Figure S8 depicts GSEA for Millstein sub-signatures in the Charité cohort. Gene sets were incrementally increased by one gene at thetime (see color code). X-axis shows the NES and only the full 101-gene signature was significantly enriched.

Figure S5 shows Candidate Genes in the Charité Cohort and Correlations among candidate genes and Literature research results for candidate genes.

Figure S6 presents Boxplots showing differences in BRCA1/2, KRAS, RB1, CCNE1 expression between LTS and STS in the Charité cohort.

PURPOSE:The late-stage diagnosis and the aggressiveness of high-grade serous tubo-ovarian carcinoma (HGSC) often result in poor survival outcomes, yet some patients exhibit an exceptionally long survival rate. This study aimed to identify molecular profiles associated with long-/short-term survival in HGSC, with the goal of better understanding protective factors and developing new treatments. EXPERIMENTAL DESIGN:To discover molecular drivers causing the aggressiveness of HGSC, tumor samples from 12 long-term HGSC survivors (>7 years overall survival) and 12 short-term survivors (<1 year overall survival) were analyzed using targeted RNA sequencing followed by computational analysis. We investigated differentially expressed genes and their functional relevance, inferred differences in cell type composition and signaling pathways, as well as mutation status. To validate our findings, we simulated our study design by using HGSC The Cancer Genome Atlas dataset samples. We evaluated differential patterns of gene expression between these two groups and developed molecular profiles of HGSC that correlate with survival phenotypes. RESULTS:Besides known molecular cancer drivers and indicators of poor prognosis, we identified specific transcriptional changes between short- and long-term survivors of HGSC, which indicate that immune processes play a fundamental role in long-term survivors. Our computational analysis reveals an important role for the ensemble of IFN-γ signaling and the RFX transcription factors, as well as the immune cell composition of the tumor microenvironment. CONCLUSIONS:Specific immunologic requirements involving IFN-γ signaling and affected pathways seem to be relevant for long-term survival in the generally considered nonimmunogenic HGSC, necessitating further research to improve diagnostic strategies and targeted therapies.

Table S4 displays the characteristics and subtypes of the Charité cohort.

Figure S2 displays the Proportions of Molecular subtypes of LTS and STS in the Charité cohort and n the TCGA cohort.

Table S3 shows the likelihoods estimated by ConsensusOV for the subtypes of Charité STS and LTS.

Figure S10 displays differences in celltypes between the cohorts A) for LTS and B) for STS based on CIBERSORT results.

Figure S4 shows the results of the DEG Analysis in the TCGA cohort A. DEGs in the TCGA (red = up in STS, blue = up in LTS) cohort B. Volcano plot

Figure S9 shows CIBERSORT estimated celltypes per sample in the Charité cohort and in the TCGA cohort.

Abstract Background: Despite recent advances in personalized medicine, conventional chemotherapy remains a backbone in breast cancer therapy. Thus, identifying markers predicting sensitivity or resistance to individual chemotherapeutics is of great importance. Methods: In the EpiTax neoadjuvant trial, enrolling patients between 1997-2003, patients with primary breast cancers (T2 >4cm, T3/T4 and/or N2/N3) were randomized to epirubicin 90mg/m2/3W or paclitaxel 200mg/m²/3W monotherapy, with cross-over in case of inferior response. Pre-treatment snap-frozen tumor biopsies from 223 patients were analyzed by targeted NGS of a 360 gene panel. Endpoint for comparison was clinical response to the first regimen, since pCR was rare due to the large tumor sizes at inclusion. For validation purposes we performed targeted sequencing of tumor samples from a total of 478 patients included in the Gepar Trio (n=132), Quattro (n=171) and Quinto (n=175) trials, in which patients with >2cm tumors received neoadjuvant anthracycline/taxane combination regimens. Here, the primary endpoint was clinical response to combined treatment, but since these tumors were smaller than in the EpiTax-trial, pCR was included as a secondary endpoint. In addition, experimental validations were performed -by CDH1 knock-down and CRISPR/Cas9 knock-out in cell line models. Results: In samples from the EpiTax-trial, CDH1 mutations predicted an inferior response (trend across response groups; cPD, cSD, cPR and cCR) in the paclitaxel arm (p=0.01) as well as the epirubicin arm (p=0.04). The predictive value was observed within the subgroup of ER-positive cases (both for paclitaxel (p=0.005) and epirubicin (p=0.003)) but not among ER-negative tumors. The majority of CDH1 mutations (24/34=71%) were observed in lobular cancers. While lobular histology predicted resistance to paclitaxel (but not epirubicin), CDH1 mutations predicted resistance also within the subgroup of lobular cases (p=0.002), demonstrating CDH1 mutations to be an independent predictor and not only a co-variate to lobular histology. As assessing functionally linked genes, mutations in GATA3, a transcriptional regulator of CDH1, were predominantly observed in ductal cancers, and were not predictive of resistance to any compound. Yet, combining GATA3 and CDH1 mutations into a composite biomarker predicted resistance to both paclitaxel (p=0.007) and epirubicin (p=0.01), especially in ER-positive cases (p=0.002 and p=0.0004, respectively). While EMT-signatures had predictive value, this effect was largely dominated by CDH1, while other EMT-related genes had limited impact on response. In the independent validation cohort from the Gepar trials, selected with enrichment for lobular cancers (34%), CDH1 mutations were not significantly associated with resistance to therapy (p=0.19) although predicted lack of pCR (p=0.01). Combining GATA3 and CDH1 mutations predicted lack of clinical response (p=0.05) and lack of pCR (p=0.0007) respectively in this cohort. In the in vitro analyses, resistance to paclitaxel was observed in three different breast cancer cell lines upon siRNA mediated knock-down of CDH1, as well as in a CRISPR/Cas9 mediated CDH1 knock-out model, as measured by growth rate, induction of apoptosis, G2 arrest, mitochondrial respiration and tubulin stability. For anthracyclines, similar effects were observed for mitochondrial respiration. Conclusions: In conclusion, mutations in CDH1 predicted resistance to paclitaxel and epirubicin. Our data suggest that CDH1 mutations should be explored further as a predictive biomarker for potential application. Citation Format: Stian Knappskog, Reham Helwa, Sivaramakrishna Rachakonda, Liv B. Gansmo, Carsten Denkert, Lucy R. Yates, Christine Solbach, Michael Untch, Bruno V. Sinn, Anne-Sophie Litmeyer, Beyhan Ataseven, Jens Huober, David C. Wedge, Thomas Karn, Oleksii Nikolaienko, Frederik Marmé, Peter A. Fasching, Hans Petter Eikesdal, Elmar Stickeler, Christian Schem, Paul Jank, Marion van Mackelenbergh, Volkmar Müller, Baerbel Felder, Johannes Holtschmidt, Peter J. Campbell, Sibylle Loibl, Per Lonning. CDH1 mutations predict resistance to neoadjuvant taxane therapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-25-12.

Introduction To optimize RNA sequencing (RNA-seq) outcomes, we investigated preanalytical variables in malignant effusions containing metastatic breast cancer. We compared 2 processing methods-Ficoll-Hypaque density gradient enrichment and CytoLyt hemolysis-focusing on their effects on RNA quality, transcript abundance, and variant detection from cytospin slides, relative to fresh-frozen samples. Additionally, we compared read-based and Unique Molecular Identifier (UMI)-based library preparation methods. Materials and methods Thirteen malignant effusion specimens from metastatic breast cancer were processed using both the Ficoll-Hypaque and Cytolyt methods. RNA was extracted from fresh-frozen samples stored in RNA preservative and from cytospin slides fixed in Carnoy's solution. RNA quality was evaluated using RNA integrity number (RIN) and the percentage of fragments >200 bases (DV200). Sequencing was conducted with both read- and UMI-based methods. Results Purified RNA was more fragmented by the Cytolyt method (mean RIN: 3.56, DV200: 78.97%), compared to the Ficoll-Hypaque method (mean RIN: 6.29, DV200: 88.08%). Sequencing data had high concordance correlation coefficient (CCC) for measurements of gene expression, whether from Cytolyt or Ficoll-Hypaque treated samples, and whether using the UMI- or read-based sequencing methods (read-based mean CCC: 0.967 from Cytolyt versus 0.974 from Ficoll-Hypaque, UMI-based mean CCC: 0.972 from Cytolyt versus 0.977 from Ficoll-Hypaque). Conclusions Despite the increased RNA fragmentation with the Cytolyt, RNA-seq data quality was comparable across Cytolyt and Ficoll-Hypaque methods. Both clearing methods are viable for short-read RNA-seq analysis, with read and UMI-based approaches performing similarly.

Background The aim of this study was to analyse transcriptomic differences between primary and recurrent high-grade serous ovarian carcinoma (HGSOC) to identify prognostic biomarkers. Methods We analysed 19 paired primary and recurrent HGSOC samples using targeted RNA sequencing. We selected the best candidates using in silico survival and pathway analysis and validated the biomarkers using immunohistochemistry on a cohort of 44 paired samples, an additional cohort of 504 primary HGSOCs and explored their function. Results We identified 233 differential expressed genes. Twenty-three showed a significant prognostic value for PFS and OS in silico. Seven markers ( AHRR, COL5A2, FABP4, HMGCS2, ITGA5, SFRP2 and WNT9B ) were chosen for validation at the protein level. AHRR expression was higher in primary tumours ( p < 0.0001) and correlated with better patient survival ( p < 0.05). Stromal SFRP2 expression was higher in recurrent samples ( p = 0.009) and protein expression in primary tumours was associated with worse patient survival ( p = 0.022). In multivariate analysis, tumour AHRR and SFRP2 remained independent prognostic markers. In vitro studies supported the anti-tumorigenic role of AHRR and the oncogenic function of SFRP2. Conclusions Our results underline the relevance of AHRR and SFRP2 proteins in aryl-hydrocarbon receptor and Wnt-signalling, respectively, and might lead to establishing them as biomarkers in HGSOC.

A patient with gastrointestinal stroma tumor (GIST) and KIT p.V559D and BRAF p.G469A alterations was referred to our institutional molecular tumor board (MTB) to discuss therapeutic implications. The patient had been diagnosed with B-cell chronic lymphocytic leukemia (CLL) years prior to the MTB presentation. GIST had been diagnosed 1 month earlier. After structured clinical annotation of the molecular alterations and interdisciplinary discussion, we considered BRAF/KIT co-mutation unlikely in a treatment-naïve GIST. Discordant variant allele frequencies furthermore suggested a second malignancy. NGS of a CLL sample revealed the identical class 2 BRAF alteration, thus supporting admixture of CLL cells in the paragastric mass, leading to the detection of 2 alterations. Following the MTB recommendation, the patient received imatinib and had a radiographic response. Structured annotation and interdisciplinary discussion in specialized tumor boards facilitate the clinical management of complex molecular findings. Coexisting malignancies and clonal hematopoiesis warrant consideration in case of complex and uncommon molecular findings.

Therapy-induced molecular adaptation of triple-negative breast cancer is crucial for immunotherapy response and resistance. We analyze tumor biopsies from three different time points in the randomized neoadjuvant GeparNuevo trial (NCT02685059), evaluating the combination of durvalumab with chemotherapy, for longitudinal alterations of gene expression. Durvalumab induces an activation of immune and stromal gene expression as well as a reduction of proliferation-related gene expression. Immune genes are positive prognostic factors irrespective of treatment, while proliferation genes are positive prognostic factors only in the durvalumab arm. We identify stromal-related gene expression as a contributor to immunotherapy resistance and poor therapy response. The results provide evidence from clinical trial cohorts suggesting a role for stromal reorganization in therapy resistance to immunotherapy and in the generation of an immune-suppressive microenvironment, which might be relevant for future therapy approaches targeting the tumor stroma parallel to immunotherapy, such as combinations of immunotherapy with anti-angiogenic therapy.

BackgroundWe have recently constructed a DNA methylation classifier that can discriminate between pancreatic ductal adenocarcinoma (PAAD) liver metastasis and intrahepatic cholangiocarcinoma (iCCA) with high accuracy (PAAD-iCCA-Classifier). PAAD is one of the leading causes of cancer of unknown primary and diagnosis is based on exclusion of other malignancies. Therefore, our focus was to investigate whether the PAAD-iCCA-Classifier can be used to diagnose PAAD metastases from other sites.MethodsFor this scope, the anomaly detection filter of the initial classifier was expanded by 8 additional mimicker carcinomas, amounting to a total of 10 carcinomas in the negative class. We validated the updated version of the classifier on a validation set, which consisted of a biological cohort (n = 3579) and a technical one (n = 15). We then assessed the performance of the classifier on a test set, which included a positive control cohort of 16 PAAD metastases from various sites and a cohort of 124 negative control samples consisting of 96 breast cancer metastases from 18 anatomical sites and 28 carcinoma metastases to the brain.ResultsThe updated PAAD-iCCA-Classifier achieved 98.21% accuracy on the biological validation samples, and on the technical validation ones it reached 100%. The classifier also correctly identified 15/16 (93.75%) metastases of the positive control as PAAD, and on the negative control, it correctly classified 122/124 samples (98.39%) for a 97.85% overall accuracy on the test set. We used this DNA methylation dataset to explore the organotropism of PAAD metastases and observed that PAAD liver metastases are distinct from PAAD peritoneal carcinomatosis and primary PAAD, and are characterized by specific copy number alterations and hypomethylation of enhancers involved in epithelial-mesenchymal-transition.ConclusionsThe updated PAAD-iCCA-Classifier (available at https://classifier.tgc-research.de/) can accurately classify PAAD samples from various metastatic sites and it can serve as a diagnostic aid.

Abstract Background Patients with pathologic complete response (pCR) to neoadjuvant chemotherapy for invasive breast cancer (BC) have better outcomes, potentially warranting less extensive surgical and systemic treatments. Early prediction of treatment response could aid in adapting therapies. Methods On-treatment biopsies from 297 patients with invasive BC in three randomized, prospective neoadjuvant trials were assessed (GeparQuattro, GeparQuinto, GeparSixto). BC quantity, tumor-infiltrating lymphocytes (TILs), and the proliferation marker Ki-67 were compared to pre-treatment samples. The study investigated the correlation between residual cancer, changes in Ki-67 and TILs, and their impact on pathologic complete response (pCR) and disease-free survival (DFS). Results Among the 297 samples, 138 (46%) were hormone receptor-positive (HR+)/human epidermal growth factor 2-negative (HER2−), 87 (29%) were triple-negative (TNBC), and 72 (24%) were HER2+. Invasive tumor cells were found in 70% of on-treatment biopsies, with varying rates across subtypes (HR+/HER2−: 84%, TNBC: 62%, HER2+: 51%; p < 0.001). Patients with residual tumor on-treatment had an 8% pCR rate post-treatment (HR+/HER2−: 3%, TNBC: 19%, HER2+: 11%), while those without any invasive tumor had a 50% pCR rate (HR+/HER2−: 27%; TNBC: 48%, HER2+: 66%). Sensitivity for predicting residual disease was 0.81, with positive and negative predictive values of 0.92 and 0.50, respectively. Increasing TILs from baseline to on-treatment biopsy (if residual tumor was present) were linked to higher pCR likelihood in the overall cohort (OR 1.034, 95% CI 1.013–1.056 per % increase; p = 0.001) and with a longer DFS in TNBC (HR 0.980, 95% CI 0.963–0.997 per % increase; p = 0.026). Persisting or increased Ki-67 was associated with with lower pCR probability in the overall cohort (OR 0.957, 95% CI 0.928–0.986; p = 0.004) and shorter DFS in TNBC (HR 1.023, 95% CI 1.001–1.047; p = 0.04). Conclusion On-treatment biopsies can predict patients unlikely to achieve pCR post-therapy. This could facilitate therapy adjustments for TNBC or HER2 + BC. They also might offer insights into therapy resistance mechanisms. Future research should explore whether standardized or expanded sampling enhances the accuracy of on-treatment biopsy procedures. Trial registration GeparQuattro (EudraCT 2005-001546-17), GeparQuinto (EudraCT 2006-005834-19) and GeparSixto (EudraCT 2011-000553-23).

GAIN-2 trial evaluated the optimal intense dose-dense (idd) strategy for high-risk early breast cancer. This study reports the secondary endpoints pathological complete response (pCR) and overall survival (OS). Patients (n = 2887) were randomized 1:1 between idd epirubicin, nab-paclitaxel, and cyclophosphamide (iddEnPC) versus leukocyte nadir-based tailored regimen of dose-dense EC and docetaxel (dtEC-dtD) as adjuvant therapy, with neoadjuvant therapy allowed after amendment. At median follow-up of 6.5 years (overall cohort) and 5.7 years (neoadjuvant cohort, N = 593), both regimens showed comparable 5-year OS rates (iddEnPC 90.8%, dtEC-dtD 90.0%, p = 0.320). In the neoadjuvant setting, iddEnPC yielded a higher pCR rate than dtEC-dtD (51.2% vs. 42.6%, p = 0.045). Patients achieving pCR had significantly improved 5-year iDFS (88.7% vs. 70.1%, HR 0.33, p < 0.001) and OS rates (93.9% vs. 83.1%, HR 0.32, p < 0.001), but OS outcomes were comparable regardless of pCR status. Thus, iddEnPC demonstrates superior pCR rates compared to dtEC-dtD, yet with comparable survival outcomes.