High Levels Of Genomic Instability Research Articles

Abstract 6487: The selective WEE1 inhibitor azenosertib shows synergistic anti-tumor activity with KRASG12C inhibitors in multiple KRASG12C models

Abstract KRASG12C is a potent oncogenic driver which results in downstream hyperactivation of MAPK signaling and unchecked oncogenic growth, while simultaneously increasing replication stress (RS) and accumulation of DNA damage. KRASG12C-specific inhibitors are approved and/or recommended in metastatic non-small cell lung cancer (NSCLC) and colorectal cancer (CRC), and while responses are robust, some patients are inherently resistant, and most responders acquire resistance. Resistance mechanisms include reactivation of MAPK signaling, and cancer cells which escape MAPKi are reported to have high levels of genomic instability. WEE1 is a kinase involved in cell cycle progression, allowing cells to repair damaged DNA before entering the next cell cycle phase. Consequently, inhibition of WEE1 leads to premature cell cycle entry and RS, thereby driving cancer cells through the cell cycle with unchecked accumulation of DNA damage, ultimately resulting in mitotic catastrophe and cell death. We hypothesized that high levels of genomic instability and RS in KRASG12C mutant and KRASG12C inhibitor-resistant tumors present a vulnerability to WEE1 inhibition and provide rationale for combining azenosertib, a novel, selective, and orally bioavailable WEE1 inhibitor, with KRASG12C inhibitors. Here, we report azenosertib in combination with KRASG12C inhibitors as an effective therapeutic strategy for KRASG12C tumors. In vitro combination of azenosertib with multiple KRASG12C inhibitors demonstrated synergistic cell growth inhibition across a panel of KRASG12C cell lines in both 2D and 3D assays. In vivo treatment with azenosertib in KRASG12C inhibitor-sensitive cell line-derived xenograft (CDX) models of NSCLC (NCI-H358, NCI-H2122, NCI-H1792), CRC (SW837, SW1463), and pancreatic cancer (MIA PaCa-2) demonstrated significant monotherapy activity as well as synergistic tumor growth inhibition (TGI) when combined with KRASG12C inhibitors, including tumor regression in some models. Importantly, the KRASG12C inhibitor-resistant SW1573 NSCLC CDX model demonstrated synergistic TGI in the combo arm (84%) when compared against monotherapy azenosertib (31%) or adagrasib (58%). Similarly, treatment of a KRASG12C inhibitor-resistant patient-derived xenograft (PDX) model of CRC resulted in synergistic TGI in the combination arm (61%) relative to monotherapy azenosertib (17%) or adagrasib (2%). Finally, analysis of biomarkers from both in vitro and in vivo tumor samples demonstrated synergistic increases in protein markers of RS, DNA damage, and apoptosis with combination therapy. Taken together, our results suggest that the combination of azenosertib with KRASG12C inhibitors enhances tumor growth inhibition over single agent therapy and may be an effective treatment option for patients with KRASG12C tumors. Citation Format: Nathan M. Jameson, Gabriel Kim, Catherine Lee, Blake Skrable, Alexandra Shea, Monah Abed, Olivier Harismendy, Jianhui Ma, Doris Kim, Mark R. Lackner. The selective WEE1 inhibitor azenosertib shows synergistic anti-tumor activity with KRASG12C inhibitors in multiple KRASG12C models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6487.

Recombination, admixture and genome instability shape the genomic landscape of Saccharomyces cerevisiae derived from spontaneous grape ferments.

Cultural exchange of fermentation techniques has driven the spread of Saccharomyces cerevisiae across the globe, establishing natural populations in many countries. Despite this, Oceania is thought to lack native populations of S. cerevisiae, only being introduced after colonisation. Here we investigate the genomic landscape of 411 S. cerevisiae isolated from spontaneous grape fermentations in Australia across multiple locations, years, and grape cultivars. Spontaneous fermentations contained highly recombined mosaic strains that exhibited high levels of genome instability. Assigning genomic windows to putative ancestral origin revealed that few closely related starter lineages have come to dominate the genetic landscape, contributing most of the genetic variation. Fine-scale phylogenetic analysis of loci not observed in strains of commercial wine origin identified widespread admixture with European derived beer yeast along with three independent admixture events from potentially endemic Oceanic lineages that was associated with genome instability. Finally, we investigated Australian ecological niches for basal isolates, identifying phylogenetically distinct S. cerevisiae of non-European, non-domesticated origin associated with admixture loci. Our results illustrate the effect commercial use of microbes may have on local microorganism genetic diversity and demonstrates the presence of non-domesticated, potentially endemic lineages of S. cerevisiae in Australian niches that are actively admixing.

Integrative multi-omics characterization reveals sex differences in glioblastoma.

Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults, with limited treatment modalities and poor prognosis. Recent studies have highlighted the importance of considering sex differences in cancer incidence, prognosis, molecular disparities, and treatment outcomes across various tumor types, including colorectal adenocarcinoma, lung adenocarcinoma, and GBM. We performed comprehensive analyses of large-scale multi-omics data (genomic, transcriptomic, and proteomic data) from TCGA, GLASS, and CPTAC to investigate the genetic and molecular determinants that contribute to the unique clinical properties of male and female GBM patients. Our results revealed several key differences, including enrichments of MGMT promoter methylation, which correlated with increased overall and post-recurrence survival and improved response to chemotherapy in female patients. Moreover, female GBM exhibited a higher degree of genomic instability, including aneuploidy and tumor mutational burden. Integrative proteomic and phosphor-proteomic characterization uncovered sex-specific protein abundance and phosphorylation activities, including EGFR activation in males and SPP1 hyperphosphorylation in female patients. Lastly, the identified sex-specific biomarkers demonstrated prognostic significance, suggesting their potential as therapeutic targets. Collectively, our study provides unprecedented insights into the fundamental modulators of tumor progression and clinical outcomes between male and female GBM patients and facilitates sex-specific treatment interventions. Highlights Female GBM patients were characterized by increased MGMT promoter methylation and favorable clinical outcomes compared to male patients. Female GBMs exhibited higher levels of genomic instability, including aneuploidy and TMB. Each sex-specific GBM is characterized by unique pathway dysregulations and molecular subtypes. EGFR activation is prevalent in male patients, while female patients are marked by SPP1 hyperphosphorylation.

Clinical outcome and molecular landscape of patients with ARID1A-loss gastric cancer.

Chromatin remodelers are commonly altered in human cancer. The mutation of AT-rich interactive domain 1A (ARID1A) in gastric cancer (GC), a component of the SWI/SNF chromatin remodeling complex, was proven associated with treatment response in our previous study. However, ARID1A loss of function was caused not only by mutations but also copy number deletions. The clinicopathologic, genomic, and immunophenotypic correlates of ARID1A loss is largely uncharacterized in GC. Here, 819 patients with clinicopathological information and sequencing data or formalin-fixed paraffin-embedded tissues from four cohorts, Zhongshan Hospital (ZSHS) cohort (n = 375), The Cancer Genome Atlas (TCGA) cohort (n = 371), Samsung Medical Center (SMC) cohort (n = 53), and ZSHS immunotherapy cohort (n = 20), were enrolled. ARID1A loss was defined by genome sequencing or deficient ARID1A expression by immunohistochemistry. We found that ARID1A mutation and copy number deletion were enriched in GC with microsatellite instability (MSI) and chromosomal-instability (CIN), respectively. In the TCGA and ZSHS cohorts, only CIN GC with ARID1A loss could benefit from fluorouracil-based adjuvant chemotherapy. In the SMC and ZSHS immunotherapy cohorts, ARID1A loss exhibited a tendency of superior responsiveness and indicated favorable overall survival after anti-PD-1 immunotherapy. ARID1A-loss tumors demonstrated elevated mutation burden, neoantigen load, and interferon gamma pathway activation. Moreover, in CIN GC, ARID1A loss was correlated with higher homologous recombination deficiency. ARID1A loss defines a distinct subtype of GC characterized by high levels of genome instability, neoantigen formation, and immune activation. These tumors show sensitivity to both chemotherapy and anti-PD-1 immunotherapy. This study provides valuable insights for precision treatment strategies in GC.

Durable Remissions in Advanced Stage and Molecularly High Risk Untreated Classic Hodgkin Lymphoma with Pembrolizumab + AVD

Introduction We previously reported results of a frontline study of pembrolizumab + AVD (Lynch et al Blood 2023) in any stage classic Hodgkin lymphoma (CHL). Despite finding surprisingly high rates of positive interim and EOT PET/CT compared to historical data, this study revealed a 2-year PFS of 97%. Separately, recent data (Alig et al. ICML 2023) suggest that CHL is characterized by two dominant genetic subtypes (H1 vs. H2). H2 tumors harbor higher levels of genomic instability, are enriched for somatic mutations in TP53, KMT2D, and BCL2, and have increased risk of upfront treatment failure with conventional regimens. To better evaluate our findings in higher risk patients, we expanded the study to include 20 additional patients with advanced stage disease. Herein we present updated clinical data for our full 50-patient cohort, and report genotype associations including novel genetic subtypes of cHL. Methods We performed a single arm study (NCT03331341) combining concurrent pembrolizumab with AVD in untreated CHL of any stage. Pembrolizumab 200 mg IV was administered every 21 days concurrently with AVD chemotherapy. AVD was administered at the standard dosing and schedule. With the cohort expansion, total planned accrual was 50 patients. The objective of the expanded study was to evaluate longer term efficacy based on concerns with the utility of PET/CT. We defined success as a 1-year PFS in advanced stage patients of at least 85%. Samples were analyzed for ctDNA at baseline, post cycle 1 (if available), post cycle 2, and end of treatment. ctDNA levels were quantified as haploid genome equivalents/mL plasma using CAPP-Seq (Newman et al Nat Biotech 2016) and PhasED-Seq (Kurtz et al. Nat Biotech 2021). Using baseline CAPP-Seq genotypes from plasma ctDNA, patients were classified as H1 vs H2 cHL by a previously validated probabilistic Latent Dirichlet Allocation (LDA) algorithm (Alig et al, 2023, in review). Results 50 patients, enrolled between Feb 1, 2019, and Apr 13, 2023, were evaluable for updated safety, response and/or exploratory analyses (one patient withdrew from study treatment prior to any response assessment). With a median follow up of 2.3 years, 2-year PFS and OS were 98% and 100%, respectively. Among advanced stage patients (n=38), 2-year PFS and OS were 97% and 100% respectively. PET2 was performed in 49 patients, with a CR rate of 73% in the early stage patients, and 58% in those with advanced stage. EOT PET was evaluable in 41 patients, with a CR rate of 91% in early stage, and 73% in those with advanced stage. To date, only one patient with a positive EOT PET has developed biopsy-proven recurrence. Four EOT PET+ patients are now > 3 years post treatment without relapse. Four additional EOT PET+ patients are less than 1 year post therapy, and one has resolved FDG uptake on repeat scan with the others undergoing surveillance. Eight (16%) patients experienced SAEs, including sepsis (one each G3 and G4), G3 Guillain Barre syndrome that resolved after permanent pembrolizumab discontinuation, and five (10%) G3 febrile neutropenia. Non-invasive genotyping using ctDNA was available in 25 patients. Among 6 patients with H2 genotypes (24%), all had advanced stage, and this group included the only patient experiencing disease recurrence. The remaining patients including all early-stage patients were classified as H1 (76%). All patients will have completed treatment and safety evaluations by October 2023, and updated safety, efficacy, and exploratory data including ctDNA will be presented at the meeting. Conclusion With longer follow up, including patients who started therapy over 4 years ago, concurrent pembrolizumab + AVD represents a highly effective frontline therapy for CHL, including for advanced stage patients and those with high risk H2 genotypes. PET2 demonstrated limited clinical utility, and no late relapses have been seen to date.

Spatial whole transcriptome profiling of primary tumor from patients with metastatic prostate cancer.

Prostate cancer (PCa) is a highly heterogeneous disease in terms of its molecular makeup and clinical prognosis. The prostate tumor microenvironment (TME) is hypothesized to play an important role in driving disease aggressiveness, but precise mechanisms remain elusive. In our study, we used spatial transcriptomics to explore for the first time the spatial gene expression heterogeneity within primary prostate tumors from patients with metastatic disease. In total, we analyzed 5459 tissue spots from three PCa patients comprising castration-resistant (CRPC) and neuroendocrine (NEPC) disease stages. Within CRPC, we identified a T cell cluster whose activity might be impaired by nearby regulatory T cells, potentially mediating the aggressive disease phenotype. Moreover, we identified Hallmark signatures of epithelial-mesenchymal transition in a cancer-associated fibroblast (CAF) cluster, indicating the aggressive characteristic of the primary TME leading to metastatic dissemination. Within NEPC, we identified active immune-stroma cross-talk exemplified by significant ligand-receptor interactions between CAFs and M2 macrophages. Further, we identified a malignant cell population that was associated with the down-regulation of an immune-related gene signature. Lower expression of this signature was associated with higher levels of genomic instability in advanced PCa patients (SU2C cohort, n = 99) and poor recurrence free survival in early-stage PCa patients (TCGA cohort, n = 395), suggesting prognostic biomarker potential. Taken together, our study reveals the importance of whole transcriptome profiling at spatial resolution for biomarker discovery and for advancing our understanding of tumor biology.

Proteomics-based clustering of lung adenocarcinoma identifies three subtypes with significantly different clinical and molecular features.

Lung adenocarcinoma (LUAD) is a predominant subtype of lung cancer. Although molecular classification of LUAD has been widely explored, proteomics-based subtyping of LUAD remains scarce. We proposed a subtyping method for LUAD based on the expression profiles of 500 proteins with the largest expression variability across LUAD. Furthermore, we comprehensively compared molecular and clinical features among the LUAD subtypes. Consensus clustering identified three subtypes of LUAD, namely MtE, DrE, and StE. We demonstrated this subtyping method to be reproducible by analyzing two independent LUAD cohorts. MtE was characterized by high enrichment of metabolic pathways, high EGFR mutation rate, low stemness, proliferation, invasion, metastasis and inflammation signatures, favorable prognosis; DrE was characterized by high enrichment of DNA repair pathways, high TP53 mutation rate, and high levels of genomic instability, stemness, proliferation, and intratumor heterogeneity (ITH); and StE was characterized by high enrichment of stroma-related pathways, high KRAS mutation rate, and low levels of genomic instability. The proteomics-based clustering analysis identified three LUAD subtypes with significantly different molecular and clinical properties. The novel subtyping method offers new perspectives on the cancer biology and holds promise in improving the clinical management of LUAD.

NR4A3 Expression Is Consistently Absent in Acinic Cell Carcinomas of the Breast: A Potential Nosologic Shift

Acinic cell carcinoma (AciCC) is a tumor that is recognized in both the breast and salivary glands. Recently, the recurrent genomic rearrangement, t(4;9)(q13;q31) was identified in salivary AciCC that results in constitutive upregulation of the nuclear transcription factor NR4A3, which can be detected by immunohistochemistry. In this study, we sought to evaluate NR4A3 expression in breast AciCC using immunohistochemistry. Strong and diffuse nuclear staining was considered a positive result. Sixteen AciCCs were studied, including 8 pure AciCCs and 8 AciCCs admixed with other types (invasive carcinoma of no special type in 5 cases and metaplastic carcinoma in 3 cases). All 16 AciCCs showed negative results for NR4A3 expression. Four cases with available material were evaluated for rearrangements of the NR4A3 gene by fluorescence in situ hybridization and no rearrangements were observed. Whole-genome sequencing of 1 AciCC revealed a TP53 splice-site mutation, high levels of genomic instability, and genomic features of homologous recombination DNA repair defects; a structural variant analysis of this case did not reveal the presence of a t(4;9) rearrangement. We conclude that breast AciCCs consistently lack NR4A3 rearrangement or overexpression, unlike most of the salivary AciCCs, and that consistent with previous results, breast AciCCs are associated with genomic alterations more similar to those seen in triple-negative breast carcinomas than salivary gland AciCCs. These results suggest that unlike other salivary gland–like tumors that occur in the breast, the molecular underpinnings of the salivary gland and breast AciCCs are different and that the salivary gland and breast AciCCs likely represent distinct entities.

Suppression of GCH1 Sensitizes Ovarian Cancer and Breast Cancer to PARP Inhibitor.

Breast and ovarian cancers are common malignancies among women, contributing to a significant disease burden, and are characterized by a high level of genomic instability, owing to the failure of homologous recombination repair (HRR). Pharmacological inhibition of poly(ADP-ribose) polymerase (PARP) could elicit the synthetic lethal effect of tumor cells in patients with homologous recombination deficiency, ultimately achieving a favorable clinical benefit. However, primary and acquired resistance remain the greatest hurdle, limiting the efficacy of PARP inhibitors; thus, strategies conferring or augmenting tumor cell sensitivity to PARP inhibitors are urgently required. Our RNA-seq data of niraparib-treated and -untreated tumor cells were analyzed by R language. Gene Set Enrichment Analysis (GSEA) was applied to assess the biological functions of GTP cyclohydrolase 1 (GCH1). Quantitative real-time PCR, Western blotting, and immunofluorescence were applied to confirm the upregulation of GCH1 upon niraparib treatment at transcriptional and translational levels. Immunohistochemistry of patient-derived xenograft (PDX)-derived tissue sections further validated that niraparib increased GCH1 expression. Tumor cell apoptosis was detected by flow cytometry, while the superiority of the combination strategy was confirmed in the PDX model. The expression of GCH1 was aberrantly enriched in breast and ovarian cancers and increased after niraparib treatment via JAK-STAT signaling. GCH1 was also demonstrated to be associated with the HRR pathway. Subsequently, the enhancement of the tumor-killing effect of PARP inhibitors induced by GCH1 suppression using siRNA and GCH1 inhibitor was validated by flow cytometry in vitro. Finally, using the PDX model, we further demonstrated that GCH1 inhibitors markedly potentiated PARP inhibitors' antitumor efficacy in vivo. Our results illustrated that PARP inhibitors promote GCH1 expression via the JAK-STAT pathway. We also elucidated the potential relationship between GCH1 and the homologous recombination repair pathway and proposed a combination regimen of GCH1 suppression with PARP inhibitors in breast and ovarian cancers.

Identification and development of TP53 mutation-associated Long non-coding RNAs signature for optimized prognosis assessment and treatment selection in hepatocellular carcinoma.

The TP53 gene is estimated to be mutated in over 50% of tumors, with the majority of tumors exhibiting abnormal TP53 signaling pathways. However, the exploration of TP53 mutation-related LncRNAs in Hepatocellular carcinoma (HCC) remains incomplete. This study aims to identify such LncRNAs and enhance the prognostic accuracy for Hepatoma patients. Differential gene expression was identified using the "limma" package in R. Prognosis-related LncRNAs were identified via univariate Cox regression analysis, while a prognostic model was crafted using multivariate Cox regression analysis. Survival analysis was conducted using Kaplan-Meier curves. The precision of the prognostic model was assessed through ROC analysis. Subsequently, the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm were executed on the TCGA dataset via the TIDE database. Fractions of 24 types of immune cell infiltration were obtained from NCI Cancer Research Data Commons using deconvolution techniques. The protein expression levels encoded by specific genes were obtained through the TPCA database. In this research, we have identified 85 LncRNAs associated with TP53 mutations and developed a corresponding signature referred to as TP53MLncSig. Kaplan-Meier analysis revealed a lower 3-year survival rate in high-risk patients (46.9%) compared to low-risk patients (74.2%). The accuracy of the prognostic TP53MLncSig was further evaluated by calculating the area under the ROC curve. The analysis yielded a 5-year ROC score of 0.793, confirming its effectiveness. Furthermore, a higher score for TP53MLncSig was found to be associated with an increased response rate to immune checkpoint blocker (ICB) therapy (p = .005). Patients possessing high-risk classification exhibited lower levels of P53 protein expression and higher levels of genomic instability. The present study aimed to identify and validate LncRNAs associated with TP53 mutations. We constructed a prognostic model that can predict chemosensitivity and response to ICB therapy in HCC patients. This novel approach sheds light on the role of LncRNAs in TP53 mutation and provides valuable resources for analyzing patient prognosis and treatment selection.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Cerebrospinal Fluid Cell-Free DNA-Based Detection of High Level of Genomic Instability Is Associated With Poor Prognosis in NSCLC Patients With Leptomeningeal Metastases.

IntroductionLeptomeningeal metastasis (LM) commonly occurs in non-small cell lung cancer (NSCLC) patients and has a poor prognosis. Due to limited access to leptomeningeal lesions, the genetic characteristics of LM have not been explored to date. Cerebrospinal fluid (CSF) may be the most representative liquid biopsy medium to obtain genomic information from LM in NSCLC.MethodsCSF biopsies and matched peripheral blood biopsies were collected from 33 NSCLC patients with LM. We profiled genetic alterations from LM by comparing CSF cell-free DNA (cfDNA) with plasma cfDNA. Somatic mutations were examined using targeted sequencing. Genomic instability was analyzed by low-coverage whole-genome sequencing (WGS).ResultsDriver mutations were detected in 100% of CSF cfDNA with much higher variant allele frequency than that in matched plasma cfDNA (57.5%). Furthermore, we found that the proportions of CSF cfDNA fragments below 150 bp were significantly higher than those in plasma cfDNA. These findings indicate enrichment of circulating tumor DNA (ctDNA) in CSF and explain the high sensitivity of mutation detection in the CSF. The absence of some mutations in CSF cfDNA—especially the first-/second-generation mutation T790M, which confers resistance to epidermal growth factor receptor (EGFR)-Tyrosine kinase inhibitors (TKIs)—that were present in plasma cfDNA samples indicates different mechanisms of cancer evolution between LM and extracranial lesions. In addition, 86.6% of CSF ctDNA samples revealed high levels of genomic instability compared with 2.5% in plasma cfDNA samples. A higher number of large-scale state transitions (LSTs) in CSF cfDNA were associated with a shorter overall survival (OS).ConclusionOur results suggest that LM and extracranial lesions develop independently. Both CSF cfDNA genetic profiling and plasma cfDNA genetic profiling are necessary for clinical decision-making for NSCLC patients with LM. Through CSF-based low-coverage WGS, a high level of LSTs was identified as a potential biomarker of poor prognosis.

Mesenchymal tumor cells drive adaptive resistance of Trp53-/- breast tumor cells to inactivated mutant Kras.

As precision medicine increases the response rate of treatment, tumors frequently bypass inhibition, and reoccur. In order for treatment to be effective long term, the mechanisms enabling treatment adaptation need to be understood. Here, we report a mouse model that, in the absence of p53 and the presence of oncogenic KrasG12D , develops breast tumors. Upon inactivation of KrasG12D , tumors initially regress and enter remission. Subsequently, the majority of tumors adapt to the withdrawal of KrasG12D expression and return. KrasG12D ‐independent tumor cells show a strong mesenchymal profile with active RAS‐RAF‐MEK‐ERK (MAPK/ERK) signaling. Both KrasG12D ‐dependent and KrasG12D ‐independent tumors display a high level of genomic instability, and KrasG12D ‐independent tumors harbor numerous amplified genes that can activate the MAPK/ERK signaling pathway. Our study identifies both epithelial‐mesenchymal transition (EMT) and active MAPK/ERK signaling in tumors that adapt to oncogenic KrasG12D withdrawal in a novel Trp53−/− breast cancer mouse model. To achieve long‐lasting responses in the clinic to RAS‐fueled cancer, treatment will need to focus in parallel on obstructing tumors from adapting to oncogene inhibition.

The Dynamic Instability of the Aneuploid Genome.

Proper partitioning of replicated sister chromatids at each mitosis is crucial for maintaining cell homeostasis. Errors in this process lead to aneuploidy, a condition in which daughter cells harbor genome imbalances. Importantly, aneuploid cells often experience DNA damage, which in turn could drive genome instability. This might be the product of DNA damage accumulation in micronuclei and/or a consequence of aneuploidy-induced replication stress in S-phase. Although high levels of genome instability are associated with cell cycle arrest, they can also confer a proliferative advantage in some circumstances and fuel tumor growth. Here, we review the main consequences of chromosome segregation errors on genome stability, with a special focus on the bidirectional relationship between aneuploidy and DNA damage. Also, we discuss recent findings showing how increased genome instability can provide a proliferation improvement under specific conditions, including chemotherapeutic treatments.

Subtyping of Human Papillomavirus-Positive Cervical Cancers Based on the Expression Profiles of 50 Genes

BackgroundHuman papillomavirus-positive (HPV+) cervical cancers are highly heterogeneous in molecular and clinical features. However, the molecular classification of HPV+ cervical cancers remains insufficiently unexplored.MethodsBased on the expression profiles of 50 genes having the largest expression variations across the HPV+ cervical cancers in the TCGA-CESC dataset, we hierarchically clustered HPV+ cervical cancers to identify new subtypes. We further characterized molecular, phenotypic, and clinical features of these subtypes.ResultsWe identified two subtypes of HPV+ cervical cancers, namely HPV+G1 and HPV+G2. We demonstrated that this classification method was reproducible in two validation sets. Compared to HPV+G2, HPV+G1 displayed significantly higher immune infiltration level and stromal content, lower tumor purity, lower stemness scores and intratumor heterogeneity (ITH) scores, higher level of genomic instability, lower DNA methylation level, as well as better disease-free survival prognosis. The multivariate survival analysis suggests that the disease-free survival difference between both subtypes is independent of confounding variables, such as immune signature, stemness, and ITH. Pathway and gene ontology analysis confirmed the more active tumor immune microenvironment in HPV+G1 versus HPV+G2.ConclusionsHPV+ cervical cancers can be classified into two subtypes based on the expression profiles of the 50 genes with the largest expression variations across the HPV+ cervical cancers. Both subtypes have significantly different molecular, phenotypic, and clinical features. This new subtyping method captures the comprehensive heterogeneity in molecular and clinical characteristics of HPV+ cervical cancers and provides potential clinical implications for the diagnosis and treatment of this disease.

DNA damage response proteins in canine cancer as potential research targets in comparative oncology

The DNA damage response (DDR) is a complex signal transduction network that is activated when endogenous or exogenous genotoxins damage or interfere with the replication of genomic DNA. Under such conditions, the DDR promotes DNA repair and ensures accurate replication and division of the genome. High levels of genomic instability are frequently observed in cancers and can stem from germline loss‐of‐function mutations in certain DDR genes, such as BRCA1, BRCA2, and p53, that form the basis of human cancer predisposition syndromes. In addition, mutation and/or aberrant expression of multiple DDR genes are frequently observed in sporadic human cancers. As a result, the DDR is considered to represent a viable target for cancer therapy in humans and a variety of strategies are under investigation. Cancer is also a significant cause of mortality in dogs, a species that offers certain advantages for experimental oncology. Domestic dogs present numerous inbred lines, many of which display predisposition to specific forms of cancer and the study of which may provide insight into the biological basis of this susceptibility. In addition, clinical trials are possible in dogs and may lead to therapeutic insights that could ultimately be extended to humans. Here we review what is known specifically about the DDR in dogs and discuss how this knowledge could be extended and exploited to advance experimental oncology in this species.

Abstract 2484: Detection of loss of heterozygosity in GBM revealed the role of a tumor suppressive gene in regulating glioma progression and tumor recurrence

Abstract Adducin 3 (ADD3) is a major cytoskeleton protein in glioma cells. Our previous studies demonstrated a significant downregulation of ADD3 in GBM when compared with lower-grade gliomas, and that ADD3 knockdown promoted malignant phenotypes. GBMs are usually characterized by high levels of genomic instability, including frequent loss of heterozygosity (LOH) of chromosome 10q, whilst ADD3 is also located in chromosome 10q25.1-25.2 where it is known to be a tumor-suppressor region. However, it is not known whether ADD3 downregulation in malignant gliomas is associated with allelic loss in 10q. To determine 10q LOH on different polymorphic DNA loci (covering ADD3 locus) on glioma tissues, specimens of Normal, Grade II, Grade III, Grade IV GBM and recurrent GBM tissue were included. DNA fragments were evaluated by using a PCR-based microsatellite capillary electrophoresis and analyzed by a fragment analyzer. LOH status were correlated with patient survival for potential prognostic and diagnostic implications. LOH on ADD3 locus was detected in high grade and recurrent GBMs. It is revealed that downregulation of ADD3 level in GBM is associated with genetic instability of LOH in 10q and associated with recurrence disease. This study would provide significant implications, particularly for understanding pathogenesis of GBM and add valuable credits on the diagnostic as well as prognostic implications. Citation Format: Mei-Yee Kiang, Stella Sun, Gilberto Ka-Kit Leung. Detection of loss of heterozygosity in GBM revealed the role of a tumor suppressive gene in regulating glioma progression and tumor recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2484.

Depletion of DNA Polymerase Theta Inhibits Tumor Growth and Promotes Genome Instability through the cGAS-STING-ISG Pathway in Esophageal Squamous Cell Carcinoma.

Simple SummaryDNA polymerase theta, encoded by the human POLQ gene, is upregulated in several cancers and is associated with poor clinical outcomes. The importance of POLQ, however, has yet to be elucidated in esophageal cancer. In this study, we explored the functional impacts of POLQ and looked into its underlying mechanisms. POLQ was overexpressed in esophageal squamous cell carcinoma (ESCC) tumors associated with unfavorable prognosis and contributed to malignant phenotypes by promoting genome stability, suggesting that targeting polymerase theta may provide a potential therapeutic approach for improving ESCC management.Overexpression of the specialized DNA polymerase theta (POLQ) is frequent in breast, colon and lung cancers and has been correlated with unfavorable clinical outcomes. Here, we aimed to determine the importance and functional role of POLQ in esophageal squamous cell carcinoma (ESCC). Integrated analysis of four RNA-seq datasets showed POLQ was predominantly upregulated in ESCC tumors. High expression of POLQ was also observed in a cohort of 25 Hong Kong ESCC patients and negatively correlated with ESCC patient survival. POLQ knockout (KO) ESCC cells were sensitized to multiple genotoxic agents. Both rH2AX foci staining and the comet assay indicated a higher level of genomic instability in POLQ-depleted cells. Double KO of POLQ and FANCD2, known to promote POLQ recruitment at sites of damage, significantly impaired cell proliferation both in vitro and in vivo, as compared to either single POLQ or FANCD2 KOs. A significantly increased number of micronuclei was observed in POLQ and/or FANCD2 KO ESCC cells. Loss of POLQ and/or FANCD2 also resulted in the activation of cGAS and upregulation of interferon-stimulated genes (ISGs). Our results suggest that high abundance of POLQ in ESCC contributes to the malignant phenotype through genome instability and activation of the cGAS pathway.

Clinical impact of copy number variation changes in bladder cancer samples.

The aim of the present study was to detect copy number variations (CNVs) related to tumour progression and metastasis of urothelial carcinoma through whole-genome scanning. A total of 30 bladder cancer samples staged from pTa to pT4 were included in the study. DNA was extracted from freshly frozen tissue via standard phenol-chloroform extraction and CNV analysis was performed on two alternative platforms (CytoChip Oligo aCGH, 4x44K and Infinium OncoArray-500K BeadChip; Illumina, Inc.). Data were analysed with BlueFuse Multi software and Karyostudio, respectively. The results highlight the role of genomic imbalances in regions containing genes with metastatic and proliferative potential for tumour invasion. A high level of genomic instability in uroepithelial tumours was observed and a total of 524 aberrations, including 175 losses and 349 gains, were identified. The most prevalent genetic imbalances affected the following regions: 1p, 1q, 2q, 4p, 4q, 5p, 5q, 6p, 6q, 7q, 8q, 9p, 9q, 10p, 10q, 11q, 13q and 17q. High-grade tumours more frequently harboured genomic imbalances (n=227) than low-grade tumours (n=103). A total of 36 CNVs in high-grade bladder tumours were detected in chromosomes 1-5, 8-11, 14, 17, 19 and 20. Furthermore, five loss of heterozygosity variants containing 176 genes were observed in high-grade bladder cancer and may be used as potential targets for precision therapy. Revealing specific chromosomal regions related to the metastatic potential of uroepithelial tumours may lay a foundation for implementing molecular CNV profiling of bladder tumours as part of a routine progression risk estimation strategy, thus expanding the personalized therapeutic approach.

Adaptation to the dietary sugar D-tagatose via genome instability in polyploid Candida albicans cells.

The opportunistic fungal pathogen Candida albicans undergoes an unusual parasexual cycle wherein diploid cells mate to form tetraploid cells that can generate genetically diverse progeny via a nonmeiotic program of chromosome loss. The genetic diversity afforded by parasex impacts clinically relevant features including drug resistance and virulence, and yet the factors influencing genome instability in C. albicans are not well defined. To understand how environmental cues impact genome instability, we monitored ploidy change following tetraploid cell growth in a panel of different carbon sources. We found that growth in one carbon source, D-tagatose, led to high levels of genomic instability and chromosome loss in tetraploid cells. This sugar is a stereoisomer of L-sorbose which was previously shown to promote karyotypic changes in C. albicans. However, while expression of the SOU1 gene enabled utilization of L-sorbose, overexpression of this gene did not promote growth in D-tagatose, indicating differences in assimilation of the two sugars. In addition, genome sequencing of multiple progenies recovered from D-tagatose cultures revealed increased relative copy numbers of chromosome 4, suggestive of chromosome-level regulation of D-tagatose metabolism. Together, these studies identify a novel environmental cue that induces genome instability in C. albicans, and further implicate chromosomal changes in supporting metabolic adaptation in this species.