Computational immunogenomic approaches to predict response to cancer immunotherapies.

Cancer immunogenomics is an emerging field that bridges genomics and immunology. The establishment of large-scale genomic collaborative efforts along with the development of new single-cell transcriptomic techniques and multi-omics approaches have enabled characterization of the mutational and transcriptional profiles of many cancer types and helped to identify clinically actionable alterations as well as predictive and prognostic biomarkers. Researchers have developed computational approaches and machine learning algorithms to accurately obtain clinically useful information from genomic and transcriptomic sequencing data from bulk tissue or single cells and explore tumours and their microenvironment. The rapid growth in sequencing and computational approaches has resulted in the unmet need to understand their true potential and limitations in enabling improvements in the management of patients with cancer who are receiving immunotherapies. In this Review, we describe the computational approaches currently available to analyse bulk tissue and single-cell sequencing data from cancer, stromal and immune cells, as well as how best to select the most appropriate tool to address various clinical questions and, ultimately, improve patient outcomes.

Genomic Landscape of Pleural Mesothelioma and Therapeutic Aftermaths.

PURPOSE OF REVIEW: In this article, we provide a comprehensive analysis of recent progress in the genetic characterisation of pleural mesothelioma, and the translation of these findings to clinical practice. RECENT FINDINGS: Advancements in sequencing technology have allowed the identification of driver mutations and improved our understanding of how these mutations may shape the mesothelioma tumour microenvironment. However, the identification of frequently mutated regions including CDKN2A, BAP1 and NF2 have, to date, not yet yielded targeted therapy options that outperform standard chemo- and immunotherapies. Similarly, the association between mutational profile and the immune microenvironment or immunotherapy response is not well characterised. Further research into the link between tumour mutational profile and response to therapy is critical for identifying targetable vulnerabilities and stratifying patients for therapy.

New Markers for Management of Mesothelioma.

In this review, we provide an update on the status of cancer biomarkers for the clinical management of pleural mesothelioma, an aggressive cancer associated with asbestos exposure. Mesothelioma can be difficult to diagnose, and response to treatment is transient, even with recently adopted immune checkpoint inhibitor (ICI) combinations. Identification of mesothelioma-specific biomarkers could facilitate early diagnosis and tailor treatment strategies. Mesothelioma is characterized by frequent loss or alteration of the tumor suppressor genes cyclin-dependent kinase inhibitor 2A (CDKN2A) and BRCA1-associated protein-1 (BAP1). Accumulating data show these genes and/or their related protein products will be valuable tissue-based biomarkers for mesothelioma. Loss of BAP1, CDKN2A, p16, or methylthioadenosine phosphorylase provide pathologists with a reliable means of differentiating between mesothelioma and reactive mesothelial cell proliferations. This can aid diagnosis in difficult cases and is requisite for the identification of the new pathological entity malignant mesothelioma in situ. However, limited progress in identifying clinically useful soluble biomarkers in this cancer type has been made, with mesothelin remaining the benchmark. To date, results from studies to identify predictive biomarkers for ICI response have been disappointing. A recent retrospective study demonstrated BAP1 loss was predictive of improved survival following combination pemetrexed- and platinum-based chemotherapy. Validation of this result could have important clinical implications. Clinical trials aimed at targeting therapy based on biomarker expression are generally in the early phase setting, with overall results being moderate. The identification of biomarkers for mesothelioma remains a key research question due to their potential to improve patient outcomes in this deadly cancer.

Immune marker expression of irradiated mesothelioma cell lines.

Background: Though immune checkpoint inhibition has recently shown encouraging clinical efficacy in mesothelioma, most patients do not respond. Combining immune checkpoint inhibition with radiotherapy presents an attractive option for improving treatment responses owing to the various immunomodulatory effects of radiation on tumors. However, the ideal dosing and scheduling of combined treatment remains elusive, as it is poorly studied in mesothelioma. The present study characterizes the dose- and time-dependent changes to expression of various immune markers and cytokines important to antitumor responses following irradiation of mesothelioma cell lines. Methods: Two murine (AB1, AE17) and two human (BYE, JU77) mesothelioma cell lines were treated with titrated gamma-radiation doses (1-8 Gy) and the expression of MHC class-I, MHC class-II and PD-L1 was measured over a series of post-irradiation timepoints (1-72 hours) by flow cytometry. Levels of cytokines IL-1α, IL-1ß, IL-6, IL-10, IL-12p70, IL-17A, IL-23, IL-27, MCP-1, IFN-ß, IFN-γ, TNF-α, and GM-CSF were measured by multiplex immunoassay in murine cell lines following 8 Gy radiation. Results: Following irradiation, a dose-dependent upregulation of MHC-I and PD-L1 was observed on three of the four cell lines studied to varying extents. For all cell lines, the increase in marker expression was most pronounced 72 hours after radiation. At this timepoint, increases in levels of cytokines IFN-ß, MCP-1 and IL-6 were observed following irradiation with 8 Gy in AB1 but not AE17, reflecting patterns in marker expression. Conclusions: Overall, this study establishes the dose- and time-dependent changes in immune marker expression of commonly studied mesothelioma cell lines following radiation and will inform future study into optimal dosing and scheduling of combined radiotherapy and immune checkpoint inhibition for mesothelioma.

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.

How Our Continuing Studies of the Pre-clinical Inbred Mouse Models of Mesothelioma Have Influenced the Development of New Therapies.

Asbestos-induced preclinical mouse models of mesothelioma produce tumors that are very similar to those that develop in humans and thus represent an ideal platform to study this rare, universally fatal tumor type. Our team and a number of other research groups have established such models as a stepping stone to new treatments, including chemotherapy, immunotherapy and other approaches that have been/are being translated into clinical trials. In some cases this work has led to changes in mesothelioma treatment practice and over the last 30 years these models and studies have led to trials which have improved the response rate in mesothelioma from less than 10% to over 50%. Mouse models have had a vital role in that improvement and will continue to play a key role in the future success of mesothelioma immunotherapy. In this review we focus only on these original inbred mouse models, the large number of preclinical studies conducted using them and their contribution to current and future clinical therapy for mesothelioma.

BAP1 Loss by Immunohistochemistry Predicts Improved Survival to First-Line Platinum and Pemetrexed Chemotherapy for Patients With Pleural Mesothelioma: A Validation Study.

INTRODUCTION: Pleural mesothelioma (PM) is an aggressive malignancy with no identified predictive biomarkers. We assessed whether tumor BAP1 status is a predictive biomarker for survival in patients receiving first-line combination platinum and pemetrexed therapy. METHODS: PM cases (n = 114) from Aalborg, Denmark, were stained for BAP1 on tissue microarrays. Demographic, clinical, and survival data were extracted from registries and medical records. Surgical cases were excluded. BAP1 status was associated with overall survival (OS) by Cox regression and Kaplan-Meier methods. Results were validated in an independent cohort from Perth, Australia (n = 234). RESULTS: BAP1 loss was found in 62% and 60.3% of all Danish and Australian samples, respectively. BAP1 loss was an independent predictor of OS in multivariate analyses corrected for histological subtype, performance status, age, sex, and treatment (hazard ratio = 2.49, p < 0.001, and 1.48, p = 0.01, respectively). First-line platinum and pemetrexed-treated patients with BAP1 loss had significantly longer median survival than those with retained BAP1 in both the Danish (20.1 versus 7.3 mo, p < 0.001) and Australian cohorts (19.6 versus 11.1 mo, p < 0.01). Survival in patients with BAP1 retained and treated with platinum and pemetrexed was similar as in those with best supportive care. There was a higher OS in patients with best supportive care with BAP1 loss, but it was significant only in the Australian cohort (16.8 versus 8.3 mo, p < 0.01). CONCLUSIONS: BAP1 is a predictive biomarker for survival after first-line combination platinum and pemetrexed chemotherapy and a potential prognostic marker in PM. BAP1 in tumor is a promising clinical tool for treatment stratification.

What's next in cancer immunotherapy? - The promise and challenges of neoantigen vaccination.

The process of tumorigenesis leaves a series of indelible genetic changes in tumor cells, that when expressed, have the potential to be tumor-specific immune targets. Neoantigen vaccines that capitalize on this potential immunogenicity have shown efficacy in preclinical models and have now entered clinical trials. Here we discuss the status of personalized neoantigen vaccines and the current major challenges to this nascent field. In particular, we focus on the types of antigens that can be targeted by vaccination and on the role that preexisting immunosuppression, and in particular T-cell exhaustion, will play in the development of effective cancer vaccines.

Analysis of early pleural fluid samples in patients with mesothelioma: A case series exploration of morphology, BAP1, and CDKN2A status with implications for the concept of mesothelioma in situ in cytology.

BACKGROUND: The concept of mesothelioma in situ has been revisited and is a new World Health Organization diagnostic entity. The definition centers on ancillary techniques used in pleural mesothelioma (PM) assessment. At the authors' institution, most PM diagnoses are made on cytologic specimens. Effusion samples obtained before definitive PM diagnosis were interrogated using BRCA1-associated protein 1 gene (BAP1), cyclin-dependent kinase inhibitor 2A gene (CDKN2A) and cytologic evaluation to assess whether early or possible in situ disease could be characterized. METHODS: All cases of PM diagnosed between January 2008 and December 2019 were identified at a tertiary referral center. Patients who had a pleural fluid sample collected 24 months before the diagnosis were selected, numbering 8 in total. The cytomorphology of each sample was reviewed; and, retrospectively, BAP1 immunohistochemistry (IHC) and CDKN2A fluorescence in situ hybridization (FISH) were performed on initial and diagnostic samples. RESULTS: The initial samples were deemed benign in 5 cases and atypical mesothelial proliferations in 3 cases. A spectrum of apparently normal to atypical cytomorphologic changes was identified. BAP1 loss was present in 6 of 8 initial cases, whereas CDKN2A homozygous deletion was identified in 1 of 7 initial cases. Either abnormality was identified in 7 of 8 initial samples. CONCLUSIONS: Detectable abnormalities of BAP1 IHC and CDKN2A FISH were present in pleural fluid specimens before the development of cytomorphologic features diagnostic of PM. This is the largest series to date describing cytology samples early in the course of PM development, thereby highlighting a possible cytological equivalent for mesothelioma in situ.

Diagnostic utility of BAP1 for malignant pleural mesothelioma in pleural fluid specimens with atypical morphology.

OBJECTIVE: To assess the utility of BRCA1-associated protein 1 (BAP1) immunohistochemistry (IHC) for the diagnosis of malignant pleural mesothelioma (MPM) in fluid samples with atypical cytology. METHODS: Pleural fluid samples with an atypical mesothelial proliferation (diagnostic categories: 'atypical' and 'suspicious') received between January 2015 and March 2018 at a tertiary referral centre were identified. Results of routine IHC testing were recorded for each case. BAP1 by IHC was performed and a final diagnosis sought from subsequent pathology specimens, medical records, or consensus clinical diagnosis. RESULTS: Of 50 cases identified, 41 were reported as atypical and 9 as suspicious. Seven (14%) demonstrated loss of BAP1 staining, 40 retained BAP1 staining, 1 had heterogeneous staining, and 2 had insufficient cells for analysis. All seven cases with BAP1 loss were diagnosed with MPM on follow-up. Of those with retained BAP1, 52.5% (21) were subsequently diagnosed with MPM, while 40% (16) had non-MPM diagnoses after a median follow-up of 24 months. Three cases were not further investigated based on patient and clinician decision. The case with heterogeneous staining was diagnosed as mesothelioma by clinical consensus. CONCLUSIONS: BAP1 IHC loss is highly specific for malignancy and has value as a rule-in test. Even in a tertiary centre with clinical interest in the cytological diagnosis of MPM this investigation was able to increase diagnostic accuracy beyond routine IHC studies. Cytological criteria remain valuable, as retained BAP1 in an atypical or suspicious mesothelial proliferation cannot exclude malignancy.

Characterization of neoantigen-specific T cells in cancer resistant to immune checkpoint therapies.

Neoantigen-specific T cells are strongly implicated as being critical for effective immune checkpoint blockade treatment (ICB) (e.g., anti-PD-1 and anti-CTLA-4) and are being targeted for vaccination-based therapies. However, ICB treatments show uneven responses between patients, and neoantigen vaccination efficiency has yet to be established. Here, we characterize neoantigen-specific CD8+ T cells in a tumor that is resistant to ICB and neoantigen vaccination. Leveraging the use of mass cytometry combined with multiplex major histocompatibility complex (MHC) class I tetramer staining, we screened and identified tumor neoantigen-specific CD8+ T cells in the Lewis Lung carcinoma (LLC) tumor model (mRiok1). We observed an expansion of mRiok1-specific CD8+ tumor-infiltrating lymphocytes (TILs) after ICB targeting PD-1 or CTLA-4 with no sign of tumor regression. The expanded neoantigen-specific CD8+ TILs remained phenotypically and functionally exhausted but displayed cytotoxic characteristics. When combining both ICB treatments, mRiok1-specific CD8+ TILs showed a stem-like phenotype and a higher capacity to produce cytokines, but tumors did not show signs of regression. Furthermore, combining both ICB treatments with neoantigen vaccination did not induce tumor regression either despite neoantigen-specific CD8+ TIL expansion. Overall, this work provides a model for studying neoantigens in an immunotherapy nonresponder model. We showed that a robust neoantigen-specific T-cell response in the LLC tumor model could fail in tumor response to ICB, which will have important implications in designing future immunotherapeutic strategies.

Increased interdigitation zone visibility on optical coherence tomography following systemic fibroblast growth factor receptor 1-3 tyrosine kinase inhibitor anticancer therapy.

BACKGROUND: To describe ocular adverse events and retinal changes during fibroblast growth factor receptor (FGFR) inhibitor (AZD4547) anticancer therapy. METHODS: This is a sub-study examining ocular adverse effects from AZD4547 therapy (single-centre, open-label, single arm phase II clinical trial). Comprehensive ocular examinations were performed 3 weekly in 24 patients. Macular optical coherence tomography (OCT) scan (300 × 250 ) was obtained at each visit and OCT parameters [central 1 mm retinal thickness (CRT) and total macular volume in central 6 mm] extracted. OCT scans were subdivided into outer (ELM to RPE) and inner (ELM to ILM) layers to compare outer and inner retinal changes. RESULTS: In 24 patients, AZD4547 was associated with eyelash elongation (n = 5, 21%) and punctate corneal erosion (n = 2, 8%). One patient developed clinically significant posterior capsular opacification during the study. OCT data were available in 23 patients, retinal changes ranged from an asymptomatic increased visibility of the interdigitation zone (IDZ) (n = 10, 43%) to multilobular subretinal fluid pockets (n = 5, 22%), which was associated with mild visual acuity loss. In a subset of patients (n = 9) with pre-AZD4547 dosing OCT baseline, CRT increased by mean (SD) of 9 (4) µm in those with IDZ change only compared with 64 (38) µm in those with other retinal changes. Retinal changes tended to be bilateral, self-limiting and improved over time without medical intervention. CONCLUSIONS: The ocular signs and symptoms did not result in dose cessation. Posteriorly, FGFR inhibition leads to outer retinal changes ranging from increased visibility of IDZ to distinct, multiple fluid pockets.

Tumour draining lymph node-generated CD8 T cells play a role in controlling lung metastases after a primary tumour is removed but not when adjuvant immunotherapy is used.

Surgical resection of cancer remains the frontline therapy for millions of patients annually, but post-operative recurrence is common, with a relapse rate of around 45% for non-small cell lung cancer. The tumour draining lymph nodes (dLN) are resected at the time of surgery for staging purposes, and this cannot be a null event for patient survival and future response to immune checkpoint blockade treatment. This project investigates cancer surgery, lymphadenectomy, onset of metastatic disease, and response to immunotherapy in a novel model that closely reflects the clinical setting. In a murine metastatic lung cancer model, primary subcutaneous tumours were resected with associated dLNs remaining intact, completely resected or partially resected. Median survival after surgery was significantly shorter with complete dLN resection at the time of surgery (49 days (95%CI)) compared to when lymph nodes remained intact (> 88 days; p < 0.05). Survival was partially restored with incomplete lymph node resection and CD8 T cell dependent. Treatment with aCTLA4 whilst effective against the primary tumour was ineffective for metastatic lung disease. Conversely, aPD-1/aCD40 treatment was effective in both the primary and metastatic disease settings and restored the detrimental effects of complete dLN resection on survival. In this pre-clinical lung metastatic disease model that closely reflects the clinical setting, we observe decreased frequency of survival after complete lymphadenectomy, which was ameliorated with partial lymph node removal or with early administration of aPD-1/aCD40 therapy. These findings have direct relevance to surgical lymph node resection and adjuvant immunotherapy in lung cancer, and perhaps other cancer, patients.

Neutrophil-to-lymphocyte ratio in malignant pleural fluid: Prognostic significance.

Predicting survival of patients with malignant pleural effusions (MPEs) is notoriously difficult. A robust prognostic marker can guide clinical decision making. The neutrophil-to-lymphocyte ratio (NLR) in blood has been shown to predict survival in many cancers. Pleural fluid bathes the malignant pleural tissues, thus the NLR of the pleural fluid may reflect more closely the local tumour environment. The objective of this study was to explore the prognostic significance of pleural effusion NLR for MPE. We analysed matched effusion and blood from 117 patients with malignant and 24 with benign pleural effusions. Those who had received recent chemotherapy or had a pleurodesis were excluded. Neutrophil and lymphocyte counts in effusions were performed by manual review of cytospin cell preparations by trained observers. Clinical data were extracted from a state-wide hospital database. We found significantly fewer neutrophils (expressed as percentage of total leukocyte count) in pleural fluid than in corresponding blood (9% vs 73%; p<0.001). The NLR was an order of magnitude lower in pleural fluid than in corresponding blood: median [IQR] = 0.20 [0.04-1.18] vs 4.9 [3.0-8.3], p<0.001. Correlation between blood and pleural fluid NLR in MPE patients was moderate (rs = 0.321, p<0.001). In univariate analysis, NLR (>0.745)) in malignant pleural fluid was predictive of poorer survival (HR = 1.698 [1.0054-2.736]; p = 0.030), and remained significant after adjustment for age, sex, presence of a chest drain, cancer type, concurrent infection and subsequent treatment with chemotherapy (HR = 1.786 [1.089-2.928]; p = 0.022). Patients with pleural fluid NLR > 0.745 had a significantly shorter median survival of 130 (95% CI 0-282) days compared to 312 (95% CI 195-428) days for pleural NLR < 0.745, p = 0.026. The NLR in blood was also predictive of poorer survival in MPE patients (HR = 1.959 [1.019-3.096]; p<0.001). The proportion of neutrophils in pleural fluid was predictive of prognosis more strongly than lymphocytes. This study provides evidence that NLR in malignant effusions can predict survival, and therefore may provide prognostic information for this cohort. This prognostic association in the fluid is driven by the presence of neutrophils.

Autoantibodies and cancer among asbestos-exposed cohorts in Western Australia.

Asbestos exposure is associated with many adverse health conditions including malignant mesothelioma and lung cancer as well as production of autoantibodies. Autoantibodies may serve as biomarkers for asbestos exposure in patients with cancer, and autoimmune dysfunction has been linked to increased rates of various cancers. The aim of this study was to examine the hypothesis that autoantibodies are more frequent in asbestos-exposed individuals with either lung cancer or mesothelioma than those without these conditions. Asbestos-exposed individuals from Western Australia who had lung cancer (n = 24), malignant mesothelioma (n = 24), or no malignancy (n = 51) were tested for antinuclear autoantibodies (ANA) using indirect immunofluorescence and specific extractable nuclear autoantibodies (ENA) employing a multiplexed addressable laser bead immunoassay. Contrary to the hypothesis, data demonstrated that individuals without malignancy were more likely to be positive for ANA compared to those with cancer. However, autoantibodies to histone and Ro-60 were found to be associated with lung cancer. These results support a possible predictive value for specific autoantibodies in the early detection of lung cancer and/or in our understanding of the role of autoimmune processes in cancer. However, further studies are needed to identify specific target antigens for the antibodies.

The Current Lung Cancer Neoantigen Landscape and Implications for Therapy.

All tumors harbor unique mutant peptides, some of which are able to elicit T-cell-mediated immune responses. These are known as neoantigens. Lung cancers bear a heavy mutational burden and hence many potential neoantigens. Neoantigens are increasingly recognized as key mediators of tumor-specific immune activation and have been identified as potential targets for personalized cancer therapies. In this review, we discuss the current data on neoantigens in lung cancer and provide an overview of the recent advances in neoantigen-based immunotherapy. Furthermore, we look ahead to highlight the major opportunities and challenges for the clinical application of neoantigen-based treatment strategies for thoracic and other malignancies.

A phase 1b clinical trial optimizing regulatory T cell depletion in combination with platinum-based chemotherapy in thoracic cancers.

Background: Single-agent cyclophosphamide can deplete regulatory T-cells (Treg). We aimed to determine optimal dosing and scheduling of oral cyclophosphamide, alongside pemetrexed-based chemotherapy, to deplete Treg in mesothelioma or non-small-cell lung cancer patients.Methods: 31 Patients received pemetrexed ± cisplatin or carboplatin on day 1 of a 21-day cycle (maximum 6 cycles). From cycle two, patients received cyclophosphamide, 50 mg/day, with intrapatient escalation to maximum 100/150 mg/day alternately. Immunological changes were examined by flow cytometry. Primary endpoint was Treg proportion of CD4+ T-cells, with doses tailored to target Treg nadir <4%.Results: Reduction in Treg proportion was observed on day 8 of all cycles, and was not augmented by cyclophosphamide. Few patients achieved the <4% Treg target. Treg proliferation reached nadir one week after chemotherapy, and peaked on day 1 of the subsequent cycle. Efficacy parameters were similar to chemotherapy alone. Seventeen percent of patients ceased cyclophosphamide due to toxicity.Conclusions: Specific Treg depletion to the degree seen with single-agent cyclophosphamide was not observed during pemetrexed-based chemotherapy. This study highlights the poor evidence basis for use of cyclophosphamide as an immunotherapeutic in combination with chemotherapy, and the importance of detailed flow cytometry studies.Trial registration: Clinical trial registration: www.anzctr.org.au identifier is ACTRN12609000260224.