0.47, GAC2 0.70) was moderate using KA and substantial using GAC2; for (type of) periductal fibrosis and lymphocytic infiltrate fair to moderate estimates were found and for mitotic activity reliability was substantial using GAC2 (0.70). Only in patients treated with BCS-RT, high mitotic activity was associated with a higher iIBC risk in univariable analysis (Hazard Ratio (HR) 2.53, 95% Confidence Interval (95% CI) 1.05-6.11); grade 3 versus 1 + 2 (HR 2.64, 95% CI 1.35-5.14) and a cribriform/solid versus flat epithelial atypia/clinging/(micro)papillary growth pattern (HR 3.70, 95% CI 1.34-10.23) were independently associated with a higher iIBC risk. CONCLUSIONS: Using majority opinion-based scores, DCIS grade, growth pattern, and mitotic activity are associated with iIBC risk in patients treated with BCS-RT, but interrater variability is substantial. Semi-quantitative grading, incorporating and separately evaluating nuclear pleomorphism, growth pattern, and mitotic activity, may improve the reliability and prognostic value of these features.

Occurrence of SARS-CoV-2 in the intraocular milieu.

The purpose of this research is to study the intraocular occurrence of SARS-CoV-2. In postmortem examinations, aqueous humor and the vitreous samples were collected. All individuals were previously positive in nasopharyngeal swabbing and cause of death was respiratory failure due to SARS-CoV-2 infection. Testing was done using quantitative RT-PCR. We included 16 aqueous humor and 16 vitreous samples for PCR testing. None of the results was positive for SARS-CoV-2. Human GAPDH genes to verify the presence of RNA was present in all aqueous humor samples (16/16, 100%) and 15/16 (93.8%) vitreous samples. In conclusion, this case series found no evidence of SARS-CoV-2 in the intraocular milieu.

Reproducibility and predictive value of scoring stromal tumour infiltrating lymphocytes in triple-negative breast cancer: a multi-institutional study.

BACKGROUND: Several studies have demonstrated a prognostic role for stromal tumour infiltrating lymphocytes (sTILs) in triple-negative breast cancer (TNBC). The reproducibility of scoring sTILs is variable with potentially excellent concordance being achievable using a software tool. We examined agreement between breast pathologists across Europe scoring sTILs on H&E-stained sections without software, an approach that is easily applied in clinical practice. The association between sTILs and response to anthracycline-taxane NACT was also examined. METHODOLOGY: Pathologists from the European Working Group for Breast Screening Pathology scored sTILs in 84 slides from 75 TNBCs using the immune-oncology biomarker working group guidance in two circulations. There were 16 participants in the first and 19 in the second circulation. RESULTS: Moderate agreement was achieved for absolute sTILs scores (intraclass correlation coefficient (ICC) = 0.683, 95% CI 0.601-0.767, p-value < 0.001). Agreement was less when a 25% threshold was used (ICC 0.509, 95% CI 0.416-0.614, p-value < 0.001) and for lymphocyte predominant breast cancer (LPBC) (ICC 0.504, 95% CI 0.412-0.610, p-value < 0.001). Intra-observer agreement was strong for absolute sTIL values (Spearman ρ = 0.727); fair for sTILs ≥ 25% (κ = 0.53) and for LPBC (κ = 0.49), but poor for sTILs as 10% increments (κ = 0.24). Increasing sTILs was significantly associated with an increased likelihood of a pathological complete response (pCR) on multivariable analysis. CONCLUSION: Increasing sTILs in TNBCs improves the likelihood of a pCR. However, inter-observer agreement is such that H&E-based assessment is not sufficiently reproducible for clinical application. Other methodologies should be explored, but may be at the cost of ease of application.

Radiation-induced morphea-a rare but severe late effect of adjuvant breast irradiation : Case report and review of the literature.

BACKGROUND: Radiation-induced morphea (RIM) is a circumscribed localized scleroderma that occurs most often in the breast. After an asymptomatic period of one month to several years, the symptoms (circumscribed inflammation, edema, sclerosis) often arise suddenly and cannot be clinically distinguished from a local recurrence in the form of inflammatory carcinoma. CASE: We present a case of a 74-year-old woman who developed this rare and serious local side-effect in connective tissue following neoadjuvant CDK 4/6 inhibitor abemaciclib (Verzenio®) and aromatase inhibitor anastrozole (Arimidex®) therapy and subsequent radiation therapy of the breast. CONCLUSIONS: Little is known about risk factors and pathogenesis of RIM. Here we describe the first case of RIM following immunotherapy. The diagnosis is based on clinical appearance and histopathological examination. Treatment should be initiated in the inflammatory stage in order to prevent or delay irreversible fibrosis and atrophy of the breast.

The misunderstanding of the R Classification-a survey amongst medical specialties treating breast cancer.

Frequent discussions in the tumour board about the Residual tumour (R) Classification of the UICC's "TNM Classification of Malignant Tumours", especially in the case of breast surgery specimens, raised the question about differing interpretations amongst different medical specialties. Thus, we designed a survey about the R Classification with a special focus on breast cancer specimens. An online survey was conducted, where a web link to the survey was distributed via email to various medical professional societies dealing with breast cancer in Austria and Germany with the request to distribute the link to their members. The study population consisted of physicians of all educational levels of different medical professions, who deal with breast carcinomas in their daily routine. Two hundred two participants, of which 160 (79.2%) have more than 10 years' professional experience, took part in the survey; 88 (43.6%) were surgeons/gynaecologists, 80 (39.6%) pathologists, 19 (9.4%) radiation oncologists/ therapists, 8 (4.0%) radiologists, and 7 (3.5%) oncologists. We show that the R Classification is not completely mastered by anyone and that there are significant differences in the interpretation of the R Classification between different medical specialties. For better differentiation between the residual tumour (R Classification) of the TNM and a pure resection margin assessment, we suggest the use of a Resection margin (Rm) Classification to avoid further misunderstandings. To assist better multidisciplinary cooperation and to ensure better patient care all medical disciplines should be educated about the actual meaning and correct application of the R Classification.

Biomechanical characterization of the passive porcine stomach.

The complex mechanics of the gastric wall facilitates the main digestive tasks of the stomach. However, the interplay between the mechanical properties of the stomach, its microstructure, and its vital functions is not yet fully understood. Importantly, the pig animal model is widely used in biomedical research for preliminary or ethically prohibited studies of the human digestion system. Therefore, this study aims to thoroughly characterize the mechanical behavior and microstructure of the porcine stomach. For this purpose, multiple quasi-static mechanical tests were carried out with three different loading modes, i.e., planar biaxial extension, radial compression, and simple shear. Stress-relaxation tests complemented the quasi-static experiments to evaluate the deformation and strain-dependent viscoelastic properties. Each experiment was conducted on specimens of the complete stomach wall and two separate layers, mucosa and muscularis, from each of the three gastric regions, i.e., fundus, body, and antrum. The significant preconditioning effects and the considerable regional and layer-specific differences in the tissue response were analyzed. Furthermore, the mechanical experiments were complemented with histology to examine the influence of the microstructural composition on the macrostructural mechanical response and vice versa. Importantly, the shear tests showed lower stresses in the complete wall compared to the single layers which the loose network of submucosal collagen might explain. Also, the stratum arrangement of the muscularis might explain mechanical anisotropy during tensile tests. This study shows that gastric tissue is characterized by a highly heterogeneous microstructure with regional variations in layer composition reflecting not only functional differences but also diverse mechanical behavior. STATEMENT OF SIGNIFICANCE: Unfortunately, only few experimental data on gastric tissue are available for an adequate material parameter and model estimation. The present study therefore combines layer- and region-specific stomach wall mechanics obtained under multiple loading conditions with histological insights into the heterogeneous microstructure. On the one hand, the extensive data sets of this study expand our understanding of the interplay between gastric mechanics, motility and functionality, which could help to identify and treat associated pathologies. On the other hand, such data sets are of high relevance for the constitutive modeling of stomach tissue, and its application in the field of medical engineering, e.g., in the development of surgical staplers and the improvement of bariatric surgical interventions.

European guidelines for the diagnosis, treatment and follow-up of breast lesions with uncertain malignant potential (B3 lesions) developed jointly by EUSOMA, EUSOBI, ESP (BWG) and ESSO.

INTRODUCTION: Breast lesions of uncertain malignant potential (B3) include atypical ductal and lobular hyperplasias, lobular carcinoma in situ, flat epithelial atypia, papillary lesions, radial scars and fibroepithelial lesions as well as other rare miscellaneous lesions. They are challenging to categorise histologically, requiring specialist training and multidisciplinary input. They may coexist with in situ or invasive breast cancer (BC) and increase the risk of subsequent BC development. Management should focus on adequate classification and management whilst avoiding overtreatment. The aim of these guidelines is to provide updated information regarding the diagnosis and management of B3 lesions, according to updated literature review evidence. METHODS: These guidelines provide practical recommendations which can be applied in clinical practice which include recommendation grade and level of evidence. All sections were written according to an updated literature review and discussed at a consensus meeting. Critical appraisal by the expert writing committee adhered to the 23 items in the international Appraisal of Guidelines, Research and Evaluation (AGREE) tool. RESULTS: Recommendations for further management after core-needle biopsy (CNB) or vacuum-assisted biopsy (VAB) diagnosis of a B3 lesion reported in this guideline, vary depending on the presence of atypia, size of lesion, sampling size, and patient preferences. After CNB or VAB, the option of vacuum-assisted excision or surgical excision should be evaluated by a multidisciplinary team and shared decision-making with the patient is crucial for personalizing further treatment. De-escalation of surgical intervention for B3 breast lesions is ongoing, and the inclusion of vacuum-assisted excision (VAE) will decrease the need for surgical intervention in further approaches. Communication with patients may be different according to histological diagnosis, presence or absence of atypia, or risk of upgrade due to discordant imaging. Written information resources to help patients understand these issues alongside with verbal communication is recommended. Lifestyle interventions have a significant impact on BC incidence so lifestyle interventions need to be suggested to women at increased BC risk as a result of a diagnosis of a B3 lesion. CONCLUSIONS: These guidelines provide a state-of-the-art overview of the diagnosis, management and prognosis of B3 lesions in modern multidisciplinary breast practice.

EUSOMA quality indicators for non-metastatic breast cancer: An update.

INTRODUCTION: Quality care in breast cancer is higher if patients are treated in a Breast Center with a dedicated and specialized multidisciplinary team. Quality control is an essential activity to ensure quality care, which has to be based on the monitoring of specific quality indicators. Eusoma has proceeded with the up-dating of the 2017 Quality indicators for non-metastatic breast cancer based on the new diagnostic, locoregional and systemic treatment modalities. METHODS: To proceed with the updating, EUSOMA setup a multidisciplinary working group of BC experts and patients' representatives. It is a comprehensive set of QIs for early breast cancer care, which are classified as mandatory, recommended, or observational. For the first time patient reported outcomes (PROMs) have been included. As used in the 2017 EUSOMA QIs, evidence levels were based on the short version of the US Agency for Healthcare Research and Quality. RESULTS: This is a set of quality indicators representative for the different steps of the patient pathway in non-metastatic setting, which allow Breast Centres to monitor their performance with referring standards, i.e minimum standard and target. CONCLUSIONS: Monitoring these Quality Indicators, within the Eusoma datacentre will allow to have a state of the art picture at European Breast Centres level and the development of challenging research projects.

Quality of screening with conventional Pap smear in Austria - a longitudinal evaluation.

BACKGROUND: In recent decades, the incidence of cervical cancer and cervical cancer mortality in Austria has declined by varying degrees. The Pap smear is to be considered a causal factor for this decline. METHODS: This longitudinal analysis is based on a data set of Pap smear assessments collected by the Committee for Quality Assurance of the Austrian Society of Cytology. Data from 15 laboratories participating in a voluntary self-monitoring program was analyzed for the time span 2004-2008. The data was analyzed in terms of smear quality and assessment quality.A rank-correlation-test for a monotonic trend analysis in the proportion of the three parameters Pap 0, "satisfactory, but limited/SBL", and Pap IIID/IV for the timespan 2004 to 2008 was carried out. RESULTS: For this study, we analyzed an average number of 730,000 smears per year over a five-year period. Specimens from all but two laboratories, i.e. < 2% of all smears, met the quality criterion for Pap 0 (Bethesda 2001 equivalent: Specimen processed and examined, but unsatisfactory for evaluation of epithelial abnormality), whilst only four laboratories, i.e. < 10% of all smears, reached the national requirement for smears classified as "satisfactory, but limited/SBL".When using the Pap IIID/IV ratio (LSIL: HSIL/AIS ratio) of 3:1 to 8:1 as a surrogate quality marker for the interpretation of smears, only five laboratories met this criterion during the survey period.The trend analysis indicated only that an increasing number of samples per year is correlated with an increased proportion of Pap 0 and "satisfactory, but limited/SBL" smears. CONCLUSIONS: Although participants get regular feedback about their results, no general improvements in smear taking or assessment were observed over the years, so mandatory quality management, including the possibility of sanctions, is suggested in order to reduce adverse health effects for women.

Global and local stiffening of ex vivo-perfused stented human thoracic aortas: A mock circulation study.

The effects of thoracic endovascular repair (TEVAR) on the biomechanical properties of aortic tissue have not been adequately studied. Understanding these features is important for the management of endograft-triggered complications of a biomechanical nature. This study aims to examine how stent-graft implantation affects the elastomechanical behavior of the aorta. Non-pathological human thoracic aortas (n=10) were subjected to long-standing perfusion (8h) within a mock circulation loop under physiological conditions. To quantify compliance and its mismatch in the test periods without and with a stent, the aortic pressure and the proximal cyclic circumferential displacement were measured. After perfusion, biaxial tension tests (stress-stretch) were carried out to examine the stiffness profiles between non-stented and stented tissue, followed by a histological assessment. Experimental evidence shows: (i) a significant reduction in aortic distensibility after TEVAR, indicating aortic stiffening and compliance mismatch, (ii) a stiffer behavior of the stented samples compared to the non-stented samples with an earlier entry into the nonlinear part of the stress-stretch curve and (iii) strut-induced histological remodeling of the aortic wall. The biomechanical and histological comparison of the non-stented and stented aortas provides new insights into the interaction between the stent-graft and the aortic wall. The knowledge gained could refine the stent-graft design to minimize the stent-induced impacts on the aortic wall and the resulting complications. STATEMENT OF SIGNIFICANCE: Stent-related cardiovascular complications occur the moment the stent-graft expands on the human aortic wall. Clinicians base their diagnosis on the anatomical morphology of CT scans while neglecting the endograft-triggered biomechanical events that compromise aortic compliance and wall mechanotransduction. Experimental replication of endovascular repair in cadaver aortas within a mock circulation loop may have a catalytic effect on biomechanical and histological findings without an ethical barrier. Demonstrating interactions between the stent and the wall can help clinicians make a broader diagnosis such as ECG-triggered oversizing and stent-graft characteristics based on patient-specific anatomical location and age. In addition, the results can be used to optimize towards more aortophilic stent grafts.

Changes in the microstructure of the human aortic adventitia under biaxial loading investigated by multi-photon microscopy.

Among the three layers of the aortic wall, the media is primarily responsible for its mechanical properties, but the adventitia prevents the aorta from overstretching and rupturing. The role of the adventitia is therefore crucial with regard to aortic wall failure, and understanding the load-induced changes in tissue microstructure is of high importance. Specifically, the focus of this study is on the changes in collagen and elastin microstructure in response to macroscopic equibiaxial loading applied to the aortic adventitia. To observe these changes, multi-photon microscopy imaging and biaxial extension tests were performed simultaneously. In particular, microscopy images were recorded at 0.02 stretch intervals. The microstructural changes of collagen fiber bundles and elastin fibers were quantified with the parameters of orientation, dispersion, diameter, and waviness. The results showed that the adventitial collagen was divided from one into two fiber families under equibiaxial loading conditions. The almost diagonal orientation of the adventitial collagen fiber bundles remained unchanged, but the dispersion was substantially reduced. No clear orientation of the adventitial elastin fibers was observed at any stretch level. The waviness of the adventitial collagen fiber bundles decreased under stretch, but the adventitial elastin fibers showed no change. These original findings highlight differences between the medial and adventitial layers and provide insight into the stretching process of the aortic wall. STATEMENT OF SIGNIFICANCE: To provide accurate and reliable material models, it is essential to understand the mechanical behavior of the material and its microstructure. Such understanding can be enhanced with tracking of the microstructural changes caused by mechanical loading of the tissue. This study provides therefore a unique dataset of structural parameters of the human aortic adventitia obtained under equibiaxial loading. The structural parameters describe orientation, dispersion, diameter, and waviness of collagen fiber bundles and elastin fibers. Eventually, the microstructural changes in the human aortic adventitia are compared with the microstructural changes in the human aortic media from a previous study. This comparison reveals the cutting-edge findings on the differences in the response to the loading between these two human aortic layers.

Explainability and causability in digital pathology.

The current move towards digital pathology enables pathologists to use artificial intelligence (AI)-based computer programmes for the advanced analysis of whole slide images. However, currently, the best-performing AI algorithms for image analysis are deemed black boxes since it remains - even to their developers - often unclear why the algorithm delivered a particular result. Especially in medicine, a better understanding of algorithmic decisions is essential to avoid mistakes and adverse effects on patients. This review article aims to provide medical experts with insights on the issue of explainability in digital pathology. A short introduction to the relevant underlying core concepts of machine learning shall nurture the reader's understanding of why explainability is a specific issue in this field. Addressing this issue of explainability, the rapidly evolving research field of explainable AI (XAI) has developed many techniques and methods to make black-box machine-learning systems more transparent. These XAI methods are a first step towards making black-box AI systems understandable by humans. However, we argue that an explanation interface must complement these explainable models to make their results useful to human stakeholders and achieve a high level of causability, i.e. a high level of causal understanding by the user. This is especially relevant in the medical field since explainability and causability play a crucial role also for compliance with regulatory requirements. We conclude by promoting the need for novel user interfaces for AI applications in pathology, which enable contextual understanding and allow the medical expert to ask interactive 'what-if'-questions. In pathology, such user interfaces will not only be important to achieve a high level of causability. They will also be crucial for keeping the human-in-the-loop and bringing medical experts' experience and conceptual knowledge to AI processes.

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.

Third International Consensus Conference on lesions of uncertain malignant potential in the breast (B3 lesions).

The heterogeneous group of B3 lesions in the breast harbors lesions with different malignant potential and progression risk. As several studies about B3 lesions have been published since the last Consensus in 2018, the 3rd International Consensus Conference discussed the six most relevant B3 lesions (atypical ductal hyperplasia (ADH), flat epithelial atypia (FEA), classical lobular neoplasia (LN), radial scar (RS), papillary lesions (PL) without atypia, and phyllodes tumors (PT)) and made recommendations for diagnostic and therapeutic approaches. Following a presentation of current data of each B3 lesion, the international and interdisciplinary panel of 33 specialists and key opinion leaders voted on the recommendations for further management after core-needle biopsy (CNB) and vacuum-assisted biopsy (VAB). In case of B3 lesion diagnosis on CNB, OE was recommended in ADH and PT, whereas in the other B3 lesions, vacuum-assisted excision was considered an equivalent alternative to OE. In ADH, most panelists (76%) recommended an open excision (OE) after diagnosis on VAB, whereas observation after a complete VAB-removal on imaging was accepted by 34%. In LN, the majority of the panel (90%) preferred observation following complete VAB-removal. Results were similar in RS (82%), PL (100%), and FEA (100%). In benign PT, a slim majority (55%) also recommended an observation after a complete VAB-removal. VAB with subsequent active surveillance can replace an open surgical intervention for most B3 lesions (RS, FEA, PL, PT, and LN). Compared to previous recommendations, there is an increasing trend to a de-escalating strategy in classical LN. Due to the higher risk of upgrade into malignancy, OE remains the preferred approach after the diagnosis of ADH.

Analysis of functional germline polymorphisms for prediction of response to anthracycline-based neoadjuvant chemotherapy in breast cancer.

To elucidate the role of predictive factors on individual's drug response, based on genetic variation, we examined the association between eight germline polymorphisms in genes involved in protection against oxidative stress, apoptosis, oncogenic transformation, proliferation, immune response and DNA repair (TP53, NQO1, IL6, TLR4 and XRCC1) and the pathological response to anthracycline-based neoadjuvant chemotherapy in 70 patients with breast cancer. The DNA was genotyped for eight polymorphisms in five genes (TP53, NQO1, IL6, TLR4 and XRCC1) by 5'-exonuclease (TaqMan™) technology. Fisher's exact test was used to evaluate the association between genotype, clinicopathological parameters and pathological response. A good pathological response, defined as a pathological complete response or residual isolated invasive tumor cells, was found significantly more frequently for estrogen (ER) and progesterone receptor (PR) negative breast carcinomas compared to ER and PR positive and ER or PR positive carcinomas, respectively (43.5 vs. 37.5 and 10.3 %, p = 0.006), and was significantly associated with high tumor grade (G3) (p = 0.002). A non-significant trend towards a good pathological response was shown in patients carrying the Arg/Arg or Arg/Pro TP53 codon 72 gene variant compared to those harboring the Pro/Pro variant (17.6 or 37.9 % vs. 0; p = 0.071). No association was found between NQO1 Pro187Ser, IL6 -174G>C, TLR4 Asp299Gly and Thr399Ile, and XRCC1 Arg194Trp, Arg399Gln and Arg280His and pathological response. The present study shows hormone receptor status and tumor grade as predictors for pathological response to neoadjuvant anthracycline-based chemotherapy. Among various functional germline polymorphisms, a potential predictive value was only found for the TP53 Arg72Pro gene variant.

Changes in the microstructure of the human aortic medial layer under biaxial loading investigated by multi-photon microscopy.

Understanding the correlation between tissue architecture, health status, and mechanical properties is essential for improving material models and developing tissue engineering scaffolds. Since structural-based material models are state of the art, there is an urgent need for experimentally obtained structural parameters. For this purpose, the medial layer of nine human abdominal aortas was simultaneously subjected to equibiaxial loading and multi-photon microscopy. At each loading interval of 0.02, collagen and elastin fibers were imaged based on their second-harmonic generation signal and two-photon excited autofluorescence, respectively. The structural alterations in the fibers were quantified using the parameters of orientation, diameter, and waviness. The results of the mechanical tests divided the sample cohort into the ruptured and non-ruptured, and stiff and non-stiff groups, which were covered by the findings from histological investigations. The alterations in structural parameters provided an explanation for the observed mechanical behavior. In addition, the waviness parameters of both collagen and elastin fibers showed the potential to serve as indicators of tissue strength. The data provided address deficiencies in current material models and bridge multiscale mechanisms in the aortic media. STATEMENT OF SIGNIFICANCE: Available material models can reproduce, but cannot predict, the mechanical behavior of human aortas. This deficiency could be overcome with the help of experimentally validated structural parameters as provided in this study. Simultaneous multi-photon microscopy and biaxial extension testing revealed the microstructure of human aortic media at different stretch levels. Changes in the arrangement of collagen and elastin fibers were quantified using structural parameters such as orientation, diameter and waviness. For the first time, structural parameters of human aortic tissue under continuous loading conditions have been obtained. In particular, the waviness parameters at the reference configuration have been associated with tissue stiffness, brittleness, and the onset of atherosclerosis.