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Aims: To investigate Epstein‐Barr virus (EBV) latency types in 19 cases of EBV‐positive nodular lymphocyte‐predominant Hodgkin lymphoma (NLPHL), as such information is currently incomplete. Methods and results: Immunohistochemistry (IHC) for CD20, CD79a, PAX5, OCT2, CD30, CD15, CD3 and programmed cell death protein 1 was performed. For EBV detection, in‐situ hybridisation (ISH) for EBV‐encoded RNA (EBER) was employed combined with IHC for EBV‐encoded latent membrane protein (LMP)‐1, EBV‐encoded nuclear antigen (EBNA)‐2, and EBV‐encoded BZLF1. In 95% of the cases, neoplastic cells with features of Hodgkin and Reed–Sternberg (HRS) cells were present, mostly showing expression of CD30. In all cases, the B‐cell phenotype was largely intact, and delineation from classic Hodgkin lymphoma (CHL) was further supported by myocyte enhancer factor 2B (MEF2B) detection. All tumour cells were EBER‐positive except in two cases. EBV latency type II was most frequent (89%) and type I was rare. Cases with latency type I were CD30‐negative. Five cases contained some BZLF1‐positive and/or EBNA‐2‐positive bystander lymphocytes. Conclusions: As HRS morphology of neoplastic cells and CD30 expression are frequent features of EBV‐positive NLPHL, preservation of the B‐cell transcription programme, MEF2B expression combined with NLPHL‐typical architecture and background composition facilitate distinction from CHL. EBER ISH is the method of choice to identify these cases. The majority present with EBV latency type II, and only rare cases present with latency type I, which can be associated with missing CD30 expression. The presence of occasional bystander lymphocytes expressing BZLF1 and/or EBNA‐2 and the partial EBV infection of neoplastic cells in some cases could indicate that EBV is either not primarily involved or is only a transient driver in the pathogenesis of EBV‐positive NLPHL. [ABSTRACT FROM AUTHOR]

In recent years, the treatment of non-small-cell lung cancer (NSCLC) has been fundamentally changed by immunotherapy where the immune system is reactivated using anti-programmed cell death protein 1/programmed death ligand 1 (PD1/PD-L1) checkpoint inhibition. With this, the immunohistological detection of PD-L1 has become one of the most important predictive biomarkers, leading pathologists to play a central role in the immuno-oncological therapy decisions. This has brought them the challenge of requiring the knowledge of relevant checkpoint inhibitors (CI), different PD-L1 scores and cut-offs as well as the choice of the right tissues and controls. Their involvement is also required in the careful validation of both clinical trial assays (CTAs) and laboratory developed tests (LDTs), in addition to the internal and external quality assessment and the interpretation and scoring of the staining based on specialist training. After the training of tumor proportion score (TPS) scoring in NSCLC, pathologists show a high level of concordance, with some variation between different cut-offs. Since not all patients benefit from immunotherapy, further research is needed to validate new predictive markers and optimize existing ones. In this context, these studies focus on a combination of PD-L1 and molecular signatures. [ABSTRACT FROM AUTHOR]

Purpose: The cell surface glycoprotein Mesothelin is overexpressed in ovarian, fallopian tube, endometrial, cervical and primary peritoneal cancer and, therefore, might become a particular interesting tumor target in gynecologic oncology. However, even in malignant tumors of the same entity the level of Mesothelin expression varies between individuals, hence it can be expected that the response to Mesothelin-targeting therapies will be variable as well. In this study we explored the therapeutic potency of a novel anti-Mesothelin antibody–drug conjugate (Anetumab ravtansine) as a function of Mesothelin expression in the targeted tumor cells. Methods: Anti-tumor activity studies were performed in human uterine xenograft tumor models that express Mesothelin at high, moderate or low levels. The antibody–drug conjugate (ADC) was applied in varying doses ranging from 2 to 15 mg/kg at variable intervals in tumor bearing SCID/beige mice and the impact on tumor growth was monitored. Results: The therapeutic response to the anti-Mesothelin ADC correlated closely with the level of Mesothelin expression in tumor cells. Within the applied dose levels complete tumor regression was achieved only in tumors which expressed Mesothelin at particularly high levels (Hela cell tumors). The application of high anti-Mesothelin ADC doses less frequently was more efficious than giving lower doses at a higher frequency. Conclusion: The studies confirm the great therapeutic potential of Anetumab ravtansine. However, a favorable treatment outcome requires strong Mesothelin expression in tumor cells. Future clinical trials may benefit from a more rigorous selection of appropriate patients based on the level of Mesothelin expression in their tumor tissue. If, in addition, it is possible to better control side effects by introducing protective measures and by doing so to increase the maximum tolerated dose, Anetumab ravtansine has the potency to become a valuable therapeutic tool, especially in the field of gynecological oncology. [ABSTRACT FROM AUTHOR]

Immunotherapy is effective in head and neck squamous cell carcinoma (HNSCC), but only a minority of patients responds to immune checkpoint blockade (ICB). To contribute to a better understanding of the underlying immune biology, we combined histomorphological evaluation and molecular analysis of the HNSCC immune microenvironment in the TCGA cohort. Analyzing digital HE-stained slides, a method for classification of tumor infiltrating lymphocytes (TILs) in the intra-epithelial compartment (ieTILs, present vs. absent) and the stromal compartment (strTILs, high vs. low) was established. We also analyzed the abundance of eight immune cell populations (estimated from RNAseq data) and PD-L1 mRNA expression. Status of ieTILs and status of strTILs were concordant for 61%, but discordant for 39% of tumors. In univariate survival analysis, ieTILs were a positive prognostic marker for DFS in the study cohort (HR = 0.66, p = 0.015) and in the HPV− subcohort (HR = 0.68, p = 0.04), but not in the HPV + subcohort. T cells were a positive prognostic marker for DFS in the study cohort (HR = 0.80, p = 0.03) and in the HPV + subcohort (HR = 0.20, p = 0.001), but not in the HPV− subcohort. In univariate survival analysis, PD-L1 mRNA expression was neither associated with DFS nor with OS. However, in bivariate and multivariate analyses including both PD-L1 mRNA levels and T cells, PD-L1 was a negative prognostic marker of DFS and OS, while T cells remained a positive prognostic marker. In conclusion, ieTILs and strTILs were non-redundant biomarkers in HNSCC and should be evaluated separately. The identified prognostic markers should be evaluated for predictivity in ICB-treated patients. [ABSTRACT FROM AUTHOR]

Purpose: The cell surface glycoprotein Mesothelin is overexpressed in several tumor entities and novel immune-based therapies are currently under the early clinical evaluation for the treatment of malignant pleura mesothelioma, ovarian cancer, and pancreatic cancer. Cervical cancer has not been recognized as a suitable target for Mesothelin-directed immune therapies so far.Methods: To exploit a possible role of Mesothelin in cervical cancer treatment, we analysed Mesothelin expression in 79 cervical carcinomas and aligned expressions patterns with tumor growth parameters. A novel anti-Mesothelin drug conjugate (Anetumab Ravtansine) was applied for dose-efficiency studies in a Mesothelin positive tumor model for cervical cancer in Scid mice.Results: In more than three-quarters (77%) of cervical adenocarcinomas, Mesothelin was expressed to high levels. Among squamous cell carcinomas of the cervix uteri expression levels were lower and expression patterns were less intense, but still ranged between 50-60% (57%). A significant correlation between Mesothelin expression levels and tumor grade, metastatic behaviour, and lymph- or hemangiosis was not found. The novel anti-Mesothelin-drug conjugate (Anetumab Ravtansine) showed a substantial dose-dependent therapeutic efficiency in a xenotransplant model for cervical cancer in SCID mice (hela cell tumors). Applying the ADC at a dose of 10 mg/kg twice weekly induced complete tumor regression in 88% of animals within 6 weeks.Conclusions: Mesothelin should be taken into account as a target in cervical cancer therapy and histological determination of Mesothelin expression should be considered in routine diagnostics of cervical carcinomas. [ABSTRACT FROM AUTHOR]

Anti-EGFR-targeted therapy is used to treat metastatic colorectal cancers with RAS wild-type. However, resistance to targeted therapy is often observed and can be primary or acquired. One reason for primary resistance is the presence of mutations that are undetected due to genetic heterogeneity, which can be expressed by differences present in primary tumor and distant metastasis or recurrence or by an intratumoral heterogeneity (presence of different subclones in the investigated tumor sample). The aim of our study was to investigate if morphological heterogeneity can be an indicator of intratumoral heterogeneity. We analysed 13 samples with homogeneous and six samples with heterogeneous morphology with NGS. We were able to demonstrate that intratumoral genetic heterogeneity is present in all studied tumor samples, independent of homogeneous or heterogeneous morphology. Moreover, one sample of our cohort with morphological and genetic heterogeneity had a genetic wild-type profile in one tumor component. Therefore, we recommend to include each morphologically identifiable tumor component in the mutational analysis to not overlook resistance-inducing or potentially targetable mutations. [ABSTRACT FROM AUTHOR]

The receptor for hyaluronan-mediated motility CD168 is associated with the processes of oncogenesis and metastasis. The objective of the present study was to determine the possible association between the expression and distribution of CD168 and the tumor stage of head-and-neck squamous cell carcinoma (SCC). Formalin-fixed and paraffinembedded tumor samples obtained from 100 patients during primary resection of SCC from the oral cavity, oropharynx, hypopharynx or larynx were included in the present study. The patients were divided into two risk groups: Low risk, representing the early stage of completely resected SCCs with good-to-moderate differentiation, and the high-risk group, representing the advanced stage SCCs with positive resection margins, vascular invasion or locoregional metastasis. All specimens were stained with a monoclonal antibody against CD168. Percentage and staining intensity of CD168-positive cells were scored, and their spatial distribution within the tumor nests was noted. The results obtained were correlated with the tumor stage. The quantification of CD168 expression revealed significant differences between the two risk groups (t-test, P=0.002), with higher scores in tumors resected from the highrisk SSC group compared with those from the low-risk group. In addition, in the high-risk group, the CD168-positive cells were present predominantly in the periphery (70.4%) of tumor nests, whereas in the low-risk group, only 56.6% were located there; however, this trend did not reach the level of statistical significance. Taken together, the results from the present study suggested that CD168 expression patterns could potentially be used as a predictor of tumor aggressiveness, and therefore they may be a prognostic factor in head-and-neck SCC. [ABSTRACT FROM AUTHOR]

We investigated 41 diffuse large B-cell lymphomas (DLBCL) diagnosed at one center harboring ≥50% of latently Epstein–Barr virus (EBV)-infected neoplastic cells occurring in 34 patients aged ≥50 years and in 7 patients younger than 50 years in the absence of any known immunodeficiency for the expression patterns of EBV latent and immediate-early proteins, for the differentiation stage of the neoplastic cells, the presence of cytogenetic alterations and a possible co-infection with the human herpes virus (HHV)-8. Here, we show that EBV-positive DLBCLs rarely arise from naïve and more frequently from post-germinal center B-cells that often contain crippling immunoglobulin gene mutations. Most of the lymphomas did not exhibit breaks in theBCL2,BCL6, andMYCgenes and none of the cases investigated contained HHV-8 sequences. Patients aged <50 years performed better than older ones while in patients aged ≥50 years only the cellular composition had an impact on overall survival. [ABSTRACT FROM AUTHOR]

Background: Although sarcoidosis and celiac disease are both chronic immunologic disorders involving multiple organ systems, reports about association of diseases in individual patients are sparse. While sarcoidosis is a chronic granulomatous disease presumably reflecting an exaggerated response to an unknown antigen, celiac disease is a T cell-driven disease triggered by ingestion of gluten, a protein composite found in wheat and related grains. Case presentation: We present three cases with a longstanding history of sarcoidosis that have been additionally diagnosed with celiac-like enteropathy. In two cases, celiac disease was established applying celiac-specific serology and duodenal histology, while one case was revealed as an AIE-75-positive autoimmune enteropathy. The HLA-DR3/DQ2 haplotype was confirmed in both celiac patients, hence confirming previous data of linkage disequilibrium as a cause for disease association. Remarkably, one celiac patient presented with granulomatous nodulae in the ileum, thus reflecting an intestinal sarcoid manifestation. In contrast the patient with an autoimmune enteropathy, was HLA-DQ9/DQ6-positive, also arguing against CD. Conclusions: Associations of sarcoidosis and celiac disease are rare but do occur. Determining the HLA status in patients with complex autoimmune associations might help classifying involved disease entities. [ABSTRACT FROM AUTHOR]

B-cell immune dysfunction contributes to the risk of severe infections after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Delayed B-cell regeneration is found in patients with systemic graft-versus-host disease (GVHD) and is often accompanied by bone marrow (BM) suppression. Little is known about human BM GVHD. We analyzed the reconstitution kinetics of B-cell subsets in adult leukemic patients within 6 months after allo-HSCT. B-cell deficiency already existed before transplant and was aggravated after transplant. Onset of B-cell reconstitution characterized by transitional B-cell recovery occurred either early (months 2-3) or late (from month 6 on) and correlated highly positively with reverse transcription-polymerase chain reaction quantified numbers of K-deleting recombination excision circles (KRECs). Delayed recovery was associated with systemic acute GVHD and full-intensity conditioning therapy. Histological analysis of BM trephines revealed increased T-cell infiltration in late recovering patients, which was associated with reduced numbers of osteoblasts. Functionally, late recovering patients displayed less pneumococcal polysaccharide-specific immunoglobin M-producing B cells on ex vivo B-cell activation than early recovering patients. Our results provide evidence for acute BM GVHD in allo-HSCT patients with infiltrating donor T cells and osteoblast destruction. This is associated with delayed B-cell reconstitution and impaired antibody response. Herein, KREC appears suitable to monitor BM B-cell output after transplant. [ABSTRACT FROM AUTHOR]

Angioimmunoblastic T-cell lymphoma (AITL) exhibits a multifaceted clinical picture and distinct architectural patterns that correlate with disease progression and the number of neoplastic cells. In this study we investigated the expression of the transcription factor T-bet and correlated it with the architectural patterns in 29 cases of AITL. Double immunolabelings for T-bet, CD20, CD3 or PD1 revealed the following patterns: predominant T-bet expression by neoplastic T-cells (A), by aggregates of small B-cells (B) or by B-immunoblasts (C). The majority of cases of AITL pattern II showed a T-bet expression pattern B (6/8 cases), while the majority of those with pattern III exhibited the T-bet pattern A (11/21 cases). We propose that T-bet expression by B-cells represents a T-cell independent immune response trying to cope with opportunistic infections, while T-bet expression by neoplastic T-cells is linked to the introduction of a Th17 response responsible for the immunologic derangements characteristic of AITL. [ABSTRACT FROM AUTHOR]

Abstract: Infection with Toxoplasma gondii (TG) presents in some individuals as a self-limited disease with a predominant lymphadenopathy characterized by prominent B-cell activation. As this is in contrast to the in vitro based concept of a Th1-immune response against TG, we investigated native lymphoid tissue and peripheral blood of a patient with serologic evidence of toxoplasmosis to verify which cells show Th1-response features. High-level expression of T-bet in monocytoid B-cells, in germinal center B-cells, and in a lesser amount in T cells could be demonstrated by immunohistochemistry. In vitro stimulation of lymph node cells with either TG, staphylococcus enterotoxin B, or phorbol 12-myristate 13-acetate/ionomycin revealed an interferon-γ expression in T-bet+ B cells only in the patient and not in controls. Similar results were found for T-bet+ T cells which were also present in controls. CD4+ peripheral blood cells stimulated with TG antigens showed a TG-specific but attenuated Th1-reactivity in the patient associated with a reduced expression of IL-2 when compared with controls. We conclude that the pathogenesis and course of toxoplasmic lymphadenopathy is based on a Th1-cell defect, which becomes compensated by the B cells mounting a Th1-like immune response. [Copyright &y& Elsevier]

Hairy cell leukemia (HCL) is an uncommon B-cell malignancy with unknown pathogenesis. In an earlier study, we demonstrated that HCL cells highly express the transcription factor T-box-expressed-in-T-cells (T-bet). T-bet is the master regulator of the T-helper (Th)1 cell response regulating interferon gamma (IFN-γ) production and also plays a central role in the T-cell independent Th1-like B-cell response. Here, we demonstrate by fluorescence activated cell sorting (FACS) analysis that neoplastic cells from the peripheral blood of five patients with HCL showed an enhanced expression of IFN-γ after stimulation. Additionally, a comparison with 55 healthy individuals revealed a significant elevation of IFN-γ in the sera of patients with HCL. Based on our recent findings that a non-neoplastic B-cell subset, the monocytoid B-cells, are T-bet positive and produce IFN-γ, we propose that monocytoid and hairy B-cells have a similar function and that the T-bet-IFN-γ axis is involved in the pathogenesis of HCL. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in which the authors talk about gene expression of Epstein-Barr virus (EBV) encoded proteins in angioimmunoblastic T-cell lymphoma and propose that T-cell lymphoma patients should be screened for serological signs of lytic reactivation and EBV viral load.

Background: Intra-tumoral CD8 + T-cell infiltration in squamous cell carcinoma of the head and neck (HNSCC) has previously been linked to the efficacy of cisplatin-based chemoradiation (CDDP-CRTX) and immune checkpoint inhibitor (ICI) monotherapy. Further detailed characterization of the tumor immune-micromilieu and its influence on outcome may guide the development of CRTX-ICI combinations. Methods: Comprehensive immune transcriptome analysis was applied to a training set of tumor specimens from oropharyngeal squamous cell carcinoma (OPSCC) patients treated with CDDP-CRTX in the ARO-0401 phase III study (n = 33). A composite immune signature risk score (ISRS) for survival prediction was developed, and subsequently validated in two independent OPSCC cohorts treated with either CDDP-CRTX (n = 36) or mitomycin-based CRTX (MMC-CRTX, n = 31). Further validation of the ISRS was performed in the OPSCC subset (n = 79) of the TCGA HNSCC cohort. Potential interference between immune signatures and HPV status was evaluated in multivariate Cox regression models. Results: Significant differences according to the 3-y OS status in the abundance of tumor-infiltrating T- and B-cells, and the expression levels of 51 immune-related genes were observed. A risk score based on 13 differentially expressed genes involved in cytokine signaling, T-cell effector functions and the TNFR pathway was established as robust predictive factor of OS. Its predictive power was superior to the 6-gene interferon-gamma signature of ICI efficacy and independent of the HPV status. Conclusions: This study further elucidates the complex interaction of the tumor immune microenvironment with the efficacy of CDDP-CRTX in OPSCC. The results suggest immune markers for selection of patients treated with CRTX-ICI combinations. [ABSTRACT FROM AUTHOR]

Stein H, Jöhrens K, Anagnostopoulos I. Non-mediastinal grey zone lymphomas and report from the workshop.Eur J Haematol 2005: 75 (Suppl. 66): 42–44.© Blackwell Munksgaard 2005.Grey zone lymphomas represent borderline lesions between classical Hodgkin lymphoma (cHL) and other morphologically and immunophenotypically related diseases and entities like nodular lymphocyte predominant HL, T-cell rich B-cell lymphoma, ALK-negative anaplastic large cell lymphoma (ALCL), diffuse large B cell lymphoma (DLBCL) anaplastic variant and primary mediastinal LBCL. The sharp definition of morphological, immunophenotypical and molecular features of the‘‘text-book cases’’ of each disease and the comparison with grey zone cases has reduced most of the latter cases. Two reports in this workshop dealt with the problematic non-mediastinal grey zone lymphomas, one with a cHL of T cell-type presenting in the skin as a ALK-negative ALCL and the other with the grey zone between cHL of B-cell type and ALK-negative ALCL. [ABSTRACT FROM AUTHOR]

• Analysis of recurrent/metastatic head and neck cancer. • Feasibility of a 231-gene NanoString panel in determining the molecular subtypes. • Tumor budding prognostically significant in recurrent/metastatic state. We aimed to evaluate the applicability of a customized NanoString panel for molecular subtyping of recurrent or metastatic head and neck squamous cell carcinoma (R/M−HNSCC). Additionally, histological analyses were conducted, correlated with the molecular subtypes and tested for their prognostic value. We conducted molecular subtyping of R/M−HNSCC according to the molecular subtypes defined by Keck et al. For molecular analyses a 231 gene customized NanoString panel (the most accurately subtype defining genes, based on previous analyses) was applied to tumor samples from R/M−HNSCC patients that were treated in the CeFCiD trial (AIO/IAG-KHT trial 1108). A total of 130 samples from 95 patients were available for sequencing, of which 80 samples from 67 patients passed quality controls and were included in histological analyses. H&E stained slides were evaluated regarding distinct morphological patterns (e.g. tumor budding, nuclear size, stroma content). Determination of molecular subtypes led to classification of tumor samples as basal (n = 46, 45 %), inflamed/mesenchymal (n = 31, 30 %) and classical (n = 26, 25 %). Expression levels of Amphiregulin (AREG) were significantly higher for the basal and classical subtypes compared to the mesenchymal subtype. While molecular subtypes did not have an impact on survival, high levels of tumor budding were associated with poor outcomes. No correlation was found between molecular subtypes and histological characteristics. Utilizing the 231-gene NanoString panel we were able to determine the molecular subtype of R/M−HNSCC samples by the use of FFPE material. The value to stratify for different treatment options remains to be explored in the future. The prognostic value of tumor budding was underscored in this clinically well annotated cohort. [ABSTRACT FROM AUTHOR]

Purpose: In recent years the tumor microenvironment and its interaction with the tumor has emerged into research focus with increased attention to the composition of Tumor-infiltrating lymphocytes. We wanted to quantify the composition of Regulatory T cells (Tregs) and T helper 17 cells (Th17 cells) and their prognostic impact in high-grade serous tubo-ovarian carcinoma. Methods: Tregs and Th17 cells were determined by immunohistochemical analysis of CD25 FoxP3 and RORγt, respectively on tissue microarrays of a cohort of 222 patients with reviewed histology and available clinical data. Expression was analyzed with Qupath for quantification and integration with clinical data enabled calculation of prognostic impact. For validation FOXP3 and RORC mRNA expression levels from 502 patients with HGSC in publicly available datasets were evaluated. Results: An average percentage of 0.93 Tregs and of 0.06 Th17 cells was detected per cells in overall tissue. Optimal cut-offs were determined and higher Tregs were associated with a better overall survival in stroma (p = 0.006), tumor area (p = 0.0012) and overall tissue (p = 0.02). After accounting for well-known prognostic factors age at diagnosis, residual tumor and FIGO stage, this association remained significant for stromal Tregs with overall survival (p = 0.02). Survival analysis for Th17 cells revealed no significant association with survival rates. Moreover, lower Th17/Treg ratios had a positive impact on patient overall survival (p = 0.025 tumor, p = 0.049 stroma and p = 0.016 overall tissue). Conclusion: Our results outline a positive prognostic effect for higher Tregs but not for Th17 in high grade serous tubo-ovarian carcinoma. [ABSTRACT FROM AUTHOR]

Background: Although sarcoidosis and celiac disease are both chronic immunologic disorders involving multiple organ systems, reports about association of diseases in individual patients are sparse. While sarcoidosis is a chronic granulomatous disease presumably reflecting an exaggerated response to an unknown antigen, celiac disease is a T cell-driven disease triggered by ingestion of gluten, a protein composite found in wheat and related grains. Case Presentation: We present three cases with a longstanding history of sarcoidosis that have been additionally diagnosed with celiac-like enteropathy. In two cases, celiac disease was established applying celiac-specific serology and duodenal histology, while one case was revealed as an AIE-75-positive autoimmune enteropathy. The HLA-DR3/DQ2 haplotype was confirmed in both celiac patients, hence confirming previous data of linkage disequilibrium as a cause for disease association. Remarkably, one celiac patient presented with granulomatous nodulae in the ileum, thus reflecting an intestinal sarcoid manifestation. In contrast the patient with an autoimmune enteropathy, was HLA-DQ9/DQ6-positive, also arguing against CD. Conclusions: Associations of sarcoidosis and celiac disease are rare but do occur. Determining the HLA status in patients with complex autoimmune associations might help classifying involved disease entities. [ABSTRACT FROM AUTHOR]

Objectives To verify retrospectively the margin status and analyse the location and characteristics of positive surgical margins ( PSMs) in patients undergoing radical prostatectomy ( RP), by a central pathology review, based on the consensus conference 2009 updated margin criteria from the International Society of Urological Pathology ( ISUP). Patients and Methods The detailed PSM characteristics of 441 patients who underwent laparoscopic RP ( LRP) between 1999 and 2007 were centrally reviewed with regard to location, number, Gleason score at the PSM and tumour width. Predictors of PSMs and the impact of several PSM characteristics on clinical outcomes were examined. Patient characteristics were compared using the chi-squared test. Differences in recurrence-free survival ( RFS) rates were analysed using the log-rank test and presented as Kaplan-Meier survival curves. Univariable and multivariable Cox regression analysis for the prediction of RFS was performed. Results Central pathology review using the updated PSM definition according to ISUP 2009, resulted in reclassification of a substantial number of patients with PSMs ( n = 113, 26.6%) as R0. Several PSM characteristics with a higher risk of biochemical recurrence ( BCR) were identified as the strongest independent predictors of RFS: pathological stage; Gleason score; and the presence of multiple PSMs (hazard ratio [ HR] 1.78; 95% confidence interval [ CI] 1.08-2.96; P = 0.025). Further analysis replacing the location of PSM by the width categories of PSM showed that a PSM >3 mm was an independent predictor of RFS ( HR 1.72; 95% CI 1.08-2.72; P = 0.022). Conclusions The impact of PSMs after LRP for prostate cancer remains unclear. PSMs in the present cohort of patients undergoing LRP had different characteristics and conferred different risks of BCR. A better understanding of PSM characteristics and a careful standardized pathological evaluation is needed to adequately counsel patients with respect to prognosis and adjuvant therapy after LRP. [ABSTRACT FROM AUTHOR]

Simple Summary: Liposarcomas (LPS) are malignancies arising from adipose tissue. Based on the histological appearance, five subtypes are distinguished: well-differentiated LPS, dedifferentiated LPS (DDLPS), myxoid LPS (MLPS), pleomorphic LPS, and myxoid pleomorphic LPS. Immune cells can infiltrate the tumor microenvironment (TME) of LPS and can either promote an efficient antitumor immune response or mediate immunosuppression paving the way for immune evasion of the tumor. The LPS subtypes display different TME characteristics and vary in regard to immune cell infiltration, ranging from the generally lowly infiltrated MLPS to the highly infiltrated DDLPS where immunological determinants predict response to novel antibody-based immunotherapy. Thus, immune cells in the TME can significantly affect response to therapy, disease progression, and patient survival. This review aims to decipher the immune contexture of LPS as well as its clinical association and highlights differences between the LPS subtypes that may have implications for the design of novel treatment strategies. Liposarcomas (LPS) are the most frequent malignancies in the soft tissue sarcoma family and consist of five distinctive histological subtypes, termed well-differentiated LPS, dedifferentiated LPS (DDLPS), myxoid LPS (MLPS), pleomorphic LPS, and myxoid pleomorphic LPS. They display variations in genetic alterations, clinical behavior, and prognostic course. While accumulating evidence implicates a crucial role of the tumor immune contexture in shaping the response to anticancer treatments, the immunological landscape of LPS is highly variable across different subtypes. Thus, DDLPS is characterized by a higher abundance of infiltrating T cells, yet the opposite was reported for MLPS. Interestingly, a recent study indicated that the frequency of pre-existing T cells in soft tissue sarcomas has a predictive value for immune checkpoint inhibitor (CPI) therapy. Additionally, B cells and tertiary lymphoid structures were identified as potential biomarkers for the clinical outcome of LPS patients and response to CPI therapy. Furthermore, it was demonstrated that macrophages, predominantly of M2 polarization, are frequently associated with poor prognosis. An improved understanding of the complex LPS immune contexture enables the design and refinement of novel immunotherapeutic approaches. Here, we summarize recent studies focusing on the clinicopathological, genetic, and immunological determinants of LPS. [ABSTRACT FROM AUTHOR]

We tested the hypothesis that gene expressions from biopsies of locally advanced head and neck squamous cell carcinoma (HNSCC) patients can supplement dose-volume parameters to predict dysphagia and xerostomia following primary radiochemotherapy (RCTx). A panel of 178 genes previously related to radiochemosensitivity of HNSCC was considered for nanoString analysis based on tumour biopsies of 90 patients with locally advanced HNSCC treated by primary RCTx. Dose-volume parameters were extracted from the parotid, submandibular glands, oral cavity, larynx, buccal mucosa, and lips. Normal tissue complication probability (NTCP) models were developed for acute, late, and for the improvement of xerostomia grade ≥2 and dysphagia grade ≥3 using a cross-validation-based least absolute shrinkage and selection operator (LASSO) approach combined with stepwise logistic regression for feature selection. The final signatures were included in a logistic regression model with optimism correction. Performance was assessed by the area under the receiver operating characteristic curve (AUC). NTCP models for acute and late xerostomia and the improvement of dysphagia resulted in optimism-corrected AUC values of 0.84, 0.76, and 0.70, respectively. The minimum dose to the contralateral parotid was selected for both acute and late xerostomia and the minimum dose to the larynx was selected for dysphagia improvement. For the xerostomia endpoints, the following gene expressions were selected: RPA2 (cellular response to DNA damage), TCF3 (salivary gland cells development), GBE1 (glycogen storage and regulation), and MAPK3 (regulation of cellular processes). No gene expression features were selected for the prediction of dysphagia. This hypothesis-generating study showed the potential of improving NTCP models using gene expression data for HNSCC patients. The presented models require independent validation before potential application in clinical practice. [ABSTRACT FROM AUTHOR]

Background: Extramedullary manifestations (EM) are rare in acute myeloid leukemia (AML) and their impact on clinical outcomes is controversially discussed. Methods: We retrospectively analyzed a large multi-center cohort of 1583 newly diagnosed AML patients, of whom 225 (14.21%) had EM. Results: AML patients with EM presented with significantly higher counts of white blood cells (p < 0.0001), peripheral blood blasts (p < 0.0001), bone marrow blasts (p = 0.019), and LDH (p < 0.0001). Regarding molecular genetics, EM AML was associated with mutations of NPM1 (OR: 1.66, p < 0.001), FLT3-ITD (OR: 1.72, p < 0.001) and PTPN11 (OR: 2.46, p < 0.001). With regard to clinical outcomes, EM AML patients were less likely to achieve complete remissions (OR: 0.62, p = 0.004), and had a higher early death rate (OR: 2.23, p = 0.003). Multivariable analysis revealed EM as an independent risk factor for reduced overall survival (hazard ratio [HR]: 1.43, p < 0.001), however, for patients who received allogeneic hematopoietic cell transplantation (HCT) survival did not differ. For patients bearing EM AML, multivariable analysis unveiled mutated TP53 and IKZF1 as independent risk factors for reduced event-free (HR: 4.45, p < 0.001, and HR: 2.05, p = 0.044, respectively) and overall survival (HR: 2.48, p = 0.026, and HR: 2.63, p = 0.008, respectively). Conclusion: Our analysis represents one of the largest cohorts of EM AML and establishes key molecular markers linked to EM, providing new evidence that EM is associated with adverse risk in AML and may warrant allogeneic HCT in eligible patients with EM. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the use of a novel battery-powered drill, enabling specimen requirement while drilling, in radiofrequency ablation of osteoid osteoma as an alternative to conventional orthopedic drills. Materials and methods: Between 2009 and 2013, altogether 33 patients underwent CT-guided radiofrequency ablation of an osteoid osteoma at our institution. To access the nidus of the clinically and radiologically suspected osteoid osteoma, a channel was drilled using the OnControl Bone Marrow Biopsy System (OBM, Arrow OnControl, Teleflex, Shavano Park, TX, USA) and a biopsy was taken. Procedure time (i.e., drilling including local anesthesia), amount of scans (i.e., single-shot fluoroscopy), radiation exposure, and the results of biopsy were investigated and compared retrospectively to a classical approach using either a manual bone biopsy system or a conventional orthopedic drill ( n = 10) after ethical review board approval. Results: Drilling the tract into the nidus was performed without problems in 22 of the 23 OBM cases. Median procedure time was 7 min compared to 13 min using the classical approach ( p < 0.001). Median amount CT scans, performed to control correct positioning of the drill was comparable with 26 compared to 24.5 ( p = NS) scans. Histologically, the diagnosis of osteoid osteomas could be determined in all 17 cases where a biopsy was taken. Radiofrequency ablation could be performed without problems in any case. Conclusions: The use of the battery-powered drill was feasible and facilitated the access to the osteoid osteoma's nidus offering the possibility to extract a specimen in the same step. [ABSTRACT FROM AUTHOR]

Despite the success of elastography in grading hepatic fibrosis by stiffness related noninvasive markers the relationship between viscoelastic constants in the liver and tissue structure remains unclear. We therefore studied the mechanical properties of 16 human liver specimens with different degrees of fibrosis, inflammation and steatosis by wideband magnetic resonance elastography (MRE) and static indentation experiments providing the specimens' static Young's modulus (E), dynamic storage modulus (G0) and dynamic loss modulus (G"). A frequency-independent shear modulus μ and a powerlaw exponent a were obtained by fitting G and G" using the two-parameter sprinpot model. The mechanical parameters were compared to the specimens' histology derived parameters such as degree of Fibrosis (F), inflammation score and fat score, amount of hydroxyproline (HYP) used for quantification of collagen, blood markers and presurgery in vivo function tests. The frequency averaged parameters G', G" and μ were significantly correlated with F (G': R=0.762, G": R=0.830; μ: R=0.744; all P < 0.01) and HYP (G': R=0.712; G": R=0.720; μ: R=0.731 ; all P < 0.01). The powerlaw exponent a displayed an inverse correlation with F (R = -0.590, P= 0.034) and a trend of inverse correlation with HYP (R = -0.470, P=0.089). The static Young's modulus E was less correlated with F (R=0.587, P=0.022) and not sensitive to HYP. Although inflammation was highly correlated with F (R = 0.773, P < 0.001), no interaction was discernable between inflammation and mechanical para-meters measured in this study. Other histological and blood markers as well as liver function test were correlated with neither F nor the measured mechanical parameters. In conclusion, viscoelastic constants measured by wideband MRE are highly sensitive to histologically proven fibrosis. Our results suggest that, in addition to the amount of connective tissue, subtle structural changes of the viscoelastic matrix determine the sensitivity of mechanical tissue properties to hepatic fibrosis. [ABSTRACT FROM AUTHOR]

This correlated with a lower incidence of chilblain-like lesions and might explain the observed decrease in chilblain-like lesions since winter 2022. Dear Editor, Exposure to Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) with mild COVID-19 disease can be followed by post-COVID cutaneous inflammation with a spectrum ranging from vasculitis to acral chilblain lesions.[[1], [3]] We have shown that chilblain-like lesions are characterized by activation of the type I interferon (IFN) pathway indicated by expression of human myxovirus resistance protein 1 (MXA) and phosphorylated Janus kinase 1 (JAK1) in lesional skin.[5] To further understand the clinical course and pathogenesis of SARS-CoV-2 associated chilblain-like lesions we followed 25 patients presenting to our department with new chilblain lesions during 2 years of the pandemic (2020-2022; Figure 1a). Symptoms ranged from mild reddish pale infiltrated to severe bullous, sporadically necrotic skin lesions at feet (17 patients) and hands (five patients) or both (three patients; Figure 2a). [Extracted from the article]

To date, most studies investigating the immune tumor microenvironment of non-small cell lung cancers (NSCLC) only consider a small number of immune cell subsets or do not reflect the distribution of these cells between different tumor compartments as they were performed on tissue microarrays (TMA). To address this, we analyzed the immune infiltrate in surgically resected NSCLCs, focusing on potential spatial heterogeneity. We evaluated 45 NSCLCs based on whole-slide sections using immunohistochemistry with eleven different antibodies (CD3, CD4, CD8, CD20, CD68, Gata3, FOXP3, T-bet, kappa, lambda, PD-L1). While most markers were relatively evenly distributed among different tumor compartments as well as within the same tumor compartment, some immune cell subsets showed a considerable variance. Notably, the immune infiltrate at the tumor invasion front was dominated by B cells. Concerning markers for T cell differentiation, FoxP3 (Th2) was predominantly expressed in stromal lymphocytes, while T-bet (Th1) was most commonly expressed in intraepithelial immune cells. Although most immune cell subtypes showed a heterogenous distribution within in the intraepithelial compartment, the results from a simulated TMA and core biopsy were mostly in line with the results from whole slide evaluation. Regarding disease specific survival, there were no clear correlations. Interestingly, patients with intraepithelial T-bet positive lymphocytes had a significantly better outcome (p = 0.039), however, this difference was not preserved in multivariate analysis. In conclusion, our study shows that the immune tumor microenvironment of NSCLCs is complex and partially heterogenous, especially concerning markers for T cell differentiation. [ABSTRACT FROM AUTHOR]

Reliable and reproducible interpretation of molecular aberrations constitutes a bottleneck of precision medicine. Evidence-based decision management systems may improve rational therapy recommendations. To cope with an increasing amount of complex molecular data in the clinical care of patients with cancer, we established a workflow for the interpretation of molecular analyses. A specialized physician screened results from molecular analyses for potential biomarkers, irrespective of the diagnostic modality. Best available evidence was retrieved and categorized through establishment of an in-house database and interrogation of publicly available databases. Annotated biomarkers were ranked using predefined evidence levels and subsequently discussed at a molecular tumour board (MTB), which generated treatment recommendations. Subsequent translation into patient treatment and clinical outcomes were followed up. One hundred patients were discussed in the MTB between January 2016 and May 2017. Molecular data were obtained for 70 of 100 patients (50 whole exome/RNA sequencing, 18 panel sequencing, 2 immunohistochemistry (IHC)/microsatellite instability analysis). The MTB generated a median of two treatment recommendations each for 63 patients. Thirty-nine patients were treated: 6 partial responses and 12 stable diseases were achieved as best responses. Genetic counselling for germline events was recommended for seven patients. The development of an evidence-based workflow allowed for the clinical interpretation of complex molecular data and facilitated the translation of personalized treatment strategies into routine clinical care. The high number of treatment recommendations in patients with comprehensive genomic data and promising responses in patients treated with combination therapy warrant larger clinical studies. • Evidence-based, flexible workflow for the clinical interpretation of molecular data. • Application to complex data including whole-exome sequencing and RNA-Sequencing in clinical routine. • Initiation of targeted treatment in 39% of patients. • Promising responses in a subset of patients. • Standardisation of precision oncology workflows could help improve prospective trials. [ABSTRACT FROM AUTHOR]

Purpose: The optimal method for detecting CMV colitis in patients with inflammatory bowel disease (IBD) has not been established. We wanted to investigate which diagnostic test would be most accurate when defining CMV colitis rather by the further clinical course than by using another diagnostic modality.Methods: All consecutive patients with moderately or severely active IBD who had been tested for CMV by PCR, histology, or antigenemia assay at the two campuses CBF and CCM of the Charité - Universitätsmedizin Berlin between September 2006 and September 2009 were included in this retrospective study. During that time, in patients with a positive CMV test, immunosuppressive treatment of any kind was immediately reduced and antiviral treatment was started. This allowed identifying patients who responded to antiviral treatment and those who only responded to later escalation of immunosuppressive therapy.Results: One hundred and nine patients were identified, out of whom nine were considered to have clinically relevant CMV colitis. Sensitivity and specificity were 1 and 0.94 for CMV PCR and 0.5 and 1 for pp65 antigen immunofluorescence assay from peripheral blood, 0.67 and 0.98 for immunohistochemistry, and 0.17 and 0.98 for hematoxylin-eosin staining. When using absence of leukocytosis, splenomegaly, and steroid refractory disease as clinical parameters to test for CMV colitis, blood CMV PCR and immunohistochemistry were able to exclude CMV colitis in negative patients with a 75% likelihood of positive patients to have clinically relevant CMV colitis.Conclusions: Blood-based CMV PCR together with simple clinical parameters can exclude clinically relevant CMV colitis at a high specificity. [ABSTRACT FROM AUTHOR]

PURPOSE: Tumor-infiltrating lymphocytes (TILs) have an established impact on the prognosis of high-grade serous ovarian carcinoma (HGSOC), however, their role in recurrent ovarian cancer is largely unknown. We therefore systematically investigated TIL densities and MHC class I and II (MHC1, 2) expression in the progression of HGSOC. EXPERIMENTAL DESIGN: CD3+, CD4+, CD8+ TILs and MHC1, 2 expression were evaluated by immunohistochemistry on tissue microarrays in 113 paired primary and recurrent HGSOC. TILs were quantified by image analysis. All patients had been included to the EU-funded OCTIPS FP7 project. RESULTS: CD3+, CD4+, CD8+ TILs and MHC1 and MHC2 expression showed significant correlations between primary and recurrent tumor levels (Spearman rho 0.427, 0.533, 0.361, 0.456, 0.526 respectively; Pb.0001 each). Paired testing revealed higher CD4+ densities and MHC1 expression in recurrent tumors (Wilcoxon P=.034 and P=.018). There was also a shift towards higher CD3+ TILs levels in recurrent carcinomas when analyzing platinum-sensitive tumors only (Wilcoxon P=.026) and in pairs with recurrent tumor tissue from first relapse only (Wilcoxon P=.031). High MHC2 expression was the only parameter to be significantly linked to prolonged progression-free survival after first relapse (PFS2, log-rank P=.012). CONCLUSIONS: This is the first study that analyzed the development of TILs density and MHC expression in paired primary and recurrent HGSOC. The level of the antitumoral immune response in recurrent tumors was clearly dependent on the one in the primary tumor. Our data contribute to the understanding of temporal heterogeneity of HGSOC immune microenvironment and have implications for selection of samples for biomarker testing in the setting of immune-targeting therapeutics. [ABSTRACT FROM AUTHOR]

Aims: Programmed death ligand 1 (PD‐L1) immunohistochemistry has become a mandatory diagnostic test in the treatment of lung cancer. Several research initiatives have started to harmonise the five PD‐L1 immunohistochemistry assays that have been used in clinical trials. Here, we report data on interlaboratory and interassay concordance for commercial assays (‘assays’) and laboratory‐developed tests (LDTs) at 10 German testing sites. Methods and results: To assess interlaboratory concordance, a tissue microarray containing 21 pulmonary carcinoma specimens was centrally prepared. Pre‐cut sections were stained at 10 sites by the use of assays 28‐8, 22C3, SP263, and SP142, as well as 11 LDTs. Assay performance was evaluated with a second tissue microarray containing 11 cell lines with defined PD‐L1 expression. Quality control was centrally performed by manual and digital analyses. The assays yielded reproducible IHC staining patterns at all sites. In agreement with previous studies, 22C3, 28‐8 and SP263 showed similar staining patterns, whereas SP142 was distinct. Among the LDTs, six of 11 protocols showed staining patterns similar to those of assays 22C3 and 28‐8. Interlaboratory concordance of tumour cell scoring by use of a six‐step system was moderate (Light's κ = 0.43–0.69), whereas the clinically approved cut‐offs of ≥1% and ≥50% showed substantial concordance (κ = 0.73–0.89). Immune cell scoring by the use of SP142 yielded moderate concordance (κ = 0.42). Conclusions: The data confirm the previously described staining patterns of the assays, and show that they can be reproducibly employed at different sites. LDTs with staining results similar to those of the assays are implementable, but have to be carefully validated. [ABSTRACT FROM AUTHOR]

Background. Even though branchial cleft cysts are currently accepted as a congenital anomaly, there is often a long delay until clinical presentation; branchial cleft cysts classically appear in the second to fourth decade of life. Our observation of their occurrence in three pregnant women encouraged us to contemplate a possible hormonal influence. Methods. Immunohistological analysis was performed for the evaluation of the estrogen receptor alpha (ERα) in paraffin-embedded tissue specimens of 16 patients with a diagnosis of branchial cleft cyst, with three of them being pregnant. Results. Expression of ERα was detected within epithelial cells only in branchial cleft cysts in pregnant females; moreover, higher growth fractions (Ki-67/Mib1) were found. Conclusion. The fact that the estrogen receptor was expressed only in pregnant women, in contrast to 13 investigated cases, may suggest that the high level of estrogen in pregnancy is a possible explanation for the spontaneous growth of branchial cleft cysts. [ABSTRACT FROM AUTHOR]

Background EGFR and its downstream signaling pathway are important targets for cancer therapy. Recently, the monoclonal anti-EGFR antibody Necitumumab in combination with gemcitabine and cisplatin was approved (EMA/14106/2016) for first-line treatment of squamous non-small cell carcinoma (SqNSCLC). Eligibility was restricted to cases with positive EGFR expression. In this context, a ring trial of the Quality Assurance Initiative for Pathology (QuIP ® ) was launched to prepare the German pathology community for a reliable and reproducible, immunohistochemically based biomarker test. Materials and methods The trial was set up by a three-step approach. Two lead institutes were nominated to organize the trial process and to select appropriate cancer samples. These were first tested by the H-score (range 0–300) to identify positive and negative cases. Seven additional pathology institutes with experience in EGFR immunohistochemistry each tested the selected panel of identical cases (internal ring trial) to confirm the suitability of samples and scoring criteria. Then the open ring trial for all institutes of pathology in German speaking countries was announced. Results For the internal trial 8 EGFR-positive and 2 negative lung sqNSCLC samples were selected. A cut-off value of cell membranous staining in ≥ 1% of tumor cells was introduced to define a case as EGFR negative or positive. Two points were attainable per correctly assessed sample leading to a maximum of 20 points, ≥ 18 points were required for a successful participation. All 7 panel institute passed this barrier, 5 with the maximum of 20 points and two with one error (18 points) being related to one case with incorrect interpretation of cytoplasmic versus membranous staining and one case with an H-score of 2 as being considered EGFR positive. A second cut-off value (H-score ≥ 3) was therefore introduced. In the open ring trial, 34 institutions participated of which 28 were successful according to the above criteria. The trial revealed a high reproducibility despite the use of different EGFR antibodies and detection systems. There was no association between technical parameters and trial failure. Again, one participant misinterpreted the subcellular EGFR localization. Conclusions The first nationwide ring test for determination of EGFR IHC expression in sqNSCLC could be successfully performed in a very tight time frame. By this, the national pathology community was prepared to incorporate this marker in the panel of predictive cancer tests in a quality assessed manner and to initiate and accompany future studies on EGFR pathway pathology. [ABSTRACT FROM AUTHOR]

Background We studied the prognostic effect of CD3-, CD8- and CD103-positive T lymphocytes in a cohort of 165 patients with resected pancreatic ductal adenocarcinomas (PDACs) of the treatment group (adjuvant gemcitabine) and the untreated control group of the CONKO-001 study. Methods Immunohistochemical stainings on tissue microarrays (TMAs) against CD3, CD8 and CD103 were performed according to standard procedures. Results A high number of CD8-positive lymphocytes were significantly and independently associated with longer disease-free survival (DFS) and overall survival (OS) in the overall study population. Median DFS/OS were 7.4/18.1 months for patients with a low number of CD8-positive intratumoural lymphocytes ( ≤ 42 per 1 mm tissue core) and 12.7/25.2 months for patients with high numbers (>42 per 1-mm tissue core; p = 0.008/0.020; HR 0.62/0.65). The ratio of intraepithelial to total CD103-positive lymphocytes, but not total numbers of CD103-positive lymphocytes or CD103-positive intraepithelial lymphocytes, was associated with significantly improved DFS and OS in the overall study population (p = 0.022/0.009). Median DFS/OS was 5.9/15.7 for patients with a ratio of intraepithelial to total CD103-positive intratumoural lymphocytes higher than 0.3 and 11.6/24.7 for patients with a lower ratio. Conclusion T-lymphocyte subpopulations might be prognostic in resectable PDAC but need standardization and verification by further studies. [ABSTRACT FROM AUTHOR]

Glycoprotein 2 (GP2) is an autoantigen in Crohn's (CD) and coeliac disease (CeD). We assessed GP2-isoform (GP2 1–4)-expression in intestinal biopsies of paediatric patients with CD, CeD, ulcerative colitis (UC), and healthy children (HC). Transcription of GP2 1–4 was elevated in proximal small intestine in CeD and CD patients (only GP2 2/4) compared to jejunum (CeD/CD) and large bowel (CD). CeD patients demonstrated higher duodenal GP2 2/4 -mRNA levels compared to HC/UC patients whereas CD patients showed higher GP2 4 -mRNA levels compared to UC patients. Duodenal synthesis of only small GP2 isoforms (GP2 3/4) was demonstrated in epithelial cells in patients/HC and in Brunner glands (also large isoforms) with a more frequent apical location in CD/CeD patients. All four GP2 isoforms interacted with gliadin and phosphopeptidomannan. Gliadin digestion improved binding to GP2 isoforms. GP2 1–4 binding to CeD/CD-related antigens, elevated duodenal GP2 1–4 -mRNA transcription, and GP2-protein secretion in Brunner glands of CeD/CD patients suggest an autoimmune CeD/CD link. [Display omitted] • Glycoprotein 2 isoforms bind to coeliac disease and Crohn's disease-related antigens. • Duodenal glycoprotein 2 mRNA transcription is elevated in coeliac disease and Crohn's disease. • Large and small glycoprotein 2 isoforms are secreted in Brunner glands. • Autoimmunity to glycoprotein 2 is a link between coeliac disease and Crohn's disease. [ABSTRACT FROM AUTHOR]

Evaluation of the RAS mutation status is necessary for patients with advanced colorectal cancer to predict the response to anti-EGFR therapy. In routine diagnostics, FFPE tissue samples are tested by sequencing (amplicon-based NGS and Sanger) to obtain the RAS status of the patient. Samples that are collected after chemotherapy occasionally contain necrotic tissue. Furthermore, colorectal cancer tissue sometimes has mucinous components. This may pose a challenge to molecular analysis because mucinous tumor samples often contain only few tumor cells compared to solid tumor samples. Therefore, the aim of this study was to explore if mucin or necrosis affect mutation analysis and if mucinous tumor samples contain enough tumor cells for reliable mutation detection. To this end, we analyzed KRAS status in 10 samples showing mucin production and 10 samples with necrosis. In all 20 samples the tissue areas with the highest amount of mucin or necrosis were used for re-evaluation. These results show no differences with those obtained during routine diagnostics, where analysis of mucinous or necrotic areas was tried to avoid. Our study thus shows that mucin and necrosis have no influence on KRAS mutation analysis. Furthermore we were able to demonstrate that mucinous adenocarcinoma contains enough tumor cells for a valid mutation analysis. In addition, we also observed only minor differences in KRAS status results when comparing Sanger sequencing with NGS. Both methods detected the KRAS mutation in 19 of 20 tested samples, even for mutated samples with low allele-frequencies. [ABSTRACT FROM AUTHOR]

Purpose: Myeloproliferative neoplasms (MPN) are clonal disorders of hematopoietic stem- and progenitor cells. Mutation of Janus-Kinase 2 (JAK2) is the most frequent genetic event detected in Philadelphia-negative MPN. In advanced phases, the clinical hallmark of the disease is a striking inflammatory syndrome. So far, the cellular and molecular basis of inflammation is not fully understood. We, therefore, sought to investigate the relationship of activating JAK2 mutation and aberrant cytokine expression in MPN. Methods: Cytokine array was performed to identify Jak2V617F-related cytokine expression and secretion. CXCL10 mRNA expression was analyzed by qPCR in peripheral blood cells. To exclude paracrine/autocrine stimulation as a potential mechanism, we generated Ba/F3-EpoR-JAK2WT or EpoR-JAK2V617F cells lacking CXCL10 receptor. Pharmacologic inhibition of JAK2 kinase was achieved by JAK-Inhibitor treatment. Signaling pathways and downstream effectors were characterized by Western blotting, immunofluorescence microscopy, luciferase reporter assays, qPCR, and chromatin-immunoprecipitation studies. Results: We identified CXCL10 as the most highly induced cytokine in JAK2-mutated cell lines. In MPN patients, CXCL10 is highly expressed in JAK2V617F but not JAK2WT MPN or healthy donor controls. Moreover, CXCL10 expression correlates with JAK2V617F allelic burden. High CXCL10 correlates with the presence of clinical risk factors but not with clinical symptoms and quality of life. Pharmacologic inhibition of mutated JAK2 kinase inhibits CXCL10 expression. NFκB signaling is activated downstream of JAK2V617F receptor and directly induces CXCL10 expression. Conclusions: Our data provide first evidence for a link between oncogenic JAK2V617F signaling and cell intrinsic induction of CXCL10 induced by activated NFkB signaling. [ABSTRACT FROM AUTHOR]

There is evidence from studies on fibrotic diseases that a TH2 polarized immune response, characterized by the production of cytokines like IL-4, IL-5, and IL-13 is important for fibrosis development. Data concerning the role of TH2 polarized immune response for fibrogenesis in lymphomas are scarce. Using immunohistochemistry, we investigated paraffin embedded tissue specimens of 58 follicular lymphomas (FL) grade 1/2 with and without fibrosis. In FL with fibrosis, we detected more interfollicular GATA-3 positive lymphocytes and a shift towards a TH2 immune response. In areas of fibrosis, which were enriched with myofibroblasts, we observed a polarization of macrophages towards a M2 phenotype with expression of CD163. Our results hold some evidence that a polarization of the adaptive immune system towards a TH2 immune response in the tumor microenvironment is associated with increased fibrosis, which may indicate its functional relevance for either development or maintenance of fibrosis in FL. [ABSTRACT FROM PUBLISHER]

Mantle cell lymphoma (MCL) is a mature B-cell lymphoma characterized by poor clinical outcome. Recent studies revealed the importance of B-cell receptor (BCR) signaling in maintaining MCL survival. However, it remains unclear which role MALT1, an essential component of the CARD11-BCL10-MALT1 complex that links BCR signaling to the NF-κB pathway, plays in the biology of MCL. Here we show that a subset of MCLs is addicted to MALT1, as its inhibition by either RNA or pharmacologic interference induced cytotoxicity both in vitro and in vivo. Gene expression profiling following MALT1 inhibition demonstrated that MALT1 controls an MYC-driven gene expression network predominantly through increasing MYC protein stability. Thus, our analyses identify a previously unappreciated regulatory mechanism of MYC expression. Investigating primary mouse splenocytes, we could demonstrate that MALT1-induced MYC regulation is not restricted to MCL, but represents a common mechanism. MYC itself is pivotal for MCL survival because its downregulation and pharmacologic inhibition induced cytotoxicity in all MCL models. Collectively, these results provide a strong mechanistic rationale to investigate the therapeutic efficacy of targeting the MALT1-MYC axis in MCL patients. [ABSTRACT FROM AUTHOR]

Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are hepatic manifestations of the metabolic syndrome. Many currently used animal models of NAFLD/NASH lack clinical features of either NASH or metabolic syndrome such as hepatic inflammation and fibrosis (e.g., high-fat diets) or overweight and insulin resistance (e.g., methionine-choline-deficient diets), or they are based on monogenetic defects (e.g., ob/ob mice). In the current study, a Western-type diet containing soybean oil with high n-6-PUFA and 0.75% cholesterol (SOD + Cho) induced steatosis, inflammation and fibrosis accompanied by hepatic lipid peroxidation and oxidative stress in livers of C57BL/6-mice, which in addition showed increased weight gain and insulin resistance, thus displaying a phenotype closely resembling all clinical features of NASH in patients with metabolic syndrome. In striking contrast, a soybean oil--containing Western-type diet without cholesterol (SOD) induced only mild steatosis but not hepatic inflammation, fibrosis, weight gain or insulin resistance. Another high-fat diet, mainly consisting of lard and supplemented with fructose in drinking water (LAD + Fru), resulted in more prominent weight gain, insulin resistance and hepatic steatosis than SOD + Cho, but livers were devoid of inflammation and fibrosis. Although both LAD + Fru- and SOD + Cho-fed animals had high plasma cholesterol, liver cholesterol was elevated only in SOD + Cho animals. Cholesterol induced expression of chemotactic and inflammatory cytokines in cultured Kupffer cells and rendered hepatocytes more susceptible to apoptosis. In summary, dietary cholesterol in the SOD + Cho diet may trigger hepatic inflammation and fibrosis. SOD + Cho--fed animals may be a useful disease model displaying many clinical features of patients with the metabolic syndrome and NASH. [ABSTRACT FROM AUTHOR]

The effect of imatinib on T and B cells in patients with chronic myelogenous leukaemia (CML) is not well understood. An upregulation of the transcription factor Broad-complex-Tramtrack-Bric-a-Brac and Cap'n'collar 1 bZip transcription factor 2 (BACH2), which is involved in the development and differentiation of B cells, was demonstrated in a CML cell line treated with imatinib. The present study retrospectively analysed the expression and distribution of cluster of differentiation (CD)3, CD20 and BACH2 (per 1,000 cells), as well as the co-expression of CD20 and BACH2, using immunohistochemistry in serial bone marrow trephines obtained from 14 CML patients treated with imatinib in comparison to 17 patients with newly diagnosed CML and 6 control trephines. Bone marrow trephines of CML patients in remission under imatinib therapy exhibited significantly higher numbers of CD3 and CD20 infiltrates (partly ordered in aggregates) compared with patients with newly diagnosed CML and control individuals. Similarly, nuclear expression of BACH2 in granulopoietic cells was increased in CML patients treated with imatinib, which may represent the histological correlate of the positive treatment effect. Furthermore, since BACH2 is involved in B cell development, its altered expression patterns by imatinib may be one explanation for high B cell numbers, as revealed by CD20/BACH2 (nuclear)-positive cells. As the present data are preliminary, future prospective studies are required to assess the prognostic and predictive role of BACH2 in patients with CML under targeted therapy. [ABSTRACT FROM AUTHOR]

Palpation is one of the most sensitive, effective diagnostic practices, motivating the quantitative and spatially resolved determination of soft tissue elasticity parameters by medical ultrasound or MRI. However, this so-called elastography often suffers from limited anatomical resolution due to noise and insufficient elastic deformation, currently precluding its use as a tomographic modality on its own. We here introduce an efficient way of processing wave images acquired by multifrequency magnetic resonance elastography (MMRE), which relies on wave number reconstruction at different harmonic frequencies followed by their amplitude-weighted averaging prior to inversion. This results in compound maps of wave speed, which reveal variations in tissue elasticity in a tomographic fashion, i.e. an unmasked, slice-wise display of anatomical details at pixel-wise resolution. The method is demonstrated using MMRE data from the literature including abdominal and pelvic organs such as the liver, spleen, uterus body and uterus cervix. Even in small regions with low wave amplitudes, such as nucleus pulposus and spinal cord, elastic parameters consistent with literature values were obtained. Overall, the proposed method provides a simple and noise-robust strategy of in-plane wave analysis of MMRE data, with a pixel-wise resolution producing superior detail to MRE direct inversion methods. [ABSTRACT FROM AUTHOR]

Background: Extrahepatic bile duct injuries remain severe complications during cholecystectomies and often require reconstruction by bilioenteric anastomosis (i.e., hepaticojejunostomy), which comes with further long-term complications (e.g., recurring ascending cholangitis, secondary biliary cirrhosis). In the case of inherent extrahepatic biliary atresia or during liver transplant, artificial or engineered bile ducts could allow novel surgical strategies without the need for hepaticojejunostomy. Methods: We present data on the implantation of in vitro-generated neo-bile ducts in 5 domestic pigs. The neo-bile ducts were engineered through decellularization of allogeneic blood vessels and recellularization with autologous cholangiocytes. On postoperative days 0, 1, 7, and 14, blood samples were taken and analyzed (aspartate aminotransferase, alanine aminotransferase, bilirubin, alkaline phosphatase, creatinine, and leukocytes). Magnetic resonance cholangiopancreatography was performed on postoperative day 14 on 1 pig. Fourteen days after implantation, the pigs were sacrificed and the bile ducts were explanted. Results: All pigs survived the complete study period without severe complications. None of the pigs showed signs of biliary leakage or peritonitis. The neo-bile ducts were infiltrated by neutrophils, and neoangiogenesis was observed around and into the implanted tissue. Conclusion: We present a novel strategy for extrahepatic bile duct replacement by implantation of an autologous neo-bile duct generated ex vivo. Whether the presented technique allows the long-term replacement of native bile ducts must be further evaluated. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Key Clinical Message We report a case of several autoimmune disorders eventually presenting as severe multi organ dysfunction syndrome caused by adult hemophagocytic lymphohistiocytosis ( HLH). Clinical and laboratory tests might lead to fatal misinterpretation without awareness of its diagnostic evaluation, as HLH shares common features with sepsis and immune-mediated systemic inflammatory response syndromes. [ABSTRACT FROM AUTHOR]

The article presents a case study of proximal muscle weakness in an 80-year-old woman with acquired necrotizing myopathy associated with extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue type (MALT) of the left parotid gland. Topics include the findings of the patient's muscle biopsy, the association between lymphoproliferative diseases and necrotizing myopathy, and the possibility of manifestations of paraneoplastic neurological disorders.

Background & Aims In liver fibrosis, activated hepatic stellate cells ( HSC) secrete excess extracellular matrix, thus, represent key targets for antifibrotic treatment strategies. Intermediate-conductance Ca2 + -activated K+-channels ( KCa3.1) are expressed in non-excitable tissues affecting proliferation, migration and vascular resistance rendering KCa3.1 potential targets in liver fibrosis. So far, no information about KCa3.1 expression and their role in HSC exists. Aim was to quantify the KCa3.1 expression in HSC depending on HSC activation and investigation of antifibrotic properties of the specific KCa3.1 inhibitor TRAM-34 in vitro and in vivo. Methods KCa3.1 expression and functionality were studied in TGF-β1-activated HSC by quantitative real time PCR, western-blot and patch-clamp analysis respectively. Effects of TRAM-34 on HSC proliferation, cell cycle and fibrosis-related gene expression were assessed by [3H]-thymidine incorporation, FACS-analysis and RT- PCR respectively. In vivo, vascular resistance and KCa3.1 gene and protein expression were determined in bile duct ligated rats by in situ liver perfusion, Taqman PCR and immunohistochemistry respectively. Results Fibrotic tissues and TGF-β1-activated HSC exhibited higher KCa3.1-expressions than normal tissue and untreated cells. KCa3.1 inhibition with TRAM-34 reduced HSC proliferation by induction of cell cycle arrest and reduced TGF-β1-induced gene expression of collagen I, alpha-smooth muscle actin and TGF-β1 itself. Furthermore, TRAM-34 blocked TGF-β1-induced activation of TGF-β signalling in HSC. In vivo, TRAM-34 reduced the thromboxane agonist-induced portal perfusion pressure. Conclusion Inhibition of KCa3.1 with TRAM-34 downregulates fibrosis-associated gene expression in vitro, and reduces portal perfusion pressure in vivo. Thus, KCa3.1 may represent novel targets for the treatment of liver fibrosis. [ABSTRACT FROM AUTHOR]

Prostate-specific membrane antigen (PSMA) is an essential molecular regulator of prostate cancer (PCa) progression coded by the FOLH1 gene. The PSMA protein has become an important factor in metastatic PCa diagnosis and radioligand therapy. However, low PSMA expression is suggested to be a resistance mechanism to PSMA-based imaging and therapy. Clinical studies revealed that androgen receptor (AR) inhibition increases PSMA expression. The mechanism has not yet been elucidated. Therefore, this study investigated the effect of activation and inhibition of androgen signaling on PSMA expression levels in vitro and compared these findings with PSMA levels in PCa patients receiving systemic therapy. To this end, LAPC4, LNCaP, and C4-2 PCa cells were treated with various concentrations of the synthetic androgen R1881 and antiandrogens. Changes in FOLH1 mRNA were determined using qPCR. Open access databases were used for ChIP-Seq and tissue expression analysis. Changes in PSMA protein were determined using western blot. For PSMA staining in patients' specimens, immunohistochemistry (IHC) was performed. Results revealed that treatment with the synthetic androgen R1881 led to decreased FOLH1 mRNA and PSMA protein. This effect was partially reversed by antiandrogen treatment. However, AR ChIP-Seq analysis revealed no canonical AR binding sites in the regulatory elements of the FOLH1 gene. IHC analysis indicated that androgen deprivation only resulted in increased PSMA expression in patients with low PSMA levels. The data demonstrate that AR activation and inhibition affects PSMA protein levels via a possible non-canonical mechanism. Moreover, analysis of PCa tissue reveals that low PSMA expression rates may be mandatory to increase PSMA by androgen deprivation. [ABSTRACT FROM AUTHOR]