Introduction: Follicular lymphoma (FL) is the most common indolent NHL. Recently, we demonstrated in limited and advanced stage FL (LSFL, ASFL), that patients with longer remissions had raised clonally expanded intratumoral CD8+ T cells (Tobin, JCO 2019). Durable remissions occur in ∼50% of LSFL patients whereas ASFL is incurable. However, little is known regarding the immunological features associated with this difference. We hypothesized HLA-I neoantigen (neoAg)-specific CD8+ T cells may play a role. Methods: The discovery cohort comprised 101 patients with diagnostic paraffin embedded tissue from the TROG99.03 LSFL clinical trial (MacManus, JCO 2018), in which stage I/II patients were randomized to involved field radiation (IFRT) only or combined modality therapy between 2000 and 2012 (PET from 2006). Contemporaneous validation cohorts were (a) AusLSFL: 60 PET staged, stage I FL patients from Australia (treatment miscellaneous), and (b) CanLSFL: 60 PET staged, stage I FL patients drawn principally from Canada (IFRT only). Digital gene expression (NanoString), targeted sequencing (330 genes), and germline HLA-typing was performed. Mutations observed by sequencing were used to predict neoAgs in TROG99.03 tissues using 8 algorithms (PVACseq) and filtered according to strong binding affinity to HLA-I over wild-type (TESLA consortium guidelines; Wells, Cell 2020). Results: CD8A gene expression was tested for prognostic significance. In TROG99.03, elevated intratumoral CD8A (by MaxStat) was associated with ∼2-fold improvement in PFS for all patients: HR 0.45 (CI: 0.77–0.26, p = 0.0053) and stage I only patients: HR 2.4 (CI: 1.2–4.6, p = 0.036). In keeping with a relationship between CD8+ T cell infiltration and tumor antigen presentation, raised expression of NLRC5 (a transcriptional HLA-I activator) was also associated with superior PFS: HR 0.48 (CI: 0.99–0.24, p = 0.024). CD8A significance was confirmed in both validation cohorts (whereas NLRC5 was validated in AusLSFL only). In keeping with recent IHC CD8 protein data (Los-de Vries, Bld Adv 2022), CD8A gene expression was raised in stage I LSFL vs. 68 ASFL patients treated with immunochemotherapy (p = 0.02). Mutational profiling was concordant with published LSFL data, with CREBBP and KMT2DA most frequent. NeoAg calling methods including functional assays for neoAg peptide binding were confirmed in a separate cohort of fresh FL tissues. 59% of TROG99.03 tissues had ≥1 neoantigens detected. Importantly, unsupervised hierarchical clustering showed 2-fold enrichment of samples with neoantigens among those with high vs. low HLA-I. Conclusions: Raised CD8A is associated with favorable prognosis in LSFL. Our data suggests disease control involves populations of expanded HLA-I neoAg-specific T cells. These findings have implications for novel immunotherapeutic strategies designed to increase the rate of durable remissions. The research was funded by: National Health and Medical Research Council, Australia; Leukaemia Foundation; Mater Foundation Keywords: Diagnostic and Prognostic Biomarkers, Indolent non-Hodgkin lymphoma, Microenvironment Conflicts of interests pertinent to the abstract. C. Keane Honoraria: Takeda, Roche, AZ, MSD, Beigene C. Cheah Consultant or advisory role: Roche, Janssen, Gilead, AstraZenecca, Lilly, TG therapeutics, Beigene, Novartis, Menarini, Daizai, Abbvie, Genmab. BMS Honoraria: BMS, Roche, Abbvie; MSD, Lilly R. Kridel Research funding: Abbvie Educational grants: Eisai M. K. Gandhi Research funding: Beigene, Janssen
Somatic mutation calling from next-generation sequencing data remains a challenge due to the difficulties of distinguishing true somatic events from artifacts arising from PCR, sequencing errors or mis-mapping. Tumor cellularity or purity, sub-clonality and copy number changes also confound the identification of true somatic events against a background of germline variants. We have developed a heuristic strategy and software (http://www.qcmg.org/bioinformatics/qsnp/) for somatic mutation calling in samples with low tumor content and we show the superior sensitivity and precision of our approach using a previously sequenced cell line, a series of tumor/normal admixtures, and 3,253 putative somatic SNVs verified on an orthogonal platform.
Abstract Background Endometrial cancer (EC) is a major gynecological cancer with increasing incidence. It comprised of four molecular subtypes with differing etiology, prognoses, and response to chemotherapy. In the future, clinical trials testing new single agents or combination therapies will be targeted to the molecular subtype most likely to respond. Pre-clinical models that faithfully represent the molecular subtypes of EC are urgently needed, we sought to develop and characterize a panel of novel EC patient-derived xenograft (PDX) models. Methods Here, we report whole exome or whole genome sequencing of 11 PDX models and the matched primary tumor. Analysis of multiple PDX lineages and passages was performed to study tumor heterogeneity across lineages and/or passages. Based on recent reports of frequent defects in the homologous recombination (HR) pathway in EC, we assessed mutational signatures and HR deficiency scores and correlated these with in vivo responses to the PARP inhibitor (PARPi) talazoparib in six PDXs representing the different molecular subtypes of EC. Results PDX models were successfully generated from all four molecular subtypes of EC and uterine carcinosarcomas, and they recapitulated morphology and the molecular landscape of primary tumors without major genomic drift. We also observed a wide range of inter-tumor and intra-tumor heterogeneity, well captured by different PDX lineages, which could lead to different treatment responses. An in vivo response to talazoparib was detected in two p53mut models consistent with stable disease, however both lacked the HR deficiency genomic signature. Conclusions EC PDX models represent the four molecular subtypes of disease and can capture intra-tumoral heterogeneity of the original primary tumor. PDXs of the p53mut molecular subtype showed sensitivity to PARPi, however, deeper and more durable responses will likely require combination of PARPi with other agents.
ABSTRACT Patients with colorectal cancer (CRC) frequently develop liver metastases during the course of their disease. A substantial proportion of them receive neoadjuvant FOLFOX (5-Fluorouracil, Oxaliplatin, Leucovorin) prior to surgery in an attempt to enable successful surgical removal of their metastases and to reduce the risk of recurrence. Yet, the majority of patients progress during treatment or recur following surgery, and molecular mechanisms that contribute to FOLFOX resistance remain poorly understood. Here, using a combination of phenotypic, transcriptomic and genomic analyses of both tumor samples derived from patients with metastatic CRC and matching patient-derived tumor organoids (PDTOs), we characterize a novel FOLFOX resistance mechanism and identify inhibitors that target this mechanism to resensitize metastatic organoids to FOLFOX. Resistant PDTOs, identified after in vitro exposure to FOLFOX, exhibited elevated expression of E2F pathway, S phase, G 2 /M and spindle assembly checkpoints (SAC) genes. Similar molecular features were detected in CRLM from patients with progressive disease while under neoadjuvant FOLFOX treatment, highlighting the relevance of this finding. FOLFOX resistant PDTOs displayed inactivating mutations of TP53 and exhibited transcriptional features of P53 pathway downregulation. We found that they accumulated in early S-phase and underwent significant DNA damage during FOLFOX exposure, thereafter arresting in G 2 /M while they repaired their DNA after FOLFOX withdrawal. In parallel, results of a large kinase inhibitor screen indicated that drugs targeting regulators of the DNA damage response, G 2 M checkpoint and SAC had cytotoxic effects on PDTOs generated from patients whose disease progressed during treatment with FOLFOX. Corroborating this finding, CHK1 and WEE1 inhibitors were found to synergize with FOLFOX and sensitize previously resistant PDTOs. Additionally, targeting the SAC master regulator MPS1 using empesertib after exposure to FOLFOX, when cells accumulate in G 2 M, was also very effective to kill FOLFOX-resistant PDTOs. Our results indicate that targeted and timely inhibition of specific cell cycle checkpoints shows great potential to improve response rates to FOLFOX in patients with metastatic CRC, for whom therapeutic alternatives remain extremely limited.
<p>Supplementary Figure S1. Representative hematoxylin and eosin stained sections from sequenced LGSC; Supplementary Figure S2. Age distribution analysis of patients from the Australian Ovarian Cancer Study. Includes patients with advanced stage, serous epithelial ovarian cancer (n = 684); Supplementary Figure S3. RAS pathway mutations in LGSC; Supplementary Figure S4. Verification of EIF1AX mutations; Supplementary Figure S5. Characterization of the AOCS2 cell line; Supplementary Figure S6. Functional effect of EIF1AX mutations and gene suppression in a LGSC cell line; Supplementary Figure S7. (a) EIF1AX and NRAS function in the mTOR and RAS/ERK signaling pathways to regulate protein translation, cell proliferation and cell survival</p>
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