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Robert C Rintoul
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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)
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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): Robert C Rintoul
- Abstract
- Presentation
Background
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.
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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): Robert C Rintoul
- Abstract
- Presentation
Background
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.
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P1.04 - Immuno-oncology (ID 164)
- Event: WCLC 2019
- Type: Poster Viewing in the Exhibit Hall
- Track: Immuno-oncology
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2019, 09:45 - 18:00, Exhibit Hall
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P1.04-22 - Potential DNA Repair Biomarker for Response to Immunotherapy of Lung Cancer Patients (ID 2600)
09:45 - 18:00 | Author(s): Robert C Rintoul
- Abstract
Background
Because only a fraction of cancer patients responds to immunotherapy, there is an urgent need for methods to predict responders, in addition to the current methods of PD-L1 expression, tumor mutation burden, and mismatch repair deficiency (MSI-H biomarker). With the goal of harnessing DNA repair to fight cancer, we have developed personalized DNA repair blood tests that measure enzymatic activity of OGG1 (8-oxoguanine DNA-glycosylase), MPG (methylpurine DNA-glycosylase) and APE1 (apurinic/apyrimidinic endonuclease-1), which repair primarily oxidative and methylation DNA damage. The three are then combined to yield a personalized DNA repair score (Cancer Prevention Res. 7, 398-406:2014; J. Natl. Cancer Inst. 104, 1765-1769:2012). The question we posed was whether there is any correlation between the DNA repair score and systemic effects on gene expression.
Method
The study included 121 non-small cell lung cancer cases and 92 controls. The DNA repair score was determined in PBMC, and gene expression was measured using RNAseq in nasal epithelial cells from all subjects. Gene expression was also measured in bronchial cells from 37 patients. Analysis using DESeq2 software was performed separately on the different tissues (nasal/bronchial) and disease state (cases/controls), with experimental batch, age, gender, smoking status (never, former and current smokers) and cancer histology (in cases) as adjusting factors. Gene Set Enrichment Analysis (GSEA) was used in order to identify whether there is an over-representation of genes belonging to specific pathways annotated by Gene Ontology.
Result
With a False Discovery Rate threshold of 0.01, we could find very few genes whose expression correlated with the DNA repair score. However, using GSEA we found that the expression of 185 immune system-related pathways exhibited negative correlation with the DNA repair score, each with q-value<0.001. This correlation was observed in lung cancer patients, but not in control subjects. The dramatic enrichment in the immune response pathways was by far the most pronounced effect, and it was robust against sub-sampling, indicating that it is not a sampling bias.
Conclusion
Low DNA repair score correlates with broad up-regulation of immune-response pathways in lung cancer patients, but not controls. Low DNA repair might cause more mutations, and increases neoantigens, contributing to the activation of the immune system, similar to tumors with a mismatch-repair deficiency. Since only about 1% of lung cancer cases exhibits a mismatch-repair deficiency, we suggest that a low DNA repair score might serve as a biomarker for response to immunotherapy of these patients, pending additional studies.
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P1.11 - Screening and Early Detection (ID 177)
- Event: WCLC 2019
- Type: Poster Viewing in the Exhibit Hall
- Track: Screening and Early Detection
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2019, 09:45 - 18:00, Exhibit Hall
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P1.11-04 - Utilizing DNA Repair Activity Biomarkers for Lung Cancer Risk Assessment and Early Detection (ID 2682)
09:45 - 18:00 | Author(s): Robert C Rintoul
- Abstract
Background
Early detection dramatically increases the 5-year survival rates of lung cancer patients. Yet, current early detection screening criteria miss most cases, highlighting the need to improve risk assessment markers. Because of the crucial role of DNA repair in eliminating DNA damage and mutations, and preventing cancer, we examined the usefulness of a panel of DNA repair blood tests for lung cancer risk assessment. The panel consisted of the DNA repair enzyme activities of OGG1 (8-oxoguanine DNA glycosylase), MPG (methylpurine DNA glycosylase) and APE1 (apurinic/apyrimidinic endonuclease 1), which act primarily on oxidative and methylation DNA damage.
Method
A case-control study with 150 non-small cell lung cancer cases and 143 controls, blinded to the experimentalists, was conducted in the UK population, with tests and analyses performed in Israel. For each study participant, DNA repair enzymes activities were measured in protein extracts prepared from peripheral blood mononuclear cells, and used to calculate an integrated personalized DNA repair score. Personal characteristic of cases and controls were compared using unpaired t-tests for continuous variables and chi-squared tests for categorical variables. Associations were calculated using logistic regression.
Result
The mean DNA repair score was lower in lung cancer cases than in control subjects, regardless of the disease stage. Individuals at the lowest tertile of DNA repair score had increased risk of lung cancer compared to individuals at the highest tertile, with OR=7.2, 95%CI 3.0-17.5; P<0.001, which was smoking-independent. ROC analysis yielded an AUC of 0.89. Integrating the DNA repair score into a commonly used risk model based on age and smoking status, led to 4-5-fold increase or decrease in the estimated 5-year risk to develop lung cancer, for low or high DNA repair score, respectively, compared to the risk model without the DNA repair score.
Conclusion
The results of this study performed in the UK population confirm and extend a similar independent study performed in the Israeli population (Sevilya et al 2014 Cancer Prevention Res. 7, 398-406; Leitner-Dagan et al 2012 J. Natl. Cancer Inst. 104, 1765-1769). It validates low DNA repair score as a mechanism-based lung cancer risk biomarker, and can significantly improve current risk models. The DNA repair score can assist in identifying high-risk individuals for early detection by CT scanning, and can perhaps aid in predicting potential malignancy of indeterminate pulmonary nodules.
