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Adult granulosa cell tumors (AGCTs) harbor a somatic FOXL2 c.402C>G mutation in ~95% of cases and are mainly surgically removed due to limited systemic treatment effect. In this study, potentially targetable genomic alterations in AGCTs were investigated by whole genome sequencing on 46 tumor samples and matched normal DNA. Copy number variant (CNV) analysis confirmed gain of chromosome 12 and 14, and loss of 22. Pathogenic TP53 mutations were identified in three patients with highest tumor mutational burden and mitotic activity, defining a high-grade AGCT subgroup. Within-patient tumor comparisons showed 29–80% unique somatic mutations per sample, suggesting tumor heterogeneity. A higher mutational burden was found in recurrent tumors, as compared to primary AGCTs. FOXL2-wildtype AGCTs harbored DICER1, TERT(C228T) and TP53 mutations and similar CNV profiles as FOXL2-mutant tumors. Our study confirms that absence of the FOXL2 c.402C>G mutation does not exclude AGCT diagnosis. The lack of overlapping variants in targetable cancer genes indicates the need for personalized treatment for AGCT patients.

Somatic mutations play a major role in tumour initiation and progression. The mutation status of a tumour may predict prognosis and guide targeted therapies. The majority of techniques to study oncogenic mutations require high quality and quantity DNA or are analytically challenging. Mass-spectrometry based mutation analysis however is a relatively simple and high-throughput method suitable for formalin-fixed, paraffin-embedded (FFPE) tumour material. Targeted gene panels using this technique have been developed for several types of cancer. These current cancer hotspot panels are not focussed on the genes that are most relevant in gynaecological cancers. In this study, we report the design and validation of a novel, mass-spectrometry based panel specifically for gynaecological malignancies and present the frequencies of detected mutations. Using frequency data from the online Catalogue of Somatic Mutations in Cancer, we selected 171 somatic hotspot mutations in the 13 most important genes for gynaecological cancers, being BRAF, CDKN2A, CTNNB1, FBXW7, FGFR2, FGFR3, FOXL2, HRAS, KRAS, NRAS, PIK3CA, PPP2R1A and PTEN. A total of 546 tumours (205 cervical, 227 endometrial, 89 ovarian, and 25 vulvar carcinomas) were used to test and validate our panel, and to study the prevalence and spectrum of somatic mutations in these types of cancer. The results were validated by testing duplicate samples and by allele-specific qPCR. The panel presented here using mass-spectrometry shows to be reproducible and high-throughput, and is usefull in FFPE material of low quality and quantity. It provides new possibilities for studying large numbers of gynaecological tumour samples in daily practice, and could be useful in guided therapy selection.

This study aimed to investigate whether molecular analysis can be used to refine risk assessment, direct adjuvant therapy, and identify actionable alterations in high-risk endometrial cancer. Trans PORTEC, an international consortium related to the PORTEC3 trial, was established for translational research in high-risk endometrial cancer. In this explorative study, routine molecular analyses were used to detect prognostic subgroups: p53 immunohistochemistry, microsatellite instability and POLE proofreading mutation. Furthermore, DNA was analyzed for hotspot mutations in 13 additional genes ( BRAF , CDKNA2 , CTNNB1 , FBXW7 , FGFR2 , FGFR3 , FOXL2 , HRAS , KRAS , NRAS , PIK3CA , PPP2R1A , and PTEN ) and protein expression of ER, PR, PTEN, and ARID1a was analyzed. Rates of distant metastasis, recurrence-free, and overall survival were calculated using the Kaplan–Meier method and log-rank test. In total, samples of 116 high-risk endometrial cancer patients were included: 86 endometrioid; 12 serous; and 18 clear cell. For endometrioid, serous, and clear cell cancers, 5-year recurrence-free survival rates were 68%, 27%, and 50% ( P =0.014) and distant metastasis rates 23%, 64%, and 50% ( P =0.001), respectively. Four prognostic subgroups were identified: (1) a group of p53-mutant tumors; (2) microsatellite instable tumors; (3) POLE proofreading-mutant tumors; and (4) a group with no specific molecular profile (NSMP). In group 3 ( POLE- mutant; n =14) and group 2 (microsatellite instable; n =19) patients, no distant metastasis occurred, compared with 50% distant metastasis rate in group 1 (p53-mutant; n =36) and 39% in group 4 (NSMP; P <0.001). Five-year recurrence-free survival was 93% and 95% for group 3 ( POLE- mutant) and group 2 (microsatellite instable) vs 42% (group 1, p53-mutant) and 52% (group 4, NSMP; P <0.001). Targetable FBXW7 and FGFR2 mutations (6%), alterations in the PI3K-AKT pathway (60%) and hormone receptor positivity (45%) were frequently found. In conclusion, molecular analysis of high-risk endometrial cancer identifies four distinct prognostic subgroups, with potential therapeutic implications. High frequencies of targetable alterations were identified and may serve as targets for individualized treatment.

Central to tumor evolution is the generation of genetic diversity. However, the extent and patterns by which de novo karyotype alterations emerge and propagate within human tumors are not well understood, especially at single-cell resolution. Here, we present 3D Live-Seq—a protocol that integrates live-cell imaging of tumor organoid outgrowth and whole-genome sequencing of each imaged cell to reconstruct evolving tumor cell karyotypes across consecutive cell generations. Using patient-derived colorectal cancer organoids and fresh tumor biopsies, we demonstrate that karyotype alterations of varying complexity are prevalent and can arise within a few cell generations. Sub-chromosomal acentric fragments were prone to replication and collective missegregation across consecutive cell divisions. In contrast, gross genome-wide karyotype alterations were generated in a single erroneous cell division, providing support that aneuploid tumor genomes can evolve via punctuated evolution. Mapping the temporal dynamics and patterns of karyotype diversification in cancer enables reconstructions of evolutionary paths to malignant fitness. Analysis of live-cell imaging and single-cell genome sequencing data of colorectal cancer organoids identifies temporal dynamics of sub-chromosomal copy-number amplifications.

MYC translocation occurs in 8–14% of diffuse large B-cell lymphoma (DLBCL), and may concur with BCL2 and/or BCL6 translocation, known as double-hit (DH) or triple-hit (TH). DLBCL- MYC / BCL2 -DH/TH are largely germinal centre B-cell like subtype, but show variable clinical outcome, with IG :: MYC fusion significantly associated with inferior survival. While DLBCL- MYC / BCL6 -DH are variable in their cell-of-origin subtypes and clinical outcome. Intriguingly, only 40-50% of DLBCL with MYC translocation show high MYC protein expression (>70%). We studied 186 DLBCLs with MYC translocation including 32 MYC/BCL2/BCL6 -TH , 75 MYC/BCL2 -DH and 26 MYC/BCL6 -DH. FISH revealed a MYC / BCL6 fusion in 59% of DLBCL- MYC/BCL2/BCL6 -TH and 27% of DLBCL- MYC / BCL6 -DH. Targeted NGS showed a similar mutation profile and LymphGen genetic subtype between DLBCL- MYC/BCL2/BCL6 -TH and DLBCL- MYC/BCL2 -DH, but variable LymphGen subtypes among DLBCL- MYC / BCL6 -DH. MYC protein expression is uniformly high in DLBCL with IG::MYC , but variable in those with non- IG::MYC including MYC / BCL6 -fusion. Translocation breakpoint analyses of 8 cases by TLC-based NGS showed no obvious genomic configuration that enables MYC transactivation in 3 of the 4 cases with non- IG: : MYC , while a typical promoter substitution or IGH super enhancer juxtaposition in the remaining cases. The findings potentially explain variable MYC expression in DLBCL with MYC translocation, and also bear practical implications in its routine assessment.

Studies in early-stage, predominantly low- and intermediate-risk endometrial cancer have demonstrated that L1 cell adhesion molecule (L1CAM) overexpression identifies patients at increased risk of recurrence, yet its prognostic significance in high-risk endometrial cancer is unclear. To evaluate this, its frequency, and the relationship of L1CAM with the established endometrial cancer biomarker p53, we analyzed the expression of both markers by immunohistochemistry in a pilot series of 116 endometrial cancers (86 endometrioid, 30 non-endometrioid subtype) with high-risk features (such as high tumor grade and deep myometrial invasion) and correlated results with clinical outcome. We used The Cancer Genome Atlas (TCGA) endometrial cancer series to validate our findings. Using the previously reported cutoff of 10% positive staining, 51/116 (44%) tumors were classified as L1CAM-positive, with no significant association between L1CAM positivity and the rate of distant metastasis ( P =0.195). However, increasing the threshold for L1CAM positivity to 50% resulted in a reduction of the frequency of L1CAM-positive tumors to 24% (28/116), and a significant association with the rate of distant metastasis ( P =0.018). L1CAM expression was strongly associated with mutant p53 in the high-risk and TCGA series ( P <0.001), although a substantial fraction (36% of endometrioid, 10% of non-endometrioid morphology) of p53-mutant endometrial cancers displayed <10% L1CAM positivity. Moreover, 30% of p53-wild-type non-endometrioid endometrial cancers demonstrated diffuse L1CAM staining, suggesting p53-independent mechanisms of L1CAM overexpression. In conclusion, the previously proposed threshold for L1CAM positivity of >10% does not predict prognosis in high-risk endometrial cancer, whereas an alternative threshold (>50%) does. L1CAM expression is strongly, but not universally, associated with mutant p53, and may be strong enough for clinical implementation as prognostic marker in combination with p53. The high frequency of L1CAM expression in high-risk endometrial cancers suggests that it may also be a promising therapeutic target in this tumor subset.

Tropomyosin receptor kinase (TRK) inhibitors have been approved for metastatic solid tumors harboring NTRK fusions, but the detection of NTRK fusions is challenging. International guidelines recommend pan-TRK immunohistochemistry (IHC) screening followed by next generation sequencing (NGS) in tumor types with low prevalence of NTRK fusions, including metastatic colorectal cancer (mCRC). RNA-based NGS is preferred, but is expensive, time-consuming, and extracting good-quality RNA from FFPE tissue is challenging. Alternatives in daily clinical practice are warranted. We assessed the diagnostic performance of RNA-NGS, FFPE-targeted locus capture (FFPE-TLC), fluorescence in situ hybridization (FISH), and the 5′/3′ imbalance quantitative RT-PCR (qRT-PCR) after IHC screening in 268 patients with microsatellite-instability-high mCRC, the subgroup in which NTRK fusions are most prevalent (1–5%). A consensus result was determined after review of all assay results. In 16 IHC positive tumors, 10 NTRK fusions were detected. In 33 IHC negative samples, no additional transcribed NTRK fusions were found, underscoring the high sensitivity of IHC. Sensitivity of RNA-NGS, FFPE-TLC, FISH, and qRT-PCR was 90%, 90%, 78%, and 100%, respectively. Specificity was 100% for all assays. Robustness, defined as the percentage of samples that provided an interpretable result in the first run, was 100% for FFPE-TLC, yet more limited for RNA-NGS (85%), FISH (70%), and qRT-PCR (70%). Overall, we do not recommend FISH for the detection of NTRK fusions in mCRC due to its low sensitivity and limited robustness. We conclude that RNA-NGS, FFPE-TLC, and qRT-PCR are appropriate assays for NTRK fusion detection, after enrichment with pan-TRK IHC, in routine clinical practice.

Introduction/BackgroundThe endometrial cancer (EC) molecular class is predictive for response to chemotherapy. Little is known about its’ predictive value for response to radiotherapy. We investigated benefit of adjuvant vaginal brachytherapy (VBT) and external beam radiotherapy (EBRT) across the four molecular classes.MethodologyParticipants of the randomized PORTEC-1 (n=714) and PORTEC-2 (n=427) trials were eligible if their EC were molecularly profiled according to the WHO 2020 classification. PORTEC-1 included intermediate risk EC and compared EBRT to no adjuvant treatment. PORTEC-2 included high-intermediate risk EC and compared VBT to EBRT. Locoregional recurrence-free survival (LRFS) was estimated and compared using Kaplan-Meier’s methodology and log-rank tests. Correction for confounding by predefined clinicopathological factors was done using Cox proportional hazards models.Abstract 2022-RA-450-ESGO Table 1Multivariable analysis of predictors of locoreginal recurrence-free survivalAbstract 2022-RA-450-ESGO Figure 1Results881 patients with a median follow-up of 11.3 years were included. Patient and tumour characteristics are presented in table 1. EC were classified as POLE-mutant (POLEmut, 7.3%) mismatch-repair deficient (MMRd, 28.0%), p53-abnormal (p53abn, 9.1%) and non-specific molecular profile (NSMP, 55.6%). Overall, adjuvant radiotherapy significantly improved LRFS (figure 1A). In POLEmut EC no LR were observed (figure 1B). In MMRd EC, VBT and EBRT did not significantly improve LRFS (figure 1C). In p53abn EC, EBRT but not VBT significantly improved LRFS (figure 1D). In NSMP EC, both EBRT and VBT significantly improved LRFS. Adjuvant radiotherapy and molecular class retained significant impact on LRFS after correction for clinicopathological risk factors (table 1).ConclusionBenefit of adjuvant radiotherapy in early-stage endometrioid EC differs between EC molecular classes. Omitting radiotherapy seems safe in early-stage POLEmut EC. Benefit of radiotherapy in MMRd EC is uncertain as only a small, non-significant reduction in LR was observed. In p53abn EC, EBRT significantly improved LRFS, in contrast to brachytherapy and no adjuvant treatment. NSMP EC seem to have a clear benefit of radiotherapy; VBT seems sufficient for locoregional tumour control.

Adult granulosa cell tumors (AGCTs) arise from the estrogen-producing granulosa cells. Treatment of recurrence remains a clinical challenge, as systemic anti-hormonal treatment or chemotherapy is only effective in selected patients. We established a method to rapidly screen for drug responses in vitro using direct patient-derived cell lines in order to optimize treatment selection. The response to 11 monotherapies and 12 combination therapies, including chemotherapeutic, anti-hormonal, and targeted agents, were tested in 12 AGCT-patient-derived cell lines and an AGCT cell line (KGN). Drug screens were performed within 3 weeks after tissue collection by measurement of cell viability 72 h after drug application. The potential synergy of drug combinations was assessed. The human maximum drug plasma concentration (Cmax) and steady state (Css) thresholds obtained from available phase I/II clinical trials were used to predict potential toxicity in patients. Patient-derived AGCT cell lines demonstrated resistance to all monotherapies. All cell lines showed synergistic growth inhibition by combination treatment with carboplatin, paclitaxel, and alpelisib at a concentration needed to obtain 50% cell death (IC50) that are below the maximum achievable concentration in patients (IC50 < Cmax). We show that AGCT cell lines can be rapidly established and used for patient-specific in vitro drug testing, which may guide treatment decisions. Combination treatment with carboplatin, paclitaxel, and alpelisib was consistently effective in AGCT cell lines and should be further studied as a potential effective combination for AGCT treatment in patients.