TERT RNAscope analysis of sub-centimetric papillary thyroid carcinomas and synchronous lymph node metastases.

BACKGROUND: Sub-centrimetric papillary thyroid carcinomas usually have a good prognosis with a cancer specific survival of > 99%, however in up to 65% of patients, lymph node metastases can be observed. Molecular alterations in BRAF, TERT and TP53 are associated with worse clinicopathological outcome in patients with papillary thyroid carcinoma. MATERIAL AND METHODS: Twenty-two cases of papillary thyroid carcinomas measuring ≤ 1 cm with synchronous lymph node metastases were examined regarding morphological patterns and immunohistochemical status of p53, Ki-67, and BRAF V600E status. TERT RNA expression in lymph node metastases were evaluated by RNAScope®. RESULTS: Morphological patterns were heterogeneous in both primary tumors and lymph node metastases. Proliferation indices measured by Ki-67 were low. Both primary and lymph node metastases were wild type for p53 by immunohistochemical analysis. No lymph node metastasis showed TERT expression by RNAScope®. CONCLUSIONS: Our data indicate that TERT expression is not involved in the development early lymph node metastasis in patients with sub-centimetric PTC.

Fast-track development and multi-institutional clinical validation of an artificial intelligence algorithm for detection of lymph node metastasis in colorectal cancer.

Lymph node metastasis (LNM) detection can be automated using artificial intelligence-based diagnostic tools. Only limited studies have addressed this task for colorectal cancer. The aim of this study was to develop of a clinical-grade digital pathology tool for LNM detection in colorectal cancer (CRC) using the original fast-track framework. The training cohort included 432 slides from one department. A segmentation algorithm detecting 8 relevant tissue classes was trained. The test cohorts consisted of materials from five pathology departments digitized by four different scanning systems. A high-quality, large training dataset was generated within 7 days, and a minimal amount of annotation work using fast-track principles. The AI tool showed very high accuracy for LNM detection in all cohorts, with sensitivity, negative predictive value, and specificity ranges of 0.980-1.000, 0.997-1.000, and 0.913-0.990, correspondingly. Only 5 of 14460 analyzed test slides with tumor cells over all cohorts were classified as false negative (3/5 representing clusters of tumor cells in lymphatic vessels). A clinical-grade tool was trained in a short time using fast-track development principles and validated using the largest international, multi-institutional, multi-scanner cohort of cases to date, showing very high precision for LNM detection in CRC. We are releasing a part of the test datasets to facilitate academic research.

Developments in predictive biomarker testing and targeted therapy in advanced stage non-small cell lung cancer and their application across European countries.

In the past two decades, the treatment of metastatic non-small cell lung cancer (NSCLC), has undergone significant changes due to the introduction of targeted therapies and immunotherapy. These advancements have led to the need for predictive molecular tests to identify patients eligible for targeted therapy. This review provides an overview of the development and current application of targeted therapies and predictive biomarker testing in European patients with advanced stage NSCLC. Using data from eleven European countries, we conclude that recommendations for predictive testing are incorporated in national guidelines across Europe, although there are differences in their comprehensiveness. Moreover, the availability of recently EMA-approved targeted therapies varies between European countries. Unfortunately, routine assessment of national/regional molecular testing rates is limited. As a result, it remains uncertain which proportion of patients with metastatic NSCLC in Europe receive adequate predictive biomarker testing. Lastly, Molecular Tumor Boards (MTBs) for discussion of molecular test results are widely implemented, but national guidelines for their composition and functioning are lacking. The establishment of MTB guidelines can provide a framework for interpreting rare or complex mutations, facilitating appropriate treatment decision-making, and ensuring quality control.

Future perspective for the application of predictive biomarker testing in advanced stage non-small cell lung cancer.

For patients with advanced stage non-small cell lung cancer (NSCLC), treatment strategies have changed significantly due to the introduction of targeted therapies and immunotherapy. In the last few years, we have seen an explosive growth of newly introduced targeted therapies in oncology and this development is expected to continue in the future. Besides primary targetable aberrations, emerging diagnostic biomarkers also include relevant co-occurring mutations and resistance mechanisms involved in disease progression, that have impact on optimal treatment management. To accommodate testing of pending biomarkers, it is necessary to establish routine large-panel next-generation sequencing (NGS) for all patients with advanced stage NSCLC. For cost-effectiveness and accessibility, it is recommended to implement predictive molecular testing using large-panel NGS in a dedicated, centralized expert laboratory within a regional oncology network. The central molecular testing center should host a regional Molecular Tumor Board and function as a hub for interpretation of rare and complex testing results and clinical decision-making.

High Concordance of Different Assays in the Determination of Homologous Recombination Deficiency-Associated Genomic Instability in Ovarian Cancer.

PURPOSE: Poly(ADP-ribose) polymerase inhibitors (PARPi) have shown promising clinical results in the treatment of ovarian cancer. Analysis of biomarker subgroups consistently revealed higher benefits for patients with homologous recombination deficiency (HRD). The test that is most often used for the detection of HRD in clinical studies is the Myriad myChoice assay. However, other assays can also be used to assess biomarkers, which are indicative of HRD, genomic instability (GI), and BRCA1/2 mutation status. Many of these assays have high potential to be broadly applied in clinical routine diagnostics in a time-effective decentralized manner. Here, we compare the performance of a multitude of alternative assays in comparison with Myriad myChoice in high-grade serous ovarian cancer (HGSOC). METHODS: DNA from HGSOC samples was extracted from formalin-fixed paraffin-embedded tissue blocks of cases previously run with the Myriad myChoice assay, and GI was measured by multiple molecular assays (CytoSNP, AmoyDx, Illumina TSO500 HRD, OncoScan, NOGGO GISv1, QIAseq HRD Panel and whole genome sequencing), applying different bioinformatics algorithms. RESULTS: Application of different assays to assess GI, including Myriad myChoice, revealed high concordance of the generated scores ranging from very substantial to nearly perfect fit, depending on the assay and bioinformatics pipelines applied. Interlaboratory comparison of assays also showed high concordance of GI scores. CONCLUSION: Assays for GI assessment not only show a high concordance with each other but also in correlation with Myriad myChoice. Thus, almost all of the assays included here can be used effectively to assess HRD-associated GI in the clinical setting. This is important as PARPi treatment on the basis of these tests is compliant with European Medicines Agency approvals, which are methodologically not test-bound.

COVID-19-associated secondary sclerosing cholangitis with liver transplantation.

We report on two cases of orthotopic liver transplantation (OLTX) due to SARS-Cov2-associated secondary sclerosing cholangitis (SSC) following long-term artificial respiration and extra-corporal membrane oxygenation in intensive care. Under these conditions, SSC is a rapidly progredient biliary disease featuring degenerative cholangiopathy, loss of bile ducts, ductular and parenchymal cholestasis, biliary fibrosis, and finally cirrhosis. Reduced perfusion and oxygenation of the peribiliary plexus, severe concurrent infections, and secondary medico-toxic effects appear to play a crucial role in the pathogenesis of the disease. A direct cytopathic effect of SARS-Cov2 on endothelial cells followed by thrombosis and fibrosing obliteration in all parts of the vascular bed of the liver may enhance the virus-associated liver disease and particularly SSC.

Corneal Infantile Myofibromatosis Caused by Novel Activating Imatinib-Responsive Variants in PDGFRB.

Purpose: To investigate the genetic cause, clinical characteristics, and potential therapeutic targets of infantile corneal myofibromatosis. Design: Case series with genetic and functional in vitro analyses. Participants: Four individuals from 2 unrelated families with clinical signs of corneal myofibromatosis were investigated. Methods: Exome-based panel sequencing for platelet-derived growth factor receptor beta gene (PDGFRB) and notch homolog protein 3 gene (NOTCH3) was performed in the respective index patients. One clinically affected member of each family was tested for the pathogenic variant detected in the respective index by Sanger sequencing. Immunohistochemical staining on excised corneal tissue was conducted. Functional analysis of the individual PDGFRB variants was performed in vitro by luciferase reporter assays on transfected porcine aortic endothelial cells using tyrosine kinase inhibitors. Protein expression analysis of mutated PDGFRB was analyzed by Western blot. Main Outcome Measures: Sequencing data, immunohistochemical stainings, functional analysis of PDGFRB variants, and protein expression analysis. Results: We identified 2 novel, heterozygous gain-of-function variants in PDGFRB in 4 individuals from 2 unrelated families with corneal myofibromatosis. Immunohistochemistry demonstrated positivity for alpha-smooth muscle actin and ß-catenin, a low proliferation rate in Ki-67 (< 5%), marginal positivity for Desmin, and negative staining for Caldesmon and CD34. In all patients, recurrence of disease occurred after corneal surgery. When transfected in cultured cells, the PDGFRB variants conferred a constitutive activity to the receptor in the absence of its ligand and were sensitive to the tyrosine kinase inhibitor imatinib. The variants can both be classified as likely pathogenic regarding the American College of Medical Genetics and Genomics classification criteria. Conclusions: We describe 4 cases of corneal myofibromatosis caused by novel PDGFRB variants with autosomal dominant transmission. Imatinib sensitivity in vitro suggests perspectives for targeted therapy preventing recurrences in the future. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

An anti-CD19/CTLA-4 switch improves efficacy and selectivity of CAR T cells targeting CD80/86-upregulated DLBCL.

Chimeric antigen receptor T cell (CAR T) therapy is a potent treatment for relapsed/refractory (r/r) B cell lymphomas but provides lasting remissions in only ∼40% of patients and is associated with serious adverse events. We identify an upregulation of CD80 and/or CD86 in tumor tissue of (r/r) diffuse large B cell lymphoma (DLBCL) patients treated with tisagenlecleucel. This finding leads to the development of the CAR/CCR (chimeric checkpoint receptor) design, which consists of a CD19-specific first-generation CAR co-expressed with a recombinant CTLA-4-linked receptor with a 4-1BB co-stimulatory domain. CAR/CCR T cells demonstrate superior efficacy in xenograft mouse models compared with CAR T cells, superior long-term activity, and superior selectivity in in vitro assays with non-malignant CD19+ cells. In addition, immunocompetent mice show an intact CD80-CD19+ B cell population after CAR/CCR T cell treatment. The results reveal the CAR/CCR design as a promising strategy for further translational study.

HoLy-Net: Segmentation of histological images of diffuse large B-cell lymphoma.

Over the last years, there has been large progress in automated segmentation and classification methods in histological whole slide images (WSIs) stained with hematoxylin and eosin (H&E). Current state-of-the-art (SOTA) techniques are based on diverse datasets of H&E-stained WSIs of different types of predominantly solid cancer. However, there is a scarcity of methods and datasets enabling segmentation of tumors of the lymphatic system (lymphomas). Here, we propose a solution for segmentation of diffuse large B-cell lymphoma (DLBCL), the most common non-Hodgkin's lymphoma. Our method applies to both H&E-stained slides and to a broad range of markers stained with immunohistochemistry (IHC). While IHC staining is an important tool in cancer diagnosis and treatment decisions, there are few automated segmentation and classification methods for IHC-stained WSIs. To address the challenges of nuclei segmentation in H&E- and IHC-stained DLBCL images, we propose HoLy-Net - a HoVer-Net-based deep learning model for lymphoma segmentation. We train two different models, one for segmenting H&E- and one for IHC-stained images and compare the test results with the SOTA methods as well as with the original version of HoVer-Net. Subsequently, we segment patient WSIs and perform single cell-level analysis of different cell types to identify patient-specific tumor characteristics such as high level of immune infiltration. Our method outperforms general-purpose segmentation methods for H&E staining in lymphoma WSIs (with an F1 score of 0.899) and is also a unique automated method for IHC slide segmentation (with an F1 score of 0.913). With our solution, we provide a new dataset we denote LyNSeC (lymphoma nuclear segmentation and classification) containing 73,931 annotated cell nuclei from H&E and 87,316 from IHC slides. Our method and dataset open up new avenues for quantitative, large-scale studies of morphology and microenvironment of lymphomas overlooked by the current automated segmentation methods.

An inducible Cd79b mutation confers ibrutinib sensitivity in mouse models of Myd88-driven diffuse large B-cell lymphoma.

ABSTRACT: Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive lymphoma and constitutes a highly heterogenous disease. Recent comprehensive genomic profiling revealed the identity of numerous molecularly defined DLBCL subtypes, including a cluster which is characterized by recurrent aberrations in MYD88, CD79B, and BCL2, as well as various lesions promoting a block in plasma cell differentiation, including PRDM1, TBL1XR1, and SPIB. Here, we generated a series of autochthonous mouse models to mimic this DLBCL cluster and specifically focused on the impact of Cd79b mutations in this setting. We show that canonical Cd79b immunoreceptor tyrosine-based activation motif (ITAM) mutations do not accelerate Myd88- and BCL2-driven lymphomagenesis. Cd79b-mutant murine DLBCL were enriched for IgM surface expression, reminiscent of their human counterparts. Moreover, Cd79b-mutant lymphomas displayed a robust formation of cytoplasmic signaling complexes involving MYD88, CD79B, MALT1, and BTK. These complexes were disrupted upon pharmacological BTK inhibition. The BTK inhibitor-mediated disruption of these signaling complexes translated into a selective ibrutinib sensitivity of lymphomas harboring combined Cd79b and Myd88 mutations. Altogether, this in-depth cross-species comparison provides a framework for the development of molecularly targeted therapeutic intervention strategies in DLBCL.

MET Fusions in NSCLC: Clinicopathologic Features and Response to MET Inhibition.

INTRODUCTION: MET fusions have been described only rarely in NSCLC. Thus, data on patient characteristics and treatment response are limited. We here report histopathologic data, patient demographics, and treatment outcome including response to MET tyrosine kinase inhibitor (TKI) therapy in MET fusion-positive NSCLC. METHODS: Patients with NSCLC and MET fusions were identified mostly by RNA sequencing within the routine molecular screening program of the national Network Genomic Medicine, Germany. RESULTS: We describe a cohort of nine patients harboring MET fusions. Among these nine patients, two patients had been reported earlier. The overall frequency was 0.29% (95% confidence interval: 0.15-0.55). The tumors were exclusively adenocarcinoma. The cohort was heterogeneous in terms of age, sex, or smoking status. We saw five different fusion partner genes (KIF5B, TRIM4, ST7, PRKAR2B, and CAPZA2) and several different breakpoints. Four patients were treated with a MET TKI leading to two partial responses, one stable disease, and one progressive disease. One patient had a BRAF V600E mutation as acquired resistance mechanism. CONCLUSIONS: MET fusions are very rare oncogenic driver events in NSCLC and predominantly seem in adenocarcinomas. They are heterogeneous in terms of fusion partners and breakpoints. Patients with MET fusion can benefit from MET TKI therapy.

TTF-1 status in early-stage lung adenocarcinoma is an independent predictor of relapse and survival superior to tumor grading.

OBJECTIVES: Thyroid transcription factor 1 (TTF-1) is a well-established independent prognostic factor in lung adenocarcinoma (LUAD), irrespective of stage. This study aims to determine if TTF-1's prognostic impact is solely based on histomorphological differentiation (tumor grading) or if it independently relates to a biologically more aggressive phenotype. We analyzed a large bi-centric LUAD cohort to accurately assess TTF-1's prognostic value in relation to tumor grade. PATIENTS AND METHODS: We studied 447 patients with resected LUAD from major German lung cancer centers (Berlin and Cologne), correlating TTF-1 status and grading with clinical, pathologic, and molecular data, alongside patient outcomes. TTF-1's impact was evaluated through univariate and multivariate Cox regression. Causal graph analysis was used to identify and account for potential confounders, improving the statistical estimation of TTF-1's predictive power for clinical outcomes. RESULTS: Univariate analysis revealed TTF-1 positivity associated with significantly longer disease-free survival (DFS) (median log HR -0.83; p = 0.018). Higher tumor grade showed a non-significant association with shorter DFS (median log HR 0.30; p = 0,62 for G1 to G2 and 0.68; p = 0,34 for G2 to G3). In multivariate analysis, TTF-1 positivity resulted in a significantly longer DFS (median log HR -0.65; p = 0.05) independent of all other parameters, including grading. Adjusting for potential confounders as indicated by the causal graph confirmed the superiority of TTF-1 over tumor grading in prognostics power. CONCLUSIONS: TTF-1 status predicts relapse and survival in LUAD independently of tumor grading. The prognostic power of tumor grading is limited to TTF-1-positive patients, and the effect size of TTF-1 surpasses that of tumor grading. We recommend including TTF1 status as a prognostic factor in the diagnostic guidelines of LUAD.

Efficiency of B-RAF-/MEK-inhibitors in B-RAF mutated Ameloblastoma: Case report and review of literature.

Background: Ameloblastoma is a benign but locally invasive and aggressive odontogenic tumor harboring activating BRAF V600E mutations in about two thirds of the cases. Case presentation: Neoadjuvant therapy with Dabrafenib and Trametinib was given to a 42-year-old male patient with recurrent ameloblastoma of the right mandible with a BRAF V600E mutation for 18 months. The patient manifested an excellent response to the therapy with remarkable reduction in tumor size from 72.6 mm to 55.9 mm. Histopathologically, the tumor underwent significant degenerative changes with only a few sparse vital residuals revealing 0 % Ki67 proliferative index. Conclusions: Neoadjuvant therapy with BRAF-inhibitors or BRAF-MEK-inhibitors is an effective means to reduce the size of mandibulary ameloblastomas. We propose the consideration of neoadjuvant therapy in future treatment modalities to minimize post-surgical morbidity and facial deformations.

Assessment of MYC and TERT copy number variations in lung cancer using digital PCR.

OBJECTIVE: Lung cancer is the second most frequent cancer type and the most common cause of cancer-related deaths worldwide. Alteration of gene copy numbers are associated with lung cancer and the determination of copy number variations (CNV) is appropriate for the discrimination between tumor and non-tumor tissue in lung cancer. As telomerase reverse transcriptase (TERT) and v-myc avian myelocytomatosis viral oncogene homolog (MYC) play a role in lung cancer the aims of this study were the verification of our recent results analyzing MYC CNV in tumor and non-tumor tissue of lung cancer patients using an independent study group and the assessment of TERT CNV as an additional marker. RESULTS: TERT and MYC status was analyzed using digital PCR (dPCR) in tumor and adjacent non-tumor tissue samples of 114 lung cancer patients. The difference between tumor and non-tumor samples were statistically significant (p < 0.0001) for TERT and MYC. Using a predefined specificity of 99% a sensitivity of 41% and 51% was observed for TERT and MYC, respectively. For the combination of TERT and MYC the overall sensitivity increased to 60% at 99% specificity. We demonstrated that a combination of markers increases the performance in comparison to individual markers. Additionally, the determination of CNV using dPCR might be an appropriate tool in precision medicine.

Testing for deficient mismatch repair and microsatellite instability : A focused update.

Testing to detect mismatch repair deficiency (dMMR) and high-grade microsatellite instability (MSI-H) has become an integral part of the routine diagnostic workup for colorectal cancer (CRC). While MSI was initially considered to be a possible indicator of a hereditary disposition to cancer (Lynch syndrome, LS), today the prediction of the therapy response to immune checkpoint inhibitors (ICI) is in the foreground. Corresponding recommendations and testing algorithms are available for use in primary diagnosis (reviewed in: Rüschoff et al. 2021).Given the increasing importance for routine use and the expanding indication spectrum of ICI therapies for non-CRCs, such as endometrial, small intestinal, gastric, and biliary tract cancers, an updated review of dMMR/MSI testing is presented. The focus is on the challenges in the assessment of immunohistochemical stains and the value of PCR-based procedures, considering the expanded ICI indication spectrum. A practice-oriented flowchart for everyday diagnostic decision-making is provided that considers new data on the frequency and type of discordances between MMR-IHC and MSI-PCR findings, and the possible role of Next Generation Sequencing in clarifying them. Reference is made to the significance of systematic quality assurance measures (e.g., QuIP MSI portal and multicenter proficiency testing), including regular continued training and education.

[Prevention, Diagnosis, Therapy, and Follow-up of Lung Cancer - Interdisciplinary Guideline of the German Respiratory Society and the German Cancer Society - Abridged Version]. / Prävention, Diagnostik, Therapie und Nachsorge des Lungenkarzinoms.

The current S3 Lung Cancer Guidelines are edited with fundamental changes to the previous edition based on the dynamic influx of information to this field:The recommendations include de novo a mandatory case presentation for all patients with lung cancer in a multidisciplinary tumor board before initiation of treatment, furthermore CT-Screening for asymptomatic patients at risk (after federal approval), recommendations for incidental lung nodule management , molecular testing of all NSCLC independent of subtypes, EGFR-mutations in resectable early stage lung cancer in relapsed or recurrent disease, adjuvant TKI-therapy in the presence of common EGFR-mutations, adjuvant consolidation treatment with checkpoint inhibitors in resected lung cancer with PD-L1 ≥ 50%, obligatory evaluation of PD-L1-status, consolidation treatment with checkpoint inhibition after radiochemotherapy in patients with PD-L1-pos. tumor, adjuvant consolidation treatment with checkpoint inhibition in patients withPD-L1 ≥ 50% stage IIIA and treatment options in PD-L1 ≥ 50% tumors independent of PD-L1status and targeted therapy and treatment option immune chemotherapy in first line SCLC patients.Based on the current dynamic status of information in this field and the turnaround time required to implement new options, a transformation to a "living guideline" was proposed.