Immune microenvironment composition in non-small cell lung cancer and its association with survival

Objectives In non-small cell lung cancer (NSCLC), the immune system and possibly its composition affect survival. In thisin silicostudy, the immune infiltrate composition in NSCLC patients was evaluated. Methods Gene expression data of tumors from early NSCLC patients were obtained from Gene Expression Omnibus (GEO). With CIBERSORT, 22 immune cell fractions were estimated. Results The immune infiltrate of 1430 pretreatment NSCLC patients contained mostly plasma cells, macrophages and CD8 T cells. Higher fractions of resting mast and CD4 T-helper cells were associated with longer overall survival (OS) (HR = 0.95,P <0.01; HR = 0.98, = 0.04, respectively) and higher fractions of M2 macrophages and active dendritic cells with shorter survival (HR = 1.02,P = 0.03; HR = 1.03,P = 0.05, respectively). Adenocarcinoma patients with survival data (n = 587) showed higher fractions of resting mast and resting CD4 T cells, and lower M0 macrophages than squamous cell carcinoma (n = 254), which were associated with OS (HR = 0.95,P = 0.04; HR = 0.97,P = 0.01; HR = 1.03,P = 0.01, respectively). Fractions of memory B cells, naive CD4 T cells and neutrophils had different associations with survival depending on the subtype. Smokers had had higher fractions of regulatory T cell, follicular helper T cell, neutrophil and M2 macrophage, which were associated with shorter survival (HR = 1.3,P <0.01; HR = 1.13,P = 0.02; HR = 1.09,P = 0.03; HR = 1.04,P = 0.02, respectively). Conclusion Pretreatment differences in immune cell composition in NSCLC are associated with survival and depend on smoking status and histological subtype. Smokers' immune composition is associated with lower survival

Genome-wide association study of cardiovascular disease in testicular cancer patients treated with platinum-based chemotherapy

Genetic variation may mediate the increased risk of cardiovascular disease (CVD) in chemotherapy-treated testicular cancer (TC) patients compared to the general population. Involved single nucleotide polymorphisms (SNPs) might differ from known CVD-associated SNPs in the general population. We performed an explorative genome-wide association study (GWAS) in TC patients. TC patients treated with platinum-based chemotherapy between 1977 and 2011, age ≤55 years at diagnosis, and ≥3 years relapse-free follow-up were genotyped. Association between SNPs and CVD occurrence during treatment or follow-up was analyzed. Data-driven Expression Prioritized Integration for Complex Trait (DEPICT) provided insight into enriched gene sets, i.e., biological themes. During a median follow-up of 11 years (range 3-37), CVD occurred in 53 (14%) of 375 genotyped patients. Based on 179 SNPs associated at p ≤ 0.001, 141 independent genomic loci associated with CVD occurrence. Subsequent, DEPICT found ten biological themes, with the RAC2/RAC3 network (linked to endothelial activation) as the most prominent theme. Biology of this network was illustrated in a TC cohort (n = 60) by increased circulating endothelial cells during chemotherapy. In conclusion, the ten observed biological themes highlight possible pathways involved in CVD in chemotherapy-treated TC patients. Insight in the genetic susceptibility to CVD in TC patients can aid future intervention strategies

Transcriptional Activity and Stability of CD39+CD103+CD8+T Cells in Human High-Grade Endometrial Cancer

Tumor-infiltrating CD8+ T cells (TIL) are of the utmost importance in anti-tumor immunity. CD103 defines tumor-resident memory T cells (T-RM cells) associated with improved survival and response to immune checkpoint blockade (ICB) across human tumors. Co-expression of CD39 and CD103 marks tumor-specific T-RM with enhanced cytolytic potential, suggesting that CD39+CD103+ T-RM could be a suitable biomarker for immunotherapy. However, little is known about the transcriptional activity of T-RM cells in situ. We analyzed CD39+CD103+ T-RM cells sorted from human high-grade endometrial cancers (n = 3) using mRNA sequencing. Cells remained untreated or were incubated with PMA/ionomycin (activation), actinomycin D (a platinum-like chemotherapeutic that inhibits transcription), or a combination of the two. Resting CD39+CD103+ T-RM cells were transcriptionally active and expressed a characteristic T-RM signature. Activated CD39+CD103+ T-RM cells differentially expressed PLEK, TWNK, and FOS, and cytokine genes IFNG, TNF, IL2, CSF2 (GM-CSF), and IL21. Findings were confirmed using qPCR and cytokine production was validated by flow cytometry of cytotoxic TIL. We studied transcript stability and found that PMA-responsive genes and mitochondrial genes were particularly stable. In conclusion, CD39+CD103+ T-RM cells are transcriptionally active T-RM cells with a polyfunctional, reactivation-responsive repertoire. Secondly, we hypothesize that differential regulation of transcript stability potentiates rapid responses upon T-RM reactivation in tumors

An mRNA expression-based signature for oncogene-induced replication-stress

Oncogene-induced replication stress characterizes many aggressive cancers. Several treatments are being developed that target replication stress, however, identification of tumors with high levels of replication stress remains challenging. We describe a gene expression signature of oncogene-induced replication stress. A panel of triple-negative breast cancer (TNBC) and non-transformed cell lines were engineered to overexpress CDC25A, CCNE1 or MYC, which resulted in slower replication kinetics. RNA sequencing analysis revealed a set of 52 commonly upregulated genes. In parallel, mRNA expression analysis of patient-derived tumor samples (TCGA, n=10,592) also revealed differential gene expression in tumors with amplification of oncogenes that trigger replication stress (CDC25A, CCNE1, MYC, CCND1, MYB, MOS, KRAS, ERBB2, and E2F1). Upon integration, we identified a six-gene signature of oncogene-induced replication stress (NAT10, DDX27, ZNF48, C8ORF33, MOCS3, and MPP6). Immunohistochemical analysis of NAT10 in breast cancer samples (n=330) showed strong correlation with expression of phospho-RPA (R=0.451, p=1.82x10(-20)) and γH2AX (R=0.304, p=2.95x10(-9)). Finally, we applied our oncogene-induced replication stress signature to patient samples from TCGA (n=8,862) and GEO (n=13,912) to define the levels of replication stress across 27 tumor subtypes, identifying diffuse large B cell lymphoma, ovarian cancer, TNBC and colorectal carcinoma as cancer subtypes with high levels of oncogene-induced replication stress

Reconsider radiation exposure from imaging during immune checkpoint inhibitor trials to reduce risk of secondary cancers in long-term survivors?

Immune checkpoint inhibitors (ICI) have improved outcomes for patients with advanced cancers, and results in increasing numbers of long-term survivors. For registration studies, progression-free survival and disease-free survival often serve as primary endpoints. This requires repeated computed tomography (CT) scans for tumour imaging which might lead to major radiation exposure. To determine this, all immune checkpoint inhibitors trials that led to FDA approval were retrieved up to July 29, 2019. From the available protocols, imaging modalities and schedules used in each trial were identified. The anticipated cumulative number of scans made after 1, 3, 5, and 10 years study participation were calculated. The percentage of lifetime attributable cancer risk was calculated using the Biological Effects of Ionizing Radiation VII report. Fifty-one trials were identified, from which 39 protocols were retrieved. Four were adjuvant trials. All protocols required repeated chest-abdomen imaging and specified CT scans as preferred imaging modality. Median calculated cumulative numbers of chest-abdomen CT scans after 1, 3, 5, and 10 years study participation were 7, 16, 24 and 46, respectively. For ages 20-70 years at study entry, the average lifetime attributable cancer risk after 1 year of study participation ranged from 1.11 to 0.40% for men and from 1.87 to 0.46% for women. At 10 years study participation, this risk increased to a range of 5.91 to 1.96% for men and 9.64 to 2.32% for women. Given high imaging radiation exposure for long-term survivors in current ICI trials an adaptive imaging interval and imaging termination rules should be considered for long-term survivors

Transcriptional effects of copy number alterations in a large set of human cancers

Copy number alterations (CNAs) can promote tumor progression by altering gene expression levels. Due to transcriptional adaptive mechanisms, however, CNAs do not always translate proportionally into altered expression levels. By reanalyzing >34,000 gene expression profiles, we reveal the degree of transcriptional adaptation to CNAs in a genome-wide fashion, which strongly associate with distinct biological processes. We then develop a platform-independent method-transcriptional adaptation to CNA profiling (TACNA profiling)-that extracts the transcriptional effects of CNAs from gene expression profiles without requiring paired CNA profiles. By applying TACNA profiling to >28,000 patient-derived tumor samples we define the landscape of transcriptional effects of CNAs. The utility of this landscape is demonstrated by the identification of four genes that are predicted to be involved in tumor immune evasion when transcriptionally affected by CNAs. In conclusion, we provide a novel tool to gain insight into how CNAs drive tumor behavior via altered expression levels

Improving gene function predictions using independent transcriptional components

The interpretation of high throughput sequencing data is limited by our incomplete functional understanding of coding and non-coding transcripts. Reliably predicting the function of such transcripts can overcome this limitation. Here we report the use of a consensus independent component analysis and guilt-by-association approach to predict over 23,000 functional groups comprised of over 55,000 coding and non-coding transcripts using publicly available transcriptomic profiles. We show that, compared to using Principal Component Analysis, Independent Component Analysis-derived transcriptional components enable more confident functionality predictions, improve predictions when new members are added to the gene sets, and are less affected by gene multi-functionality. Predictions generated using human or mouse transcriptomic data are made available for exploration in a publicly available web portal. Our understanding of the function of many transcripts is still incomplete, limiting the interpretability of transcriptomic data. Here the authors use consensus-independent component analysis, together with a guilt-by-association approach, to improve the prediction of gene function

Serotonin and Dopamine Receptor Expression in Solid Tumours Including Rare Cancers

In preclinical studies serotonin stimulates and dopamine inhibits tumour growth and angiogenesis. Information regarding serotonin and dopamine receptor (5-HTR and DRD) expression in human cancers is limited. Therefore, we screened a large tumour set for receptor mRNA overexpression using functional genomic mRNA (FGmRNA) profiling, and we analysed protein expression and location of 5-HTR1B, 5-HTR2B, DRD1, and DRD2 with immunohistochemistry in different tumour types. With FGmRNA profiling 11,756 samples representing 43 tumour types were compared to 3,520 normal tissue samples to analyse receptor overexpression. 5-HTR2B overexpression was present in many tumour types, most frequently in uveal melanomas (56%). Receptor overexpression in rare cancers included 5-HTR1B in nasopharyngeal carcinoma (17%), DRD1 in ependymoma (30%) and synovial sarcoma (21%), and DRD2 in astrocytoma (13%). Immunohistochemistry demonstrated high 5-HTR2B protein expression on melanoma and gastro-intestinal stromal tumour cells and endothelial cells of colon, ovarian, breast, renal and pancreatic tumours. 5-HTR1B expression was predominantly low. High DRD2 protein expression on tumour cells was observed in 48% of pheochromocytomas, and DRD1 expression ranged from 14% in melanoma to 57% in renal cell carcinoma. In conclusion, 5-HTR1B, 5-HTR2B, DRD1, and DRD2 show mRNA overexpression in a broad spectrum of common and rare cancers. 5-HTR2B protein is frequently highly expressed in human cancers, especially on endothelial cells. These findings support further investigation of especially 5HTR2B as a potential treatment target

Overexpression of Cyclin E1 or Cdc25A leads to replication stress, mitotic aberrancies, and increased sensitivity to replication checkpoint inhibitors

Oncogene-induced replication stress, for instance as a result of Cyclin E1 overexpression, causes genomic instability and has been linked to tumorigenesis. To survive high levels of replication stress, tumors depend on pathways to deal with these DNA lesions, which represent a therapeutically actionable vulnerability. We aimed to uncover the consequences of Cyclin E1 or Cdc25A overexpression on replication kinetics, mitotic progression, and the sensitivity to inhibitors of the WEE1 and ATR replication checkpoint kinases. We modeled oncogene-induced replication stress using inducible expression of Cyclin E1 or Cdc25A in non-transformed RPE-1 cells, either in a TP53 wild-type or TP53-mutant background. DNA fiber analysis showed Cyclin E1 or Cdc25A overexpression to slow replication speed. The resulting replication-derived DNA lesions were transmitted into mitosis causing chromosome segregation defects. Single cell sequencing revealed that replication stress and mitotic defects upon Cyclin E1 or Cdc25A overexpression resulted in genomic instability. ATR or WEE1 inhibition exacerbated the mitotic aberrancies induced by Cyclin E1 or Cdc25A overexpression, and caused cytotoxicity. Both these phenotypes were exacerbated upon p53 inactivation. Conversely, downregulation of Cyclin E1 rescued both replication kinetics, as well as sensitivity to ATR and WEE1 inhibitors. Taken together, Cyclin E1 or Cdc25A-induced replication stress leads to mitotic segregation defects and genomic instability. These mitotic defects are exacerbated by inhibition of ATR or WEE1 and therefore point to mitotic catastrophe as an underlying mechanism. Importantly, our data suggest that Cyclin E1 overexpression can be used to select patients for treatment with replication checkpoint inhibitors

Considering the biology of late recurrences in selecting patients for extended endocrine therapy in breast cancer

Extended endocrine therapy can reduce recurrences occurring more than 5 years after diagnosis (late recurrences) in estrogen receptor (ER)-positive breast cancer. Given the side effects of endocrine therapy, optimal patient selection for extended treatment is crucial. Enhanced understanding of late recurrence biology could optimize patient selection in this setting. We therefore summarized the current knowledge of late recurrence biology, clinical trials on extended endocrine therapy, and tools for predicting late recurrence and benefit from treatment extension. Extending 5 years of tamoxifen therapy with 5 years of tamoxifen or an aromatase inhibitor (AI) reduces late recurrence risk by 2-5%, but results of extending AI-based therapy are inconsistent. Although several clinicopathological parameters and multigene assays are prognostic for late recurrence, selection tools predicting benefit from extended endocrine therapy are sparse. Therefore, we additionally performed a pooled analysis using 2231 mRNA profiles of patients with ER-positive/human epidermal growth factor receptor 2 negative breast cancer. Gene Set Enrichment Analysis was applied on genes ranked according to their association with early and late recurrence risk. Higher expression of estrogen-responsive genes was associated with a high recurrence risk beyond 5 years after diagnosis when patients had received no systemic therapy. Although 5 years of endocrine therapy reduced this risk, this effect disappeared after treatment cessation. This suggests that late recurrences of tumors with high expression of estrogen-responsive genes are likely ER-driven. Long-term intervention in this pathway by means of extended endocrine therapy might reduce late recurrences in patients with tumors showing high expression of estrogen-responsive genes