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Pitx2 is a transcription factor responsible for establishment of the right-left axis and development of the gut and pituitary. In mouse embryos, Pitx2 is expressed in the greater curvature of the stomach and midgut. Previously, Pitx2 was studied in pituitary neuroendocrine tumours but not in other NETs. Pitx2 expression was immunohistochemically assessed in whole sections and tissue microarrays in a cohort of 224 neuroendocrine neoplasms, and was analysed in 29 cases. The cohort included 18 cauda equina NETs, 38 paragangliomas, 98 cases of primary visceral NETs from different organs, 23 metastases of visceral NETs and 47 neuroendocrine carcinomas (NECs). Pitx2 expression was observed in 29.5% (29 of 98) NETs and 14.9% (7 of 47) NECs, but was not observed in any paraganglioma or cauda equina NET. Pitx2 was observed only in tumours of midgut-derived organs, including the small intestine (100%, 20 of 20), appendix (88.9%, eight of nine) and large intestine (9.1%, one of 11 - only caecal NET). The NETs of remaining locations were negative. Pitx2 was 96.7% sensitive and 100% specific for NETs of midgut origin. In NECs, Pitx2 positivity was observed in goblet cell adenocarcinoma (75%, three of four), medullary thyroid carcinoma (42.9, three of seven) and one Merkel cell carcinoma (25%, one of four). In metastatic NETs, Pitx2 was observed in all the tumours originating in the small intestine (n = 17) or caecum (n = 1). No positivity was observed in tumours from other locations (four pancreas, one lung). We observed no correlation between immunoreactivity and mRNA expression. Thus, Pitx2 immunohistochemistry can be helpful in assessing the midgut origin of NETs.

PURPOSE Triple-negative breast cancer (TNBC) is considered aggressive, and therefore, virtually all young patients with TNBC receive (neo)adjuvant chemotherapy. Increased stromal tumor-infiltrating lymphocytes (sTILs) have been associated with a favorable prognosis in TNBC. However, whether this association holds for patients who are node-negative (N0), young (< 40 years), and chemotherapy-naïve, and thus can be used for chemotherapy de-escalation strategies, is unknown. METHODS We selected all patients with N0 TNBC diagnosed between 1989 and 2000 from a Dutch population–based registry. Patients were age < 40 years at diagnosis and had not received (neo)adjuvant systemic therapy, as was standard practice at the time. Formalin-fixed paraffin-embedded blocks were retrieved (PALGA: Dutch Pathology Registry), and a pathology review including sTILs was performed. Patients were categorized according to sTILs (< 30%, 30%-75%, and ≥ 75%). Multivariable Cox regression was performed for overall survival, with or without sTILs as a covariate. Cumulative incidence of distant metastasis or death was analyzed in a competing risk model, with second primary tumors as competing risk. RESULTS sTILs were scored for 441 patients. High sTILs (≥ 75%; 21%) translated into an excellent prognosis with a 15-year cumulative incidence of a distant metastasis or death of only 2.1% (95% CI, 0 to 5.0), whereas low sTILs (< 30%; 52%) had an unfavorable prognosis with a 15-year cumulative incidence of a distant metastasis or death of 38.4% (32.1 to 44.6). In addition, every 10% increment of sTILs decreased the risk of death by 19% (adjusted hazard ratio: 0.81; 95% CI, 0.76 to 0.87), which are an independent predictor adding prognostic information to standard clinicopathologic variables (χ2 = 46.7, P < .001). CONCLUSION Chemotherapy-naïve, young patients with N0 TNBC with high sTILs (≥ 75%) have an excellent long-term prognosis. Therefore, sTILs should be considered for prospective clinical trials investigating (neo)adjuvant chemotherapy de-escalation strategies.

Formalin, an aqueous solution of formaldehyde, has been the gold standard for fixation of histological samples for over a century. Despite its considerable advantages, growing evidence points to objective toxicity, particularly highlighting its carcinogenicity and mutagenic effects. In 2016, European Union proposed a ban, but a temporary permission was granted in consideration of its fundamental role in the medical-diagnostic field.In the present study, we tested an innovative fixative, Glyoxal Acid-Free (GAF) (a glyoxal solution deprived of acids), which allows optimal tissue fixation at structural and molecular level combined with the absence of toxicity and carcinogenic activity. An open label, non-inferiority, multicentric trial was performed comparing fixation of histological specimens with GAF fixative vs standard Phosphate Buffered Formalin (PBF), evaluating the morphological preservation and the diagnostic value with four binary score questions answered by both the central pathology reviewer and local centre reviewers. The mean of total score in the GAF vs PBF fixative groups was 3.7 ± 0.5 vs 3.9 ±0.3 for the central reviewer and 3.8 ± 0.5 vs 4.0 ±0.1 for the local pathologist reviewers, respectively. In terms of median value, similar results were observed between the two fixative groups, with a median value of 4.0. Data collected indicate the non-inferiority of GAF as compared to PBF for all organ tested. The present clinical performance study, performed following the international standard for performance evaluation ofin vitrodiagnostic medical devices, highlights the capability of GAF to ensure both, structural preservation and diagnostic value of the preparations.

Purpose:The successful implementation of immune checkpoint inhibitors (ICI) in the clinical management of various solid tumors has raised considerable expectations for patients with epithelial ovarian carcinoma (EOC). However, EOC is poorly responsive to ICIs due to immunologic features including limited tumor mutational burden (TMB) and poor lymphocytic infiltration. An autologous dendritic cell (DC)-based vaccine (DCVAC) has recently been shown to be safe and to significantly improve progression-free survival (PFS) in a randomized phase II clinical trial enrolling patients with EOC (SOV01, NCT02107937).Patients and Methods:We harnessed sequencing, flow cytometry, multispectral immunofluorescence microscopy, and IHC to analyze (pretreatment) tumor and (pretreatment and posttreatment) peripheral blood samples from 82 patients enrolled in SOV01, with the aim of identifying immunologic biomarkers that would improve the clinical management of patients with EOC treated with DCVAC.Results:Although higher-than-median TMB and abundant CD8+ T-cell infiltration were associated with superior clinical benefits in patients with EOC receiving standard-of-care chemotherapy, the same did not hold true in women receiving DCVAC. Conversely, superior clinical responses to DCVAC were observed in patients with lower-than-median TMB and scarce CD8+ T-cell infiltration. Such responses were accompanied by signs of improved effector functions and tumor-specific cytotoxicity in the peripheral blood.Conclusions:Our findings suggest that while patients with highly infiltrated, “hot” EOCs benefit from chemotherapy, women with “cold” EOCs may instead require DC-based vaccination to jumpstart clinically relevant anticancer immune responses.

Supplementary Figure from An Autologous Dendritic Cell Vaccine Promotes Anticancer Immunity in Patients with Ovarian Cancer with Low Mutational Burden and Cold Tumors

Supplementary Data from An Autologous Dendritic Cell Vaccine Promotes Anticancer Immunity in Patients with Ovarian Cancer with Low Mutational Burden and Cold Tumors

Purpose:In multiple oncological settings, expression of the coinhibitory ligand PD-L1 by malignant cells and tumor infiltration by immune cells expressing coinhibitory receptors such as PD-1, CTLA4, LAG-3, or TIM-3 conveys prognostic or predictive information. Conversely, the impact of these features of the tumor microenvironment on disease outcome among high-grade serous carcinoma (HGSC) patients remains controversial.Experimental Design:We harnessed a retrospective cohort of 80 chemotherapy-naïve HGSC patients to investigate PD-L1 expression and tumor infiltration by CD8+ T cells, CD20+ B cells, DC-LAMP+ dendritic cells as well as by PD-1+, CTLA4+, LAG-3+, and TIM-3+ cells in relation with prognosis and function orientation of the tumor microenvironment. IHC data were complemented with transcriptomic and functional studies on a second prospective cohort of freshly resected HGSC samples. In silico analysis of publicly available RNA expression data from 308 HGSC samples was used as a confirmatory approach.Results:High levels of PD-L1 and high densities of PD-1+ cells in the microenvironment of HGSCs were strongly associated with an immune contexture characterized by a robust TH1 polarization and cytotoxic orientation that enabled superior clinical benefits. Moreover, PD-1+TIM-3+CD8+ T cells presented all features of functional exhaustion and correlated with poor disease outcome. However, although PD-L1 levels and tumor infiltration by TIM-3+ cells improved patient stratification based on the intratumoral abundance of CD8+ T cells, the amount of PD-1+ cells failed to do so.Conclusions:Our data indicate that PD-L1 and TIM-3 constitute prognostically relevant biomarkers of active and suppressed immune responses against HGSC, respectively.

Purpose: The successful implementation of immune checkpoint inhibitors (ICI) in the clinical management of various solid tumors has raised considerable expectations for patients with epithelial ovarian carcinoma (EOC). However, EOC is poorly responsive to ICIs due to immunologic features including limited tumor mutational burden (TMB) and poor lymphocytic infiltration. An autologous dendritic cell (DC)-based vaccine (DCVAC) has recently been shown to be safe and to significantly improve progression-free survival (PFS) in a randomized phase II clinical trial enrolling patients with EOC (SOV01, NCT02107937). Patients and Methods: We harnessed sequencing, flow cytometry, multispectral immunofluorescence microscopy, and IHC to analyze (pretreatment) tumor and (pretreatment and posttreatment) peripheral blood samples from 82 patients enrolled in SOV01, with the aim of identifying immunologic biomarkers that would improve the clinical management of patients with EOC treated with DCVAC. Results: Although higher-than-median TMB and abundant CD8+ T-cell infiltration were associated with superior clinical benefits in patients with EOC receiving standard-of-care chemotherapy, the same did not hold true in women receiving DCVAC. Conversely, superior clinical responses to DCVAC were observed in patients with lower-than-median TMB and scarce CD8+ T-cell infiltration. Such responses were accompanied by signs of improved effector functions and tumor-specific cytotoxicity in the peripheral blood. Conclusions: Our findings suggest that while patients with highly infiltrated, “hot” EOCs benefit from chemotherapy, women with “cold” EOCs may instead require DC-based vaccination to jumpstart clinically relevant anticancer immune responses.

In somatotroph pituitary tumours, somatostatin analogue (SSA) therapy outcomes vary throughout the studies. We performed an analysis of cohort of patients with acromegaly from the Czech registry to identify new prognostic and predictive factors. Clinical data of patients were collected, and complex immunohistochemical assessment of tumour samples was performed (SSTR1‐5, dopamine D2 receptor, E‐cadherin, AIP). The study included 110 patients. In 31, SSA treatment outcome was evaluated. Sparsely granulated tumours (SGST) differed from the other subtypes in expression of SSTR2A, SSTR3, SSTR5 and E‐cadherin and occurred more often in young. No other clinical differences were observed. Trouillas grading system showed association with age, tumour size and SSTR2A expression. Factors significantly associated with SSA treatment outcome included age, IGF1 levels, tumour size and expression of E‐cadherin and SSTR2A. In the group of SGST, poor SSA response was observed in younger patients with larger tumours, lower levels of SSTR2A and higher Ki67. We observed no relationship with expression of other proteins including AIP. No predictive value of E‐cadherin was observed when tumour subtype was considered. Multiple additional factors apart from SSTR2A expression can predict treatment outcome in patients with acromegaly.

Background MicroRNAs (miRNAs) are non-coding regulatory molecules 18-25 nucleotides in length that act as post-transcriptional regulators of gene expression. MiRNAs affect various biological processes including carcinogenesis. Deregulation of miRNAa expression has been described in a variety of tumors including papillary thyroid carcinoma (PTC). The aim of the present study was to investigate the role of selected miRNAs in PTC and find associations between miRNA expression and the BRAF (V600E) mutation. Materials and methods The study group comprised a total of 62 patients with surgically treated PTC. The control group consisted of 30 patients with nodular goitre that were surgically treated in the same time period. The expression status of miR-146b, miR-181a, miR-187, miR-221 and miR-222 was determined using quantitative real-time PCR. BRAF mutation analysis was performed by PCR with reverse hybridization. Results MiR-146b, miR-181a, miR-187, miR-221 and miR-222 were up-regulated in PTC compared to normal thyroid gland tissue of the same patient. MiR-146b, miR-187, miR-221 and miR-222 were also up-regulated in PTC compared to nodular goitre. The recurrent tumors were statistically significantly associated with up-regulation of miR-221. The mutation V600E of BRAF gene was significantly associated with up-regulation of miR-146b and with down-regulation of miR-187. Conclusion Over-expression of selected miRNAs in PTC compared to normal thyroid gland tissue and nodular goitre was found. Moreover, miR-221 may serve as a prognostic marker as its over-expression was significantly associated with recurrent tumors.

Selection of non-small-cell lung cancer patients for treatment relies on the detection of expression of anaplastic lymphoma kinase (ALK) and ROS proto-oncogene 1 (ROS1) protein by immunohistochemistry (IHC). We evaluated staining performance for different IHC protocols and laboratory characteristics, and their influence on ALK and ROS1 interpretation during external quality assessment schemes between 2015 and 2018. Participants received five formalin-fixed, paraffin-embedded cases for staining by their routine protocol, whereafter at least two pathologists scored them simultaneously under a multihead microscope and awarded a graded expert staining score (ESS) from 1 to 5 points based on staining quality. European Conformity in Vitro Diagnostic kits (such as D5F3) revealed a better ALK ESS compared with laboratory-developed tests. ESS was indifferent to the applied antibody dilution or a recent protocol change. Lower ESSs were observed for higher antibody incubation times and temperatures. ESS for various ROS1 protocols were largely similar. Overall, for both markers, ESS improved over time and for repeated external quality assessment participation but was independent of laboratory setting or experience. Except for ROS1, ESS positively correlated with laboratory accreditation. IHC stains with lower ESS correlated with increased error rates in ALK and ROS1 interpretation and analysis failures. Laboratory characteristics differently affected staining quality and interpretation, and laboratories should assess both aspects, and less common protocols need improvement in staining performance. ispartof: JOURNAL OF MOLECULAR DIAGNOSTICS vol:22 issue:12 pages:1438-1452 ispartof: location:United States status: published

Diagnostics-related errors made by medical students represent a considerable issue for the field, as they very often have negative consequences that reduce learning effectiveness. This study propos...

Background/Aim: The study aimed to evaluate differences in the overall survival of HER2+ breast cancer patients treated with regard to their hormone receptors negativity or positivity. We evaluated a cohort of patients treated with trastuzumab in the Czech Republic. Patients and Methods: The present study is a retrospective analysis of patients whose data were recorded in a nationwide non-interventional, post-authorisation database BREAST. After propensity score matching of data, the cohort included 4,532 patients. Results: A significant difference in overall survival (OS) of the entire cohort was found between patients with and without hormone dependence. The OS was significantly higher in the group of patients with hormone receptor-positive (HR+) tumours in the following cohorts: patients treated with neoadjuvant therapy, patients with advanced disease, G2 tumours, stage III and IV and in patients with stage II and III of G2 tumours. Conclusion: Increased OS rates were found in several subgroups of patients with HR+/HER2+ tumours compared to those with HR-/HER2+ tumours. Better outcomes of HR+/HER2+ patients were only observed in the first four/five years of follow-up, and the differences disappeared over time

Breast cancer with high expression of human epidermal growth factor receptor (HER)-2 represents a biologically and clinically heterogeneous group of neoplastic disorders. Importantly, hormone receptor expression has an effect on biological properties and affects the selection of therapies. On the basis of molecular genetics, four principal subtypes, including luminal A, luminal B, HER2-enriched (HER-2-E), and basal-like can be distinguished. Breast tumors characterized by HER-2 positivity and simultaneous expression of hormone receptors, triple positive breast cancers (TPBC) are of increasing interest owing to the unique biological characteristics associated with complex interactions between HER-2 and hormone receptor signaling pathways. Interactions between hormone receptors and HER-2 explain the decreased efficacy of hormonal therapy in comparison with HER-2-negative patients. The expression of estrogen receptors in HER-2 positive tumors may also be associated with resistance to anti-HER-2 treatment. Multiple available therapeutic options, including hormonal therapy, anti-HER-2 agents and cytotoxic drugs explain favorable prognosis of TPBC. Escalation and de-escalation therapeutic strategies that could result in lower toxicities are being investigated as well as combinations of anti-HER-2 agents with hormonal therapy, immunotherapy, cyclin dependent kinase 4/6 and phosphatidyl inositol-3-kinase inhibitors. Distinction between subtypes of HER-2-positive breast cancer and treatment diversification may result in improved outcomes in TPBC. A response to neoadjuvant therapy may serve in the tailoring of therapy management.

Stromal tumour infiltrating lymphocytes (sTILs) are a strong prognostic marker in triple negative breast cancer (TNBC). Consistency scoring sTILs is good and was excellent when an internet-based scoring aid developed by the TIL-WG was used to score cases in a reproducibility study. This study aimed to evaluate the reproducibility of sTILs assessment using this scoring aid in cases from routine practice and to explore the potential of the tool to overcome variability in scoring. Twenty-three breast pathologists scored sTILs in digitized slides of 49 TNBC biopsies using the scoring aid. Subsequently, fields of view (FOV) from each case were selected by one pathologist and scored by the group using the tool. Inter-observer agreement was good for absolute sTILs (ICC 0.634, 95% CI 0.539–0.735, p &lt, 0.001) but was poor to fair using binary cutpoints. sTILs heterogeneity was the main contributor to disagreement. When pathologists scored the same FOV from each case, inter-observer agreement was excellent for absolute sTILs (ICC 0.798, 95% CI 0.727–0.864, p &lt, 0.001) and good for the 20% (ICC 0.657, 95% CI 0.561–0.756, p &lt, 0.001) and 40% (ICC 0.644, 95% CI 0.546–0.745, p &lt, 0.001) cutpoints. However, there was a wide range of scores for many cases. Reproducibility scoring sTILs is good when the scoring aid is used. Heterogeneity is the main contributor to variance and will need to be overcome for analytic validity to be achieved.

Background Standard treatment of oropharyngeal squamous cell carcinoma (OPSCC) is associated with high morbidity, whereas immunotherapeutic approaches using PD-1:PD-L1 checkpoint blockade only show moderate response rates in OPSCC patients. Therefore, a better stratification of patients and the development of novel therapeutic protocols are crucially needed. The importance of tumor-infiltrating B cells (TIL-Bs) in shaping antitumor immunity remains unclear; therefore, we analyzed frequency, phenotype, prognostic value and possible roles of TIL-Bs in OPSCC. Methods We utilized transcriptomic analysis of immune response-related genes in 18 OPSCC samples with respect to human papillomavirus (HPV) status. The density and localization of CD20+, CD8+ and DC-LAMP+ cells were subsequently analyzed in 72 tissue sections of primary OPSCC samples in relation to patients’ prognosis. The immunohistochemical approach was supplemented by flow cytometry-based analysis of phenotype and functionality of TIL-Bs in freshly resected primary OPSCC tissues. Results We observed significantly higher expression of B cell-related genes and higher densities of CD20+ B cells in HPV-associated OPSCC samples. Interestingly, CD20+ TIL-Bs and CD8+ T cells formed non-organized aggregates with interacting cells within the tumor tissue. The densities of both intraepithelial CD20+ B cells and B cell/CD8+ T cell interactions showed prognostic significance, which surpassed HPV positivity and CD8+ TIL density in stratification of OPSCC patients. High density of TIL-Bs was associated with an activated B cell phenotype, high CXCL9 production and high levels of tumor-infiltrating CD8+ T cells. Importantly, the abundance of direct B cell/CD8+ T cell interactions positively correlated with the frequency of HPV16-specific CD8+ T cells, whereas the absence of B cells in tumor-derived cell cultures markedly reduced CD8+ T cell survival. Conclusions Our results indicate that high abundance of TIL-Bs and high density of direct B cell/CD8+ T cell interactions can predict patients with excellent prognosis, who would benefit from less invasive treatment. We propose that in extensively infiltrated tumors, TIL-Bs might recruit CD8+ T cells via CXCL9 and due to a highly activated phenotype contribute by secondary costimulation to the maintenance of CD8+ T cells in the tumor microenvironment. Electronic supplementary material The online version of this article (10.1186/s40425-019-0726-6) contains supplementary material, which is available to authorized users.

Chronically inflamed mucosa in inflammatory bowel disease (IBD) is associated with an increased risk of cancer. Besides IBD-associated dysplasia, there are non-conventional mucosal changes that may act as potential precursors. The aim of the study was to retrospectively review samples from IBD patients focusing on detection of such lesions with evaluation of their immunohistochemical and molecular properties. Surgical specimens and/or endoscopical biopsy samples of IBD patients examined during a 10-year period were reviewed. Detected mucosal lesions were divided into three groups-group 1 (non-conventional or putative precursor lesions - PPLs) with serrated or villous hypermucinous morphology, group 2 (true serrated polyps fulfilling valid criteria), and group 3 (IBD-associated neoplasia). Lesions from all groups were analyzed with antibodies against MLH1, p53, and MGMT and by molecular testing of KRAS, NRAS, and BRAF mutation. Samples from 309 IBD patients were reviewed. A total of 88 mucosal lesions were found in 51 patients. Most common were lesions from group 1 with superficial serrated epithelial change seen in 41 samples (46.6%) and villous hypermucinous change in 6 (6.8%). Group 2 consisted of 15 true serrated polyps. Six conventional IBD-dysplasia cases and 11 carcinomas were seen in group 3. Six lesions from group 1 were associated with invasive carcinoma whereas two shared the same mutation in KRAS or BRAF. Lesions from group 1 were characterized by loss of MGMT expression in 44.6%, aberrant p53 expression, and by mutations in KRAS gene in 42.9% of cases. This study proves the existence of mucosal changes other than conventional IBD-dysplasia and extends the knowledge about their immunohistochemical and molecular properties and relation to carcinoma further supporting their potential role in IBD-related carcinogenesis.

Purpose: In multiple oncological settings, expression of the coinhibitory ligand PD-L1 by malignant cells and tumor infiltration by immune cells expressing coinhibitory receptors such as PD-1, CTLA4, LAG-3, or TIM-3 conveys prognostic or predictive information. Conversely, the impact of these features of the tumor microenvironment on disease outcome among high-grade serous carcinoma (HGSC) patients remains controversial. Experimental Design: We harnessed a retrospective cohort of 80 chemotherapy-naïve HGSC patients to investigate PD-L1 expression and tumor infiltration by CD8+ T cells, CD20+ B cells, DC-LAMP+ dendritic cells as well as by PD-1+, CTLA4+, LAG-3+, and TIM-3+ cells in relation with prognosis and function orientation of the tumor microenvironment. IHC data were complemented with transcriptomic and functional studies on a second prospective cohort of freshly resected HGSC samples. In silico analysis of publicly available RNA expression data from 308 HGSC samples was used as a confirmatory approach. Results: High levels of PD-L1 and high densities of PD-1+ cells in the microenvironment of HGSCs were strongly associated with an immune contexture characterized by a robust TH1 polarization and cytotoxic orientation that enabled superior clinical benefits. Moreover, PD-1+TIM-3+CD8+ T cells presented all features of functional exhaustion and correlated with poor disease outcome. However, although PD-L1 levels and tumor infiltration by TIM-3+ cells improved patient stratification based on the intratumoral abundance of CD8+ T cells, the amount of PD-1+ cells failed to do so. Conclusions: Our data indicate that PD-L1 and TIM-3 constitute prognostically relevant biomarkers of active and suppressed immune responses against HGSC, respectively.

The paper examines the development and testing of an electro-pneumatic device for wound healing therapy after surgery in the neck area. The device generates air pressure values in a miniaturized cuff using electronic circuitry to drive an electro-valve and air compressor. The device works in two distinct modes: continuous pressure mode and pulsating pressure mode. The pressure value setting can vary from 3 to 11 mmHg, and the pulsating pressure mode’s operating frequency range is approximately 0.1 to 0.3 Hz. Laboratory measurements were conducted to evaluate the device’s correct functioning in both continuous and pulsating pressure modes. A four-day prospective study with animals (n = 10) was also conducted to evaluate neck wound healing therapy using the electro-pneumatic device. Out of the twelve histological parameters analysed to reveal the differences between the experimental and control wounds, only one demonstrated a significant difference. Out of the ten animals treated with the device, three showed a significant difference in terms of benefit after therapy. We can therefore conclude that the device potentially improves the wound healing process in the neck area if the pre-set air pressure value does not exceed 8 mmHg.

Background Patients with inflammatory bowel disease (IBD) – ulcerative colitis (UC) and Crohn’s disease (CD) have an elevated risk of developing colorectal carcinoma (CRC). Major risk factor in IBD patients is the continuous chronic inflammation leading to development of dysplasia and carcinoma. Nevertheless, other types of non-conventional but suspicious mucosal changes serrated change/dysplasia, NOS and villous hypermucinous change, have also been reported in IBD patients. Preneoplastic potential of these lesions is still not well elucidated. Aims The aim of this study was identification of IBD-associated CRCs focusing on finding related precursor lesions in the surgical specimen or in archival biopsy samples followed by a detailed morphological, immunohistochemical and molecular evaluation. For the purpose of the study the mucosal lesions were divided into conventional IBD-associated dysplasia and non-conventional lesions that were merged under a provisory term of putative preneoplastic lesions (PPL). Methods A total of 309 consecutive IBD colectomy specimens diagnosed during a 10-year period were reviewed. Detailed morphological evaluation, immunohistochemical analysis of mismatch repair (MMR) proteins, p53 and O6-methylguanine DNA methyltransferase (MGMT) expression and molecular analysis for KRAS, NRAS and BRAF gene mutation were performed in the retrieved CRC cases as well as in the detected dysplasia and PPLs of these patients. Results We identified 11 cases of morphologically heterogenous IBD-associated CRCs, occurring in 5 males and 6 females, aged 26–79 years (mean 44 years). A total of 22 mucosal lesions were revealed in 8 CRC patients comprising conventional IBD-associated dysplasia (4 lesions), PPLs as serrated change/dysplasia NOS (11 lesions), villous hypermucinous change (5 lesions), and two true serrated lesions (one sessile serrated adenoma and one traditional serrated adenoma). More than one type of lesion was found in 6 patients. Seven CRC cases harbored mutation of KRAS/NRAS and one case of BRAF. Two patients with KRAS-mutated CRC showed the same mutation in PPL in the same specimen (one serrated change NOS and one TSA with high-grade dysplasia). Similarly, one BRAF-mutated carcinoma case presented the same mutation in serrated change/dysplasia, NOS in the same specimen. Of the CRCs, two showed deficient MMR system profile, six presented with loss of MGMT expression, and six showed aberrant p53 expression. PPLs showed deficient MGMT expression (14 cases) and aberrant p53 (10 cases) as well. Conclusion IBD-associated CRCs are very heterogeneous entities. Besides conventional IBD-related dysplasia, other types of mucosal lesions may be associated with long lasting IBD and CRC e.g. villous hypermucinous change and serrated change/dysplasia, NOS. Since these lesions share certain genetic or immunohistochemical changes with the related CRC, a suspicion is raised that these lesions may also have preneoplastic potential. Awareness of these changes is necessary to prevent their missing and under-reporting, and further studies of these lesions should be carried out.

AIMS: Results from external quality assessment revealed considerable variation in neoplastic cell percentages (NCP) estimation in samples for biomarker testing. As molecular biology tests require a minimal NCP, overestimations may lead to false negative test results. We aimed to develop recommendations to improve the NCP determination in a prototypical entity - colorectal carcinoma - that can be adapted for other cancer types. METHODS AND RESULTS: A modified Delphi study was conducted to reach consensus by 10 pathologists from 10 countries with experience in determining the NCP for colorectal adenocarcinoma. This study included two online surveys and a decision-making meeting. Consensus was defined a priori as an agreement of > 80%. All pathologists completed both surveys. Consensus was reached for 8 out of 19 and 2 out of 13 questions in the first and second surveys, respectively. Remaining issues were resolved during the meeting. Twenty-four recommendations were formulated. Major recommendations resulted as follows: only pathologists should conduct the morphological evaluation; nevertheless molecular biologists/technicians may estimate the NCP, if specific training has been performed and a pathologist is available for feedback. The estimation should be determined in the area with the highest density of viable neoplastic cells and lowest density of inflammatory cells. Other recommendations concerned: the determination protocol itself, needs for micro- and macro-dissection, reporting and interpreting, referral practices and applicability to other cancer types. CONCLUSION: We believe these recommendations may lead to more accurate NCP estimates, ensuring the correct interpretation of test results, and might help in validating digital algorithms in the future. ispartof: HISTOPATHOLOGY vol:75 issue:3 pages:312-319 ispartof: location:England status: published

Sentinel lymph node biopsy (SLNB) has become the preferred method of surgical pathological nodal staging of early breast cancer by the end of the nineties. As the most likely sites of metastasis, the SLNs allow a more precise staging, and indeed gross sectioning, step sectioning, immunohistochemistry, and molecular staging methods have been used to disclose metastatic involvement of these lymph nodes. This review summarizes the backgrounds of SLNB, trends in related surgery and pathology. It also gives an insight into European National recommendations related to SLN and divergent daily practices in European pathology departments, on the basis of replies to questionnaires from 84 pathologists from 38 European countries. The questionnaires revealed the post-neoadjuvant setting as an area where a significant minority of pathologists report less confidence in classifying residual nodal involvement into TNM categories. The review also summarizes the neoadjuvant therapy-related aspects of SLNB.

AIMS In somatotroph pituitary neuroendocrine tumours (adenomas), a pattern of cytokeratin (CK) 18 expression is used for tumour subclassification, with possible clinical implications. Rare somatotroph tumours do not express CK 18. We aimed to characterise this subset clinically and histologically. METHODS AND RESULTS Clinical and pathological data for the study were derived from a previously published data set of a cohort of 110 patients with acromegaly. Data included serum levels of insulin-like growth factor 1 (IGF1), growth hormone (GH), prolactin and thyroid-stimulating hormone (TSH), tumour diameter, tumour invasion defined by Knosp grade and immunohistochemical data concerning the expression of Ki67, p53, E-cadherin, somatostatin receptor (SSTR)1, SSTR2A, SSTR3, SSTR5 and D2 dopamine receptor. Additional immunohistochemical analysis (AE1/3, CK 8/18, vimentin, neurofilament light chain, internexin-α) was performed. CK 18 was negative in 10 of 110 (9.1%) tumours. One of these tumours was immunoreactive with CK 8/18 antibody, while the remainder expressed only internexin-α intermediate filament in patterns similar to CK 18 (perinuclear fibrous bodies). CK-negative tumours showed no significant differences with respect to biochemical, radiological or pathological features. They showed significantly higher expression of SSTR2A compared to the sparsely granulated subtype and significantly lower expression of E-cadherin compared to the non-sparsely granulated subtypes of tumours. The tumours showed divergent morphology and hormonal expression: two corresponded to densely granulated tumours and three showed co-expression of prolactin and morphology of either mammosomatotroph or somatotroph-lactotroph tumours. Four tumours showed morphology and immunoprofile compatible with plurihormonal Pit1-positive tumours. CONCLUSIONS CK-negative somatotroph tumours do not represent a distinct subtype of somatotroph tumours, and can be further subdivided according to their morphology and immunoprofile.

Tumor biology plays a crucial role in the systemic treatment, specifically in HER2-positive tumors. Distinct biological behavior of breast cancer subtypes is associated with different rates of locoregional recurrence (LRR). HER2- positive breast cancer patients treated with surgery in combination with radiation, without trastuzumab have poor outcome, including high LRR. The efficacy of radiotherapy in HER-2-positive breast cancer appears to be associated with the expression of estrogen receptors. In patients with HER-2-positive breast cancer, studies conducted before the introduction of trastuzumab indicated higher benefit of adjuvant radiation in patients with hormone receptor-positive tumors compared to patients with tumors not expressing hormone receptors. The introduction of agents targeting HER-2 has transformed the management of these patients, resulting in improved outcomes. The data of clinical studies show that the administration of trastuzumab as part of a multimodality approach (with radiation based on standard guidelines) results in improved outcomes, including lower locoregional recurrence. The risk of cardiac toxicity associated with radiation to the heart and administration of potential cardiotoxic trastuzumab is not clear. In patients treated concomitantly with regional lymph node irradiation and anti-HER-2 agents after prior anthracycline-based chemotherapy minimizing the dose to the myocardium, e.g. respiratory gating or proton beam radiotherapy, have been suggested. Biologie nádorů hraje klíčovou roli v systémové léčbě, konkrétně v NÁDORECH POZITIVNÍCH NA HER2. Odlišné biologické chování podtypů rakoviny prsu je spojeno s různými mírami locoregionální recidivy (LRR). HER2- pozitivní pacienti s karcinomem prsu léčení chirurgicky v kombinaci s ozařováním, bez trastuzumabu mají špatný výsledek, včetně vysoké LRR. Účinnost radioterapie u HER-2 pozitivního karcinomu prsu se zdá být spojena s expresí estrogenních receptorů. U pacientů s HER- 2-pozitivním karcinomem prsu studie provedené před zavedením trastuzumabu ukázaly vyšší přínos adjuvans záření u pacientů s nádory pozitivními na hormonální receptory ve srovnání s pacienty s nádory, kteří nevyjadřují hormonální receptory. Zavedení látek zaměřených na HER-2 změnilo řízení těchto pacientů, což vedlo ke zlepšení výsledků. Údaje z klinických studií ukazují, že podávání trastuzumabu v rámci multimodality přístupu (s radiačním zářením založeným na standardních pokynech) vede ke zlepšení výsledků, včetně nižší locoregionální recidivy. Riziko srdeční toxicity spojené s ozářením srdce a podáním potenciálního kardiotoxické trastuzumabu není jasné. U pacientů léčených současně s ozářením regionálních lymfatických uzlin a anti-HER- 2 přípravky po předchozí chemoterapii na bázi antracyklinu, která minimalizovala dávku myokardu, např.

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.

BackgroundThe immunological microenvironment of primary high-grade serous carcinomas (HGSCs) has a major impact on disease outcome. Conversely, little is known on the microenvironment of metastatic HGSCs and its potential influence on patient survival. Here, we explore the clinical relevance of the immunological configuration of HGSC metastases.MethodsRNA sequencing was employed on 24 paired primary tumor microenvironment (P-TME) and metastatic tumor microenvironment (M-TME) chemotherapy-naive HGSC samples. Immunohistochemistry was used to evaluate infiltration by CD8+ T cells, CD20+ B cells, DC-LAMP+ (lysosomal-associated membrane protein 3) dendritic cells (DCs), NKp46+ (natural killer) cells and CD68+CD163+ M2-like tumor-associated macrophages (TAMs), abundance of PD-1+ (programmed cell death 1), LAG-3+ (lymphocyte-activating gene 3) cells, and PD-L1 (programmed death ligand 1) expression in 80 samples. Flow cytometry was used for functional assessments on freshly resected HGSC samples.Results1468 genes were differentially expressed in the P-TME versus M-TME of HGSCs, the latter displaying signatures of extracellular matrix remodeling and immune infiltration. M-TME infiltration by immune effector cells had little impact on patient survival. Accordingly, M-TME-infiltrating T cells were functionally impaired, but not upon checkpoint activation. Conversely, cytokine signaling in favor of M2-like TAMs activity appeared to underlie inhibited immunity in the M-TME and poor disease outcome.ConclusionsImmunosuppressive M2-like TAM infiltrating metastatic sites limit clinically relevant immune responses against HGSCs.

Background Correct identification of the EGFR c.2369C>T p.(Thr790Met) variant is key to decide on a targeted therapeutic strategy for patients with acquired EGFR TKI resistance in non-small cell lung cancer. The aim of this study was to evaluate the correct detection of this variant in 12 tumor tissue specimens tested by 324 laboratories participating in External Quality Assessment (EQA) schemes. Methods Data from EQA schemes were evaluated between 2013 and 2018 from cell lines (6) and resections (6) containing the EGFR c.2369C>T p.(Thr790Met) mutation. Adequate performance was defined as the percentage of tests for which an outcome was available and correct. Additional data on the used test method were collected from the participants. Chi-squared tests on contingency tables and a biserial rank correlation were applied by IBM SPSS Statistics version 25 (IBM, Armonk, NY, USA). Results In 26 of the 1190 tests (2.2%) a technical failure occurred. For the remaining 1164 results, 1008 (86.6%) were correct, 151 (12.9%) were false-negative and 5 (0.4%) included incorrect mutations. Correct p.(Thr790Met) detection improved over time and for repeated scheme participations. In-house non-next-generation sequencing (NGS) techniques performed worse (81.1%, n = 293) compared to non-NGS commercial kits (85.2%, n = 656) and NGS (97.0%, n = 239). Over time there was an increase in the users of NGS. Resection specimens performed worse (82.6%, n = 610 tests) compared to cell line material (90.9%, n = 578 tests), except for NGS (96.3%, n = 344 for resections and 98.6%, n = 312 for cell lines). Samples with multiple mutations were more difficult compared to samples with the single p.(Thr790Met) variant. A change of the test method was shown beneficial to reduce errors but introduced additional analysis failures. Conclusions A significant number of laboratories that offer p.(Thr790Met) testing did not detect this relevant mutation compared to the other EQA participants. However, correct identification of this variant is improving over time and was higher for NGS users. Revising the methodology might be useful to resolve errors, especially for resection specimens with low frequency or multiple variants. EQA providers should include challenging resections in the scheme.

Programmed death ligand 1 (PD-L1) immunohistochemistry (IHC) is accepted as a predictive biomarker for the selection of immune checkpoint inhibitors. We evaluated the staining quality and estimation of the tumor proportion score (TPS) in non-small-cell lung cancer during two external quality assessment (EQA) schemes by the European Society of Pathology. Participants received two tissue micro-arrays with three (2017) and four (2018) cases for PD-L1 IHC and a positive tonsil control, for staining by their routine protocol. After the participants returned stained slides to the EQA coordination center, three pathologists assessed each slide and awarded an expert staining score from 1 to 5 points based on the staining concordance. Expert scores significantly (p < 0.01) improved between EQA schemes from 3.8 (n = 67) to 4.3 (n = 74) on 5 points. Participants used 32 different protocols: the majority applied the 22C3 (56.7%) (Dako), SP263 (19.1%) (Ventana), and E1L3N (Cell Signaling) (7.1%) clones. Staining artifacts consisted mainly of very weak or weak antigen demonstration (63.0%) or excessive background staining (19.8%). Participants using CE-IVD kits reached a higher score compared with those using laboratory-developed tests (LDTs) (p < 0.05), mainly attributed to a better concordance of SP263. The TPS was under- and over-estimated in 20/423 (4.7%) and 24/423 (5.7%) cases, respectively, correlating to a lower expert score. Additional research is needed on the concordance of less common protocols, and on reasons for lower LDT concordance. Laboratories should carefully validate all test methods and regularly verify their performance. EQA participation should focus on both staining concordance and interpretation of PD-L1 IHC.

Additional file 2 Supplemental Table S2: Overview of applied test methods and reasons reported for the technical failures observed in the EQA schemes.

Additional file 1 Supplemental Table S1: Preparation of the in-house cell lines distributed in the 2013 and 2014 ESP EQA schemes

Additional file 4 Supplemental Table S4: Performance over time for the 11 most frequently used c.2369C > T p.(Thr790Met) detection methods.

Additional file 6 Supplemental Figure S1: Ranked biserial correlation between variant allele frequencies and incorrect outcomes (panel A) or technical failures (panel B).

Additional file 3 Supplemental Table S3: Performance of different technique types related to time, number of EQA participations, sample type and variant allele frequency.

Additional file 5 Supplemental Table S5: Use and performance of the different methods for c.2369C > T p.(Thr790Met) detection between 2013 and 2018.

A questionnaire on biomarker testing previously used in central European countries was extended and distributed in Western and Central European countries to the pathologists participating at the Pulmonary Pathology Society meeting 26-28 June 2019 in Dubrovnik, Croatia. Each country was represented by one responder. For recent biomarkers the availability and reimbursement of diagnoses of molecular alterations in non-small cell lung carcinoma varies widely between different, also western European, countries. Reimbursement of such assessments varies widely between unavailability and payments by the health care system or even pharmaceutical companies. The support for testing from alternative sources, such as the pharmaceutical industry, is no doubt partly compensating for the lack of public health system support, but it is not a viable or long-term solution. Ideally, a structured access to testing and reimbursement should be the aim in order to provide patients with appropriate therapeutic options. As biomarker enabled therapies deliver a 50% better probability of outcome success, improved and unbiased reimbursement remains a major challenge for the future. ispartof: TRANSLATIONAL LUNG CANCER RESEARCH vol:9 issue:3 pages:887-897 ispartof: location:China status: published

This article analyzes the availability of different diagnostic procedures of non-small cell lung cancer (NSCLC) and the reimbursement landscape of drugs for NSCLC in countries of central and southeastern Europe (CEE). A survey was conducted by the Central European Cooperative Oncology Group. Results of the survey show that both availability and reimbursement of diagnoses of molecular alterations in NSCLC, the detection of which is essential for therapeutic decisions, varies widely between countries of CEE. Not only is “reflex” testing often substituted by analyses performed only “on demand,” but reimbursement of such assessments varies widely between unavailability and payments by the health care system or even pharmaceutical companies. It was concluded that a structured access to testing and reimbursement should be the aim in order to provide patients with appropriate therapeutic options. Implications for Practice This article provides an overview of the limitations in lung cancer treatment in countries of central and southeastern Europe, as well as the reimbursement status of various lung cancer treatment regimens in these countries, which directly impacts treatment options.