Comprehensive genomic and tumour immune profiling reveals potential therapeutic targets in malignant pleural mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) has a poor overall survival with few treatment options. Whole genome sequencing (WGS) combined with the immune features of MPM offers the prospect of identifying changes that could inform future clinical trials. METHODS: We analysed somatic mutations from 229 MPM samples, including previously published data and 58 samples that had undergone WGS within this study. This was combined with RNA-seq analysis to characterize the tumour immune environment. RESULTS: The comprehensive genome analysis identified 12 driver genes, including new candidate genes. Whole genome doubling was a frequent event that correlated with shorter survival. Mutational signature analysis revealed SBS5/40 were dominant in 93% of samples, and defects in homologous recombination repair were infrequent in our cohort. The tumour immune environment contained high M2 macrophage infiltrate linked with MMP2, MMP14, TGFB1 and CCL2 expression, representing an immune suppressive environment. The expression of TGFB1 was associated with overall survival. A small subset of samples (less than 10%) had a higher proportion of CD8 T cells and a high cytolytic score, suggesting a 'hot' immune environment independent of the somatic mutations. CONCLUSIONS: We propose accounting for genomic and immune microenvironment status may influence therapeutic planning in the future.

Characterization of the immune cell landscape in CRC: Clinical implications of tumour-infiltrating leukocytes in early- and late-stage CRC.

Introduction: Colorectal cancer (CRC) is the third leading cause of cancer-related deaths globally. Tumour-infiltrating leukocytes play an important role in cancers, including CRC. We therefore sought to characterize the impact of tumour-infiltrating leukocytes on CRC prognosis. Methods: To determine whether the immune cell profile within CRC tissue could influence prognosis, we employed three computational methodologies (CIBERSORT, xCell and MCPcounter) to predict abundance of immune cell types, based on gene expression. This was done using two patient cohorts, TCGA and BC Cancer Personalized OncoGenomics (POG). Results: We observed significant differences in immune cell composition between CRC and normal adjacent colon tissue, as well as differences in based on method of analysis. Evaluation of survival based on immune cell types revealed dendritic cells as a positive prognostic marker, consistently across methodologies. Mast cells were also found to be a positive prognostic marker, but in a stage-dependent manner. Unsupervised cluster analysis demonstrated that significant differences in immune cell composition has a more pronounced effect on prognosis in early-stage CRC, compared to late-stage CRC. This analysis revealed a distinct group of individuals with early-stage CRC which have an immune infiltration signature that indicates better survival probability. Conclusions: Taken together, characterization of the immune landscape in CRC has provided a powerful tool to assess prognosis. We anticipate that further characterization of the immune landscape will facilitate use of immunotherapies in CRC.

Identification of a CD8+ T-cell response to a predicted neoantigen in malignant mesothelioma.

Neoantigens present unique and specific targets for personalized cancer immunotherapy strategies. Given the low mutational burden yet immunotherapy responsiveness of malignant mesothelioma (MM) when compared to other carcinogen-induced malignancies, identifying candidate neoantigens and T cells that recognize them has been a challenge. We used pleural effusions to gain access to MM tumor cells as well as immune cells in order to characterize the tumor-immune interface in MM. We characterized the landscape of potential neoantigens from SNVs identified in 27 MM patients and performed whole transcriptome sequencing of cell populations from 18 patient-matched pleural effusions. IFNγ ELISpot was performed to detect a CD8+ T cell responses to predicted neoantigens in one patient. We detected a median of 68 (range 7-258) predicted neoantigens across the samples. Wild-type non-binding to mutant binding predicted neoantigens increased risk of death in a model adjusting for age, sex, smoking status, histology and treatment (HR: 33.22, CI: 2.55-433.02, p = .007). Gene expression analysis indicated a dynamic immune environment within the pleural effusions. TCR clonotypes increased with predicted neoantigen burden. A strong activated CD8+ T-cell response was identified for a predicted neoantigen produced by a spontaneous mutation in the ROBO3 gene. Despite the challenges associated with the identification of bonafide neoantigens, there is growing evidence that these molecular changes can provide an actionable target for personalized therapeutics in difficult to treat cancers. Our findings support the existence of candidate neoantigens in MM despite the low mutation burden of the tumor, and may present improved treatment opportunities for patients.

Malignant cells from pleural fluids in malignant mesothelioma patients reveal novel mutations.

OBJECTIVES: Malignant mesothelioma (MM) is an asbestos related tumour affecting cells of serosal cavities. More than 70% of MM patients develop pleural effusions which contain tumour cells, representing a readily accessible source of malignant cells for genetic analysis. Although common somatic mutations and losses have been identified in solid MM tumours, the characterization of tumour cells within pleural effusions could provide novel insights but is little studied. MATERIALS AND METHODS: DNA and RNA were extracted from cells from short term cultures of 27 human MM pleural effusion samples. Whole exome and transcriptome sequencing was performed using the Ion Torrent platform. Somatic mutations were identified using VarScan2 and SomaticSniper. Copy number alterations were identified using ExomeCNV in R. Significant copy number alterations were identified across all samples using GISTIC2.0. The association between tumour intrinsic properties and survival was analyzed using the Cox proportional hazards regression model. RESULTS: We identified BAP1, CDKN2A and NF2 alterations in the cells from MM pleural effusions at a higher frequency than what is typically seen in MM tumours from surgical series. The median mutation rate was 1.09 mutations/Mb. TRAF7 and LATS2 alterations were also identified at a high frequency (66% and 59% respectively). Novel regions of interest were identified, including alterations in FGFR3, and the regions 19p13.3, 8p23.1 and 1p36.32. CONCLUSION: Short term cultures of tumour cells from MM pleural effusions offer an accessible alternative to surgical tumour biopsies in the study of MM genomics and reveal novel mutations of interest. Pleural effusion tumour cells provide an opportunity for the monitoring of tumour dynamics, treatment response and the clonal evolution of MM tumours.

The use of whole exome sequencing and murine patient derived xenografts as a method of chemosensitivity testing in sarcoma.

BACKGROUND: Soft tissue and bone sarcoma represent a broad spectrum of different pathology and genetic variance. Current chemotherapy regimens are derived from randomised trials and represent empirical treatment. Chemosensitivity testing and whole exome sequencing (WES) may offer personalized chemotherapy treatment based on genetic mutations. METHODS: A pilot, prospective, non-randomised control experimental study was conducted. Twelve patients with metastatic bone or soft tissue sarcoma that had failed first line chemotherapy treatment were enrolled for this study. Human tissue taken at surgical biopsy under general anaesthetic was divided between two arms of the trial. Subsections of the tumour were used for WES and the remainder was implanted subcutaneously in immunodeficient mice (PDX). Results of WES were analysed using a bioinformatics pipeline to identify mutations conferring susceptibility to kinase inhibitors and common chemotherapeutic agents. PDX models exhibiting successful growth underwent WES of the tumour and subsequent chemosensitivity testing. RESULTS: WES was successful in all 12 patients, with successful establishment PDX tumours models in seven patients. WES identified potential actionable therapeutics in all patients. Significant variation in predicted therapeutics was demonstrated between three PDX samples and their matched tumour samples. CONCLUSION: Analysis of WES of fresh tumour specimens via a bioinformatics pipeline may identify potential actionable chemotherapy agents. Further research into this field may lead to the development of personalized cancer therapy for sarcoma.

Whole exome sequencing of an asbestos-induced wild-type murine model of malignant mesothelioma.

BACKGROUND: Malignant mesothelioma (MM) is an aggressive cancer of the pleural and peritoneal cavities caused by exposure to asbestos. Asbestos-induced mesotheliomas in wild-type mice have been used extensively as a preclinical model because they are phenotypically identical to their human counterpart. However, it is not known if the genetic lesions in these mice tumours are similar to in the human disease, a prerequisite for any new preclinical studies that target genetic abnormalities. METHODS: We performed whole exome sequencing of fifteen asbestos-induced murine MM tumour cell lines from BALB/c, CBA and C57BL/6 mouse strains and compared the somatic mutations and copy number variations with those recurrently reported in human MM. We then catalogued and characterised the mutational landscape of the wild-type murine MM tumours. Quantitative RT-PCR was used to interrogate the expression of key MM genes of interest in the mRNA. RESULTS: Consistent with human MM tumours, we identified homozygous loss of the tumour suppressor Cdkn2a in 14/15 tumours. One tumour retained the first exon of both of the p16INK4a and p19ARF isoforms though this tumour also contained genetic amplification of Myc resulting in increased expression of the c-Myc proto-oncogene in the mRNA. There were no chromosomal losses in either the Bap1 or Nf2 regions. One tumour harbored homozygous loss of Trp53 in the DNA. Mutation rates were similar in tumours generated in the CBA and C57BL/6 strains when compared to human MM. Interestingly, all BALB/c tumour lines displayed high mutational loads, consistent with the known mutator phenotype of the host strain. The Wnt, MAPK and Jak-STAT signaling pathways were found to be the most commonly affected biological pathways. Mutations and copy number deletions also occurred in the Hedgehog and Hippo pathways. CONCLUSIONS: These data suggest that in the wild-type murine model asbestos causes mesotheliomas in a similar way to in human MM. This further supports the notion that the murine model of MM represents a genuine homologue of the human disease, something uncommon in cancer, and is thus a valuable tool to provide insight into MM tumour development and to aide the search for novel therapeutic strategies.

Strong spontaneous tumor neoantigen responses induced by a natural human carcinogen.

A key to improving cancer immunotherapy will be the identification of tumor-specific "neoantigens" that arise from mutations and augment the resultant host immune response. In this study we identified single nucleotide variants (SNVs) by RNA sequencing of asbestos-induced murine mesothelioma cell lines AB1 and AB1-HA. Using the NetMHCpan 2.8 algorithm, the theoretical binding affinity of predicted peptides arising from high-confidence, exonic, non-synonymous SNVs was determined for the BALB/c strain. The immunoreactivity to 20 candidate mutation-carrying peptides of increased affinity and the corresponding wild-type peptides was determined using interferon-γ ELISPOT assays and lymphoid organs of non-manipulated tumor-bearing mice. A strong endogenous immune response was demonstrated to one of the candidate neoantigens, Uqcrc2; this response was detected in the draining lymph node and spleen. Antigen reactive cells were not detected in non-tumor bearing mice. The magnitude of the response to the Uqcrc2 neoantigen was similar to that of the strong influenza hemagglutinin antigen, a model tumor neoantigen. This work confirms that the approach of RNAseq plus peptide prediction and ELISPOT testing is sufficient to identify natural tumor neoantigens.

Absence of germline mutations in BAP1 in sporadic cases of malignant mesothelioma.

Malignant mesothelioma (MM) is a uniformly fatal tumour caused predominantly by exposure to asbestos. It is not known why some exposed individuals get mesothelioma and others do not. There is some epidemiological evidence of host susceptibility. BAP1 gene somatic mutations and allelic loss are common in mesothelioma and recently a BAP1 cancer syndrome was described in which affected individuals and families had an increased risk of cancer of multiple types, including MM. To determine if BAP1 mutations could underlie any of the sporadic mesothelioma cases in our cohort of patients, we performed targeted deep sequencing of the BAP1 exome on the IonTorrent Proton sequencer in 115 unrelated MM cases. No exonic germline BAP1 mutations of known functional significance were observed, further supporting the notion that sporadic germline BAP1 mutations are not relevant to the genetic susceptibility of MM.

Comparison of the diagnostic accuracy of the MSLN gene products, mesothelin and megakaryocyte potentiating factor, as biomarkers for mesothelioma in pleural effusions and serum.

The MSLN gene products, soluble mesothelin and megakaryocyte potentiating factor (MPF), are being investigated as biomarkers for the asbestos-related cancer malignant mesothelioma (MM). Pleural fluid biomarkers of MM can be elevated when serum levels remain normal. The aim of this study was to determine if this was true for MPF and to compare levels of mesothelin. Biomarker concentrations were compared in 66 MM patients, 39 patients with other malignancies, 37 with benign disease, 18 asbestos-exposed healthy individuals, and 53 patients with chronic kidney disease. In pleural effusions, MPF and soluble mesothelin concentrations were both significantly elevated in MM patients relative to controls. No significant difference between the area under the receiver operator curve (AUC) for MPF (0.945 ± 0.02) and mesothelin (0.928 ± 0.03) when distinguishing MM from all other causes of effusion was observed. MPF and mesothelin serum concentrations were highly correlated and of equivalent diagnostic accuracy with AUCs of 0.813 ± 0.04 and 0.829 ± 0.03, respectively. Serum levels of both markers increased with decreasing kidney function. In conclusion, MPF is elevated in the pleural effusions of MM patients similar to that of mesothelin. Mesothelin and MPF convey equivalent diagnostic information for distinguishing MM from other diseases in pleural effusions as well as serum.