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MA23 - Preclinical Models and Genetics of Malignant Pleural Mesothelioma (ID 353)
- Event: WCLC 2019
- Type: Mini Oral Session
- Track: Mesothelioma
- Presentations: 2
- Now Available
- Moderators:Ramon Palmero Sánchez, Raphael Bueno
- Coordinates: 9/10/2019, 14:30 - 16:00, Copenhagen (1980)
MA23.10 - Low Number of Mutations and Frequent Co-Deletions of CDKN2A and IFN Type I Characterize Malignant Pleural Mesothelioma (Now Available) (ID 1627)
14:30 - 16:00 | Author(s): Matthew Edwards
Malignant pleural mesothelioma (MPM) is an aggressive tumour with dismal prognosis and overall survival.
To expand our understanding of molecular background of MPM and to identify novel targetable aberrations we report an integrated genomic analysis of 121 tumour samples.Method
Fresh-frozen tumour samples (obtained from Mesobank UK,the BLF funded Mick Knighton Mesothelioma Tissue Bank, Respiratory BRU Biobank Diagnostic Archive, Royal Brompton Hospital and an Imperial College London prospective study) were analysed by whole exome sequencing (WES, n=50), SNP genotyping (n=118) and targeted capture sequencing (n=119) for 57 genes.
Sequencing libraries were prepared using Target Enrichment Systems for the Illumina Multiplexed Sequencing platform. Somatic mutations were called using VarScan after recalibration of alignments by Genome Analysis Toolkit (GATK). SNP genotyping was performed with the Human Infinium Omni-Express-Exome v1.3/1.4 Bead Chips arrays. Segmentation and copy number calling was performed using a combination of Allelic specific copy number analysis of tumour (ASCAT), DNACopy and GISTIC softwares.Result
Analysis of WES paired samples revealed a median of 31 non-synonymous somatic mutations per tumour, lower than melanoma (315 somatic mutations) or lung cancer (187.5 for squamous and 158 for adenocarcinoma), two types of tumours linked to known carcinogen exposure.
Investigation of copy number showed significant frequent deletion (q-value>0.05) of 9p21 locus where CDKN2A, MTAP and IFN type I genes are located. Deletion of CDKN2A was seen in 71/121 patients with homozygous deletion in 58/71 patients. Homozygous co-deletion of CDKN2A and IFN type I was seen in 38/58 patients, homozygous codeletion with MTAP in 49/58 patients while 37 patients showed all three as homozygous co-deleted.
Patients with CDKN2A and IFN type I deletions had worse overall survival compared with the CDKN2A wild type and patients CDKN2A only deleted patients (median 8.3 months vs 13.1 months, p-value=0.016).
Deletion of 3p21.1 locus and mutations in BAP1 were detected in 54.5% of the patients, making BAP1 the second most commonly altered gene. RB1 (13q14.2) was commonly altered mainly by deletion in 25.6% of the patients. NF2 and TP53 were affected by mutations in 19.8% and 7.4% of the patients, repectively. Patients with mutations in TP53 had worse overall survival compared with TP53 wild type patients (p-value=0.0005).Conclusion
Co-deletion of CDKN2A, MTAP and IFN type I genes could have therapeutic implications for the patients. Deletion of IFN type I may have direct implications for patient responses to immunotherapy. In the contex of multiple vulnerabilities, the presence of both CDKN2A and RB1 loss might define an important group of patients susceptible to CDK4/6i targeted therapies.
MA23.11 - Analysis of Immune Phenotype Composition in Malignant Pleural Mesothelioma (MPM) Using Bulk RNA Sequencing (Now Available) (ID 2326)
14:30 - 16:00 | Author(s): Matthew Edwards
Exploiting the immune status of the tumour microenvironment (TME) is increasingly being adopted for many cancer types. Investigation into immune phenotype composition of the TME is at present lacking for malignant pleural mesothelioma (MPM) but critically important in light of the cancer’s overall poor prognosis and lack of targeted therapy as clinical standard of care. In this study, CD8+ve tumour infiltrating lymphocyte (TIL) level has been used as a starting point to compare differences in mutational patterns, histology and survival in MPM.Method
Bulk RNA sequencing of tumour tissue from 35 MPM patients (in-house cohort) was performed. Sequencing read alignment and gene count estimation were performed using STAR (v.2.5.2b). To increase the sample size, raw data from Bueno et al. (n=211 subjects) was accessed and gene count estimations performed. In addition, the TCGA-MESO cohort (n=86 subjects) count data was included from the GDC (Genomic Data Commons) website. All count data were normalized cohort-wise using the ‘voom’ method implemented in limma package. Deconvolution of constituent immune phenotypes in the TME from the bulk RNA-sequencing data was performed by applying CIBERSORT (v.1.04) on normalized count data sets. For assessing the genetic context of observed immune phenotypes, somatic mutations were profiled using targeted sequencing of a custom gene panel for the in-house cohort. For the Bueno et al. and the TCGA-MESO cohorts, somatic mutations were either available from an overlap of whole-exome sequencing (WES) and targeted gene panel, or from WES only.Result
A total of 27 samples (3 of 35 (8.6%), 21 of 211 (9.9%) and 3 of 86 (3.5%) from the in-house, Bueno et al. and TCGA-MESO cohorts respectively) were identified with immune phenotype enriched for CD8+ve TIL. Histological subtype distribution in the CD8+ve enriched samples was seen to be almost equivalently split between Epithelioid and Biphasic subtypes (51.85% and 48.15% respectively). Interestingly, BAP1 mutation was found to be present in only 7.7% of the samples. Considering in addition the genes NF2, SETD2, SETD6, SETDB1, TP53 and LATS1/2, mutations were only found to be present in 57.7% of the samples in total. As such >40% of samples with CD8+ve TIL do not have any mutations detected in known hotspot genes for MPM. Histological subtype is not significantly different between these ‘wild-type’ and hotspot gene(s) mutated samples. Median survival for the groups was found to be 1.85 and 0.73 years respectively.Conclusion
In the present study, approximately 3-10% of MPM samples were found to have enrichment for CD8+ve TIL. Nonetheless on closer examination of the genetic context, mutation patterns emerge that warrant further investigation. For samples that have TP53 (n=3) mutation or mutations in multiple hotspot genes (BAP1, NF2, SETD2, LATS2; n=1), survival understandably is lowest (0.27 years average). This raises a number of further questions including what sustains a tumour despite high CD8+ve TIL population? And more importantly with lack of tumour mutational burden what other TME signals draw effector immune cells? Further investigations, by comparing additional immune markers with copy number changes that might be present in hotspot genes, are therefore required.
P2.03 - Biology (ID 162)
- Event: WCLC 2019
- Type: Poster Viewing in the Exhibit Hall
- Track: Biology
- Presentations: 1
- Coordinates: 9/09/2019, 10:15 - 18:15, Exhibit Hall
P2.03-10 - Comprehensive Molecular Profiling and Comparison of Common and Rarer Subtypes of Lung Cancer (ID 1870)
10:15 - 18:15 | Author(s): Matthew Edwards
Genomic profiling of tumours has become a crucial component of precision cancer medicine. In order to comprehensively characterize molecular alterations in different lung cancer subtypes, we analysed a total number of 327 samples by using Whole-Exome Sequencing (WES) and SNP genotyping arrays. Additionally, we used Targeted Capture Sequencing (TCS) for scanning a selected panel of genes (n=52) at high sequencing depth.Method
We WES (McGill University Innovation Centre in Montreal) 153 paired tumour-normal samples, with a further 174 paired tumour-normal samples undergoing TCS. Sequencing data were processed and mutations identified using BWA (v.0.7.15), Picard (v.2.17.11), GATK (v.3.7), VarScan (v.2.4.2) and VEP (v. 92) softwares. Illumina OmniExpressExome (v1.6) arrays were used for genotyping all samples and copy number alterations (CNAs) were identified using ASCAT (v.2.5.2), DNACopy (v.1.56.0) and GISTIC (v.2.0) softwares.Result
The analysed samples had a tumour content varying from 20 to 90%. The age range of patients was between 28 to 89 years. Out of 159 lung cancer patients, 89 patients had lung adenocarcinoma (LUAD), 36 squamous cell carcinoma (LUSC) and 34 lung neuroendocrine (NET), of which 22 were subclassified as lung carcinoid (LC), 6 small cell carcinoma (LSCLC), 5 large cell carcinoma (LCNEC) and 1 combined NET subtype.
TP53 appeared as the most mutated gene in LUSCs (82%), non-carcinoid NETs (58%) and LUADs (47%), but not in the LC subtype, while the chromatin remodelling gene ARID1A was altered across all subtypes (9%). Other mutated genes in LUAD were KRAS (31%), STK11 (22%), RBM10 (15%), EGFR (14%) and KEAP1 (14%); in LUSC were PTEN (26%), CDKN2A (21%), KEAP1 (21%) and NF1 (15%); in NET, non-carcinoid top mutated genes included RB1 (42%), ENPP2 (33%), ERBB4 and STK11 (17% for each), while ARID1A and ACKR3 were each present in 9.5 % of LC.
In LUADs, mutations in EGFR and KRAS appeared as mutually-exclusive (P=0.007), while gene pairs NFE2L2 - AKT1 (P=0.012) and STK11- ALK (P=0.029) were co-mutated in LUAD and LUSC, respectively. Deletions in exons 19 and 20 of EGFR correlated with longer survival time compared to patients with wild-type EGFR (P=0.058). In NET, patients with mutated RB1 showed lower survival time compared to patients with wild-type RB1 (P=0.022).
Examination of CNAs showed TERT amplifications (5p15.33 cytoband) were commonly found at high frequencies across all subtypes, especially in non-carcinoid NET (71.4%). Other recurrent CNAs included amplifications in MYC in 37% of LUAD and 40% of LUSC, and in EGFR in 33% of LUAD and 14% of LUSC. Deletions in the CDKN2A locus were seen at frequencies of 31% and 28% in LUAD and LUSC, respectively.
LC patients showed longer survival time compared to other tumours (P=0.015). COSMIC mutational signatures 18 (of unknown aetiology) and 24 (associated with exposure to aflatoxin) were exclusively found in LC.Conclusion
The results confirm that lung cancer is a group of heterogenous diseases. In addition to the known effects of EFGR mutations, possible therapeutic avenues could be suggested for TERT amplifications, for which nucleoside analogues have shown to promote cancer cell death or EZH2 inhibitors for ARID1A-mutated cancers.