BAG6 restricts pancreatic cancer progression by suppressing the release of IL33-presenting extracellular vesicles and the activation of mast cells.

Recent studies reveal a critical role of tumor cell-released extracellular vesicles (EVs) in pancreatic cancer (PC) progression. However, driver genes that direct EV function, the EV-recipient cells, and their cellular response to EV uptake remain to be identified. Therefore, we studied the role of Bcl-2-associated-anthanogene 6 (BAG6), a regulator of EV biogenesis for cancer progression. We used a Cre recombinase/LoxP-based reporter system in combination with single-cell RNA sequencing to monitor in vivo EV uptake and tumor microenvironment (TME) changes in mouse models for pancreatic ductal adenocarcinoma (PDAC) in a Bag6 pro- or deficient background. In vivo data were validated using mouse and human organoids and patient samples. Our data demonstrated that Bag6-deficient subcutaneous and orthotopic PDAC tumors accelerated tumor growth dependent on EV release. Mechanistically, this was attributed to mast cell (MC) activation via EV-associated IL33. Activated MCs promoted tumor cell proliferation and altered the composition of the TME affecting fibroblast polarization and immune cell infiltration. Tumor cell proliferation and fibroblast polarization were mediated via the MC secretome containing high levels of PDGF and CD73. Patients with high BAG6 gene expression and high protein plasma level have a longer overall survival indicating clinical relevance. The current study revealed a so far unknown tumor-suppressing activity of BAG6 in PDAC. Bag6-deficiency allowed the release of EV-associated IL33 which modulate the TME via MC activation promoting aggressive tumor growth. MC depletion using imatinib diminished tumor growth providing a scientific rationale to consider imatinib for patients stratified with low BAG6 expression and high MC infiltration. EVs derived from BAG6-deficient pancreatic cancer cells induce MC activation via IL33/Il1rl1. The secretome of activated MCs induces tumor proliferation and changes in the TME, particularly shifting fibroblasts into an inflammatory cancer-associated fibroblast (iCAF) phenotype. Blocking EVs or depleting MCs restricts tumor growth.

NGS-Guided Precision Oncology in Breast Cancer and Gynecological Tumors-A Retrospective Molecular Tumor Board Analysis.

Background: Precision oncology treatments are being applied more commonly in breast and gynecological oncology through the implementation of Molecular Tumor Boards (MTBs), but real-world clinical outcome data remain limited. Methods: A retrospective analysis was conducted in patients with breast cancer (BC) and gynecological malignancies referred to our center's MTB from 2018 to 2023. The analysis covered patient characteristics, next-generation sequencing (NGS) results, MTB recommendations, therapy received, and clinical outcomes. Results: Sixty-three patients (77.8%) had metastatic disease, and forty-four patients (54.3%) had previously undergone three or more lines of systemic treatment. Personalized treatment recommendations were provided to 50 patients (63.3%), while 29 (36.7%) had no actionable target. Ultimately, 23 patients (29.1%) underwent molecular-matched treatment (MMT). Commonly altered genes in patients with pan-gyn tumors (BC and gynecological malignancies) included TP53 (n = 42/81, 51.9%), PIK3CA (n = 18/81, 22.2%), BRCA1/2 (n = 10/81, 12.3%), and ARID1A (n = 9/81, 11.1%). Patients treated with MMT showed significantly prolonged progression-free survival (median PFS 5.5 vs. 3.5 months, p = 0.0014). Of all patients who underwent molecular profiling, 13.6% experienced a major clinical benefit (PFSr ≥ 1.3 and PR/SD ≥ 6 months) through precision oncology. Conclusions: NGS-guided precision oncology demonstrated improved clinical outcomes in a subgroup of patients with gynecological and breast cancers.

Systematic Analysis of Homologous Recombination Deficiency Testing in Ovarian Cancer-Development of Recommendations for Optimal Assay Performance.

Homologous recombination deficiency (HRD) assays are an important element of personalized oncology in ovarian carcinomas, but the optimal tissue requirements for these complex molecular assays remain unclear. As a result, a considerable percentage of assays are not successful, leading to suboptimal diagnoses for these patients. In this study, we have systematically analyzed tumor and tissue parameters for HRD analysis in a large cohort of real-world cancer samples. The aim of this study is to give recommendations for pathologists and gynecologic oncologists for selection of tissue samples to maximize the success rate of HRD analyses. Tumor samples from 2702 patients were sent to the Institute of Pathology of the Philipps-University Marburg between October 2020 and September 2022, of which 2654 were analyzed using the Myriad MyChoice HRD+ CDx assay. A total of 2396 of 2654 samples (90.3%) were successfully tested, of which 984 of 2396 (41.1%) were HRD positive and 1412 (58.9%) were HRD negative. Three hundred sixty-three of 2396 samples (15.2%) were BRCA1/2-mutated; 27 samples had a BRCA1/2 mutation and a genomic instability score (GIS) < 42. Twenty-two samples (0.9%) failed GIS measurement but displayed a BRCA1/2 mutation. BRCA1/2-mutated samples showed significantly (P < .0001) higher GIS values than those with a wild-type BRCA1/2 status. Tumor cell content, tumor area, and histology significantly (P < .0001) affected the probability of successfully analyzing a sample. Based on a systematic analysis of tumor cell content and tumor area, we recommend selecting patient high-grade serous ovarian cancer samples that display a tumor cell content ≥30% and a tumor area ≥0.5 cm2 (based on their hematoxylin and eosin) for HRD testing to allow for optimal chances of a successful analysis and conclusive results. Considering histologic and sample conditions, success rates of up to 98% can be achieved. Our comprehensive evaluation contributes to further standardization of recommendations on HRD testing in ovarian cancer, which will have a large impact on personalized therapeutic strategies in this highly aggressive tumor type.

Mesenteric fibrosis in patients with small intestinal neuroendocrine tumors is associated with enrichment of alpha-smooth muscle actin-positive fibrosis and COMP-expressing stromal cells.

Neuroendocrine tumors of the small intestine (SI-NETs) often develop lymph node metastasis (LNM)-induced mesenteric fibrosis (MF). MF can cause intestinal obstruction as well as ischemia and render surgical resection technically challenging. The underlying pathomechanisms of MF are still not well understood. We examined mesenteric LNM and the surrounding stroma compartment from 24 SI-NET patients, including 11 with in situ presentation of strong MF (MF+) and 13 without MF (MF-). Differential gene expression was assessed with the HTG EdgeSeq Oncology Biomarker Panel comparing MF+ with MF- within LNM and paired stromal samples, respectively. Most interesting differentially expressed genes were validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in combination with validation of associated protein levels utilizing immunohistochemistry (IHC) staining of MF+ and MF- formalin-fixed, paraffin-embedded (FFPE) patient samples. Overall, 14 genes measured with a 2549-gene expression panel were differentially expressed in MF+ patients compared to MF-. Of those, nine were differentially expressed genes in LNM and five genes in the stromal tissue (>2-fold change, p < .05). The top hits included increased COMP and COL11A1 expression in the stroma of MF+ patients compared to MF-, as well as decreased HMGA2, COL6A6, and SLC22A3 expression in LNM of MF+ patients compared to LNM of MF- patients. RT-qPCR confirmed high levels of COMP and COL11A1 in stroma samples of MF+ compared to MF- patients. IHC staining confirmed the enrichment of α-smooth muscle actin-positive fibrosis in MF+ compared to MF- patients with corresponding increase of COMP-expressing stromal cells in MF+. Since COMP is associated with the known driver for fibrosis development transforming growth factor beta and with a cancer-associated fibroblasts enriched environment, it seems to be a promising new target for MF research.

Intratumoral CD8+ T cells with a tissue-resident memory phenotype mediate local immunity and immune checkpoint responses in breast cancer.

CD8+ tumor-infiltrating lymphocytes with a tissue-resident memory T (TRM) cell phenotype are associated with favorable prognosis in patients with triple-negative breast cancer (TNBC). However, the relative contribution of CD8+ TRM cells to anti-tumor immunity and immune checkpoint blockade efficacy in breast cancer remains unknown. Here, we show that intratumoral CD8+ T cells in murine mammary tumors transcriptionally resemble those from TNBC patients. Phenotypic and transcriptional studies established two intratumoral sub-populations: one more enriched in markers of terminal exhaustion (TEX-like) and the other with a bona fide resident phenotype (TRM-like). Treatment with anti-PD-1 and anti-CTLA-4 therapy resulted in expansion of these intratumoral populations, with the TRM-like subset displaying significantly enhanced cytotoxic capacity. TRM-like CD8+ T cells could also provide local immune protection against tumor rechallenge and a TRM gene signature extracted from tumor-free tissue was significantly associated with improved clinical outcomes in TNBC patients treated with checkpoint inhibitors.

[Clinically relevant molecular pathological diagnostics in breast cancer]. / Klinisch relevante molekularpathologische Diagnostik beim Mammakarzinom.

In breast cancer, the current guideline for pathological workup includes recommendations for advanced molecular analysis of certain predictive molecular markers in addition to basic immunohistochemical diagnostics. These markers are determined depending on tumor stage, including sequencing techniques and immunohistochemical methods. This comprises the systematic investigation of molecular alterations such as PIK3CA or BRCA1,2 mutations, NTRK fusions, or microsatellite instability as a basis for targeted therapy. Further alterations, for example in the PI3K pathway, ESR1 alterations, or ERBB2 mutations, may also be relevant for individual therapy decisions especially in the context of resistant or relapsed disease. Thus, particularly in advanced stages, a more comprehensive molecular characterization of the tumor may reveal genetic alterations that act as tumor drivers and provide targets for personalized therapies. Due to the large number of potential molecular targets, NGS panel diagnostics are a suitable approach in this conjunction with immunohistochemical characterization and the individual clinical situation. Molecular based therapeutical strategies outside of entity-specific approvals should be discussed in an interdisciplinary team within the framework of a molecular tumor board.

Homologous Recombination Deficiency as an Ovarian Cancer Biomarker in a Real-World Cohort: Validation of Decentralized Genomic Profiling.

The diagnostic evaluation of homologous recombination deficiency (HRD) is central to define targeted therapy strategies for patients with ovarian carcinoma. We evaluated HRD in 514 ovarian carcinoma samples by next-generation sequencing of DNA libraries, including BRCA1/BRCA2 and 26,523 single-nucleotide polymorphisms using the standardized Myriad HRD assay, with the predefined cut point of ≥42 for a positive genomic instability score (GIS). All samples were measured in the central Myriad laboratory and in an academic molecular pathology laboratory. A positive GIS was detected in 196 (38.1%) of tumors, whereas 318 (61.9%) were GIS negative. Combining GIS and BRCA mutations, a total of 200 (38.9%) of the 514 tumors were HRD positive. A positive GIS was significantly associated with high-grade serous histology (P < 0.000001), grade 3 tumors (P = 0.001), and patient age <60 years (P = 0.0003). The concordance between both laboratories for the GIS status was 96.9% (P < 0.000001), with a sensitivity of 94.6% and a specificity of 98.4%. Concordance for HRD status was 97.1% (499 of 514 tumors). The percentage of HRD-positive tumors in our real-life cohort was similar to the proportion observed in the recently published PAOLA-1 trial, with high concordance between central and local laboratories. Our results support introduction of the standardized HRD assay in academic molecular pathology laboratories, thus broadening access to personalized oncology strategies for patients with ovarian cancer worldwide.

Combined Focused Next-Generation Sequencing Assays to Guide Precision Oncology in Solid Tumors: A Retrospective Analysis from an Institutional Molecular Tumor Board.

BACKGROUND: Increasing knowledge of cancer biology and an expanding spectrum of molecularly targeted therapies provide the basis for precision oncology. Despite extensive gene diagnostics, previous reports indicate that less than 10% of patients benefit from this concept. METHODS: We retrospectively analyzed all patients referred to our center's Molecular Tumor Board (MTB) from 2018 to 2021. Molecular testing by next-generation sequencing (NGS) included a 67-gene panel for the detection of short-sequence variants and copy-number alterations, a 53- or 137-gene fusion panel and an ultra-low-coverage whole-genome sequencing for the detection of additional copy-number alterations outside the panel's target regions. Immunohistochemistry for microsatellite instability and PD-L1 expression complemented NGS. RESULTS: A total of 109 patients were referred to the MTB. In all, 78 patients received therapeutic proposals (70 based on NGS) and 33 were treated accordingly. Evaluable patients treated with MTB-recommended therapy (n = 30) had significantly longer progression-free survival than patients treated with other therapies (n = 17) (4.3 vs. 1.9 months, p = 0.0094). Seven patients treated with off-label regimens experienced major clinical benefits. CONCLUSION: The combined focused sequencing assays detected targetable alterations in the majority of patients. Patient benefits appeared to lie in the same range as with large-scale sequencing approaches.

Terpene Synthase Genes in Quercus robur - Gene Characterization, Expression and Resulting Terpenes Due to Cockchafer Feeding.

Root herbivory caused by larvae of the forest cockchafer (Melolontha hippocastani) enhances the impact of drought on trees, particularly in oak forest rejuvenations. In Germany, geographically distant oak stands show differences in infestation strength by the forest cockchafer. While in Southwestern Germany this insect causes severe damage, oak forests in northern Germany are rarely infested. It is known that root-released volatile organic compounds (VOCs) are perceived by soil herbivores, thus guiding the larvae toward the host roots. In this work, we exposed seedlings of two distant oak provenances to forest cockchafer larvae and studied their population genetic properties, their root-based VOC chemotypes, their attraction for larvae and terpene synthase gene expression. Based on nuclear and chloroplast marker analysis, we found both oak populations to be genetically highly variable while showing typical patterns of migration from different refugial regions. However, no clear association between genetic constitution of the different provenances and the abundance of cockchafer populations on site was observed. In contrast to observations in the field, bioassays revealed a preference of the larvae for the northeastern oak provenance. The behavior of larvae was most likely related to root-released volatile terpenes and benzenoids since their composition and quantity differed between oak populations. We assume repellent effects of these compounds because the populations attractive to insects showed low abundance of these compounds. Five different oak terpene synthase (TPS) genes were identified at the genomic level which can be responsible for biosynthesis of the released terpenes. TPS gene expression patterns in response to larval feeding revealed geographic variation rather than genotypic variation. Our results support the assumption that root-released VOC are influencing the perception of roots by herbivores.