Genomic Instability Characteristic Research Articles

Characterization of RNA Processing Genes in Colon Cancer for Predicting Clinical Outcomes.

Colon cancer is associated with multiple levels of molecular heterogeneity. RNA processing converts primary transcriptional RNA to mature RNA, which drives tumourigenesis and its maintenance. The characterisation of RNA processing genes in colon cancer urgently needs to be elucidated. In this study, we obtained 1033 relevant samples from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases to explore the heterogeneity of RNA processing phenotypes in colon cancer. Firstly, Unsupervised hierarchical cluster analysis detected 4 subtypes with specific clinical outcomes and biological features via analysis of 485 RNA processing genes. Next, we adopted the least absolute shrinkage and selection operator (LASSO) as well as Cox regression model with penalty to characterise RNA processing-related prognostic features. An RNA processing-related prognostic risk model based on 10 genes including FXR1, MFAP1, RBM17, SAGE1, SNRPA1, SRRM4, ADAD1, DDX52, ERI1, and EXOSC7 was identified finally. A composite prognostic nomogram was constructed by combining this feature with the remaining clinical variables including TNM, age, sex, and stage. Genetic variation, pathway activation, and immune heterogeneity with risk signatures were also analysed via bioinformatics methods. The outcomes indicated that the high-risk subgroup was associated with higher genomic instability, increased proliferative and cycle characteristics, decreased tumour killer CD8+ T cells and poorer clinical prognosis than the low-risk group. This prognostic classifier based on RNA-edited genes facilitates stratification of colon cancer into specific subgroups according to TNM and clinical outcomes, genetic variation, pathway activation, and immune heterogeneity. It can be used for diagnosis, classification and targeted treatment strategies comparable to current standards in precision medicine. It provides a rationale for elucidation of the role of RNA editing genes and their clinical significance in colon cancer as prognostic markers.

Effect of homologous recombination deficient (HRD) breast cancers on a distinct immune marker phenotype by comprehensive genomic and immune profiling (CGIP).

1029 Background: Preclinical evidence suggests that breast cancer with features of genomic instability may upregulate the host antitumor immune response by producing neoantigens through DNA damage and increasing interferon production through the stimulator of interferon genes (STING) pathway. In this study, we evaluated the association between features of genomic instability and immune response in a real-world breast cancer population. Methods: We analyzed retrospective comprehensive genomic and immune profiling (CGIP) results from 529 breast tumors tested in the real-world clinical setting. We defined the HRD phenotype as tumor with any single nucleotide variants (SNV), indels, copy number variations (CNV) or fusions in the following genes: ARID1A, ATM, ATRX, BAP1, BARD1, BLM, BRCA1/2, BRIP1, CHEK1/2, FANCA, MRE11A, NBN, PALB2, RAD50 and RAD51. Otherwise, they were considered HR-proficient (HRP). PD-L1 expression (CPS positive ≥10) and TMB (high ≥10 Mut/Mb) were determined using IHC and DNA sequencing, respectively. mRNA expression signatures of tumor inflammation (TIGS, strong/moderate/weak), cell proliferation (CP, high/moderate/poor) and cancer testis antigen burden (CTAB, high/low) were determined by RNA-sequencing from a 395-gene panel. We used over-representation and proportion analysis using chi-squared test to determine association of HRD to immune correlates. Results: Among 529 cases, the median patient age was 63.2 years (25.5-93.5). The majority of patients were female (519, 98%), and the most common tumor histology was invasive ductal carcinoma (287, 54%). A total of 405 (77%) and 124 (23%) of patients had HRD and HRP phenotypes, respectively. A greater proportion of HRD tumors (16%) had higher TMB compared to HRP (5.6%, p=0.003). HRP tumors showed a relatively low CP index, whereas HRD tumors were associated with a moderate CP index score (p = 0.007). HRD phenotype was associated with a significantly higher proportion of weakly inflamed tumors, as represented by lower TIGS scores (p=0.007). No significant difference in PD-L1 or CTAB was found between HRD and HRP phenotypes. Conclusions: Breast tumors with mutations in the HR genes demonstrated greater TMB and moderate cellular proliferation index of both tumor and immune cells, which suggest susceptibility to immune checkpoint inhibitors. Interestingly, this cohort lacked elevated markers of immune infiltration (TIGS), indicating a mechanism of potential tumor immune evasion. These results suggest that CGIP may allow for more informed treatment decisions in HRD breast cancers. Furthermore, assessment of HR status and immunotherapy susceptibility may support clinical trial selections for therapies targeting the complex interplay of genomic and immune components of breast cancer (e.g., a combination of PARP inhibitor and immunotherapy).

Controversies and management of deficient mismatch repair gastrointestinal cancers in the neoadjuvant setting.

High microsatellite instability (MSI-H)/deficient mismatch repair (dMMR) phenotype is a distinct molecular signature across gastrointestinal cancers characterized by high tumor mutational burden and high neoantigen load. Tumors harboring dMMR are highly immunogenic and heavily infiltrated by immune cells; consequently, they are uniquely vulnerable to therapeutic strategies enhancing immune antitumor response such as checkpoint inhibitors. The MSI-H/dMMR phenotype arose as a powerful predictor of response to immune checkpoint inhibitors with evidence supporting significantly improved outcomes in the metastatic setting. On the other hand, the genomic instability characteristic of MSI-H/dMMR tumors appears to be associated with decreased sensitivity to chemotherapy, and the benefits of standard adjuvant or neoadjuvant chemotherapy approaches in this subtype are being increasingly questioned. Here, we review the prognostic and predictive impact of MMR status in localized gastric and colorectal cancers, and highlight the emerging clinical data incorporating checkpoint inhibitors in the neoadjuvant setting.

Role of Sirtuins in the Pathobiology of Onco-Hematological Diseases: A PROSPERO-Registered Study and In Silico Analysis.

Simple SummaryThe aging of the hematological system can cause physiological disorders such as anemia, reduced immunity, and the increased incidence of blood cancer. Patients diagnosed with hematologic malignancies comprise nearly 10% of all cancer deaths identified in international epidemiologic studies. Therefore, it is considered a public health problem worldwide. Scientific evidence demonstrates the important involvement of sirtuins (SIRTs) in the pathogenesis of several types of solid tumors. However, the role of SIRTs in the pathobiology of malignant hematological diseases has not yet been systematically reviewed. In this systematic review, we highlight the role of different SIRTs in the pathogenesis of acute and chronic leukemias, lymphoma and myeloma. Also, we performed a bioinformatic analysis to identify whether the expression of SIRTs is altered in onco-hematological diseases, such as lymphomas and leukemias. The advent of new applicability of SIRTs in the process of aging and hematological carcinogenesis may allow the development of new diagnostic and therapeutic approaches for these diseases.The sirtuins (SIRT) gene family (SIRT1 to SIRT7) contains the targets implicated in cellular and organismal aging. The role of SIRTs expression in the pathogenesis and overall survival of patients diagnosed with solid tumors has been widely discussed. However, studies that seek to explain the role of these pathways in the hematopoietic aging process and the consequences of their instability in the pathogenesis of different onco-hematological diseases are still scarce. Therefore, we performed a systematic review (registered in PROSPERO database #CRD42022310079) and in silico analysis (based on GEPIA database) to discuss the role of SIRTs in the advancement of pathogenesis and/or prognosis for different hematological cancer types. In summary, given recent available scientific evidence and in silico gene expression analysis that supports the role of SIRTs in pathobiology of hematological malignances, such as leukemias, lymphomas and myeloma, it is clear the need for further high-quality research and clinical trials that expands the SIRT inhibition knowledge and its effect on controlling clonal progression caused by genomic instability characteristics of these diseases. Finally, SIRTs represent potential molecular targets in the control of the effects caused by aging on the failures of the hematopoietic system that can lead to the involvement of hematological neoplasms.

Nasopharyngeal Carcinoma Progression: Accumulating Genomic Instability and Persistent Epstein-Barr Virus Infection.

Genomic instability facilitates the evolution of cells, tissues, organs, and species. The progression of human malignancies can be regarded as the accumulation of genomic instability, which confers a high evolutionary potential for tumor cells to adapt to continuous changes in the tumor microenvironment. Nasopharyngeal carcinoma (NPC) is a head-and-neck squamous-cell carcinoma closely associated with Epstein–Barr virus (EBV) infection. NPC progression is driven by a combination of accumulated genomic instability and persistent EBV infection. Here, we present a review of the key characteristics of genomic instability in NPC and the profound implications of EBV infection. We further discuss the significance of profiling genomic instability for the assessment of disease progression and treatment efficacy, as well as the opportunities and challenges of targeted therapies for NPC based on its unique genomic instability.

Mitotic DNA synthesis is caused by transcription-replication conflicts in BRCA2-deficient cells

Aberrant replication causes cells lacking BRCA2 to enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS). Here, we identify genome-wide the sites where MiDAS reactions occur when BRCA2 is abrogated. High-resolution profiling revealed that these sites are different from MiDAS at aphidicolin-induced common fragile sites in that they map to genomic regions replicating in the early S-phase, which are close to early-firing replication origins, are highly transcribed, and display R-loop-forming potential. Both transcription inhibition in early S-phase and RNaseH1 overexpression reduced MiDAS in BRCA2-deficient cells, indicating that transcription-replication conflicts (TRCs) and R-loops are the source of MiDAS. Importantly, the MiDAS sites identified in BRCA2-deficient cells also represent hotspots for genomic rearrangements in BRCA2-mutated breast tumors. Thus, our work provides a mechanism for how tumor-predisposing BRCA2 inactivation links transcription-induced DNA damage with mitotic DNA repair to fuel the genomic instability characteristic of cancer cells.

Abstract 541: Evaluation of low-pass whole genome sequencing for the diagnosis and monitoring of patients with pediatric solid tumors

Abstract Liquid biopsy assays based on sequence analysis of cell-free circulating tumor DNA (cfDNA) have the potential to transform patient care. Recent advances, however, have primarily focused on adult patients with solid tumors from whom access to sufficient volumes of blood, urine or other bodily fluids is not a barrier to testing. For pediatric cancer patients, noninvasive procedures are of paramount importance for early diagnosis as well as effective monitoring of disease, but clinical diagnostic tests need to be optimized for low volumes of blood. Furthermore, most childhood solid tumors have a limited number of driver mutations that can be interrogated by targeted next generation sequencing (NGS) panels. We evaluated analysis of liquid biopsy using Low-Pass Whole Genome Sequencing (LP-WGS) of plasma cfDNA as a potential approach for diagnosing and monitoring pediatric solid tumors. We profiled samples from 42 patients with a variety of solid tumors including sarcoma (n = 28), germ-cell (n = 4), and renal tumors ( n = 10). cfDNA was extracted with the MagMax cell-free total nucleic acid extraction kit from 4-6ml peripheral blood collected in EDTA tubes at the time of diagnosis (if available), during and after chemotherapy, and when available, at relapse. Whole-genome sequencing libraries were constructed with the xGen Prism DNA Library Prep Kit using 5ng of cfDNA and paired-end sequenced on an Illumina NextSeq 500 at an average of 1x depth of coverage. Data was analyzed using iChor software. The LP-WGS data from the cfDNA samples was compared with copy number alteration (CNA) profiles of the primary tumors generated from clinical chromosomal microarray analysis (CMA) (CytoScanHD or OncoScan). The CNA profiles detected in plasma using LP-WGS were very similar to those of the primary tumors observed by CMA. Of 42 patients analyzed to date, 19 had non-informative profiles either at diagnosis or relapse. The loss of 9p and 17p, including CDKN2A and TP53 respectively, were the most common pathogenic CNAs detected in the abnormal cases. One patient’s germ cell tumor profiled with our OncoKids NGS panel had a confirmed KRAS mutation and 12p amplification that were also detected by LP-WGS in the liquid biopsy. Marked genomic instability characteristic of osteosarcoma was readily detectable by LP-WGS and an indication of response to therapy was mirrored by the absence of CNAs in the liquid biopsy specimen after treatment. Combined, these data suggest that liquid biopsy with LP-WGS may be used to monitor pediatric patients with solid tumors, from diagnosis through treatment and recurrence. Citation Format: Eirini Christodoulou, Dejerianne Ostrow, Anya Zdanowicz, Venkata Yellapantula, Cameron O'Connell, Moiz Bootwalla, Pan Yachen, Jennifer Cotter, James Amatruda, Leo Mascarenhas, Fariba Navid, Gai Xiaowu, Jaclyn Biegel. Evaluation of low-pass whole genome sequencing for the diagnosis and monitoring of patients with pediatric solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 541.

6q deletion is frequent but unrelated to patient prognosis in breast cancer

BackgroundDeletions involving the long arm of chromosome 6 have been reported to occur in breast cancer, but little is known about the clinical relevance of this alteration.MethodsWe made use of a pre-existing tissue microarray with 2197 breast cancers and employed a 6q15/centromere 6 dual-labeling probe for fluorescence in situ (FISH) analysisResultsHeterozygous 6q15 deletions were found in 202 (18%) of 1099 interpretable cancers, including 19% of 804 cancers of no special type (NST), 3% of 29 lobular cancers, 7% of 41 cribriform cancers, and 28% of 18 cancers with papillary features. Homozygous deletions were not detected. In the largest subset of NST tumors, 6q15 deletions were significantly linked to advanced tumor stage and high grade (p < 0.0001 each). 6q deletions were also associated with estrogen receptor negativity (p = 0.0182), high Ki67 proliferation index (p < 0.0001), amplifications of HER2 (p = 0.0159), CCND1 (p = 0.0069), and cMYC (p = 0.0411), as well as deletions of PTEN (p = 0.0003), 8p21 (p < 0.0001), and 9p21 (p = 0.0179). However, 6q15 deletion was unrelated to patient survival in all cancers, in NST cancers, or in subsets of cancers defined by the presence or absence of lymph-node metastases.ConclusionOur data demonstrate that 6q deletion is a frequent event in breast cancer that is statistically linked to unfavorable tumor phenotype and features of genomic instability. The absence of any prognostic impact argues against a clinical applicability of 6q15 deletion testing in breast cancer patients.

Identification of a Somatic Mutation-Derived Long Non-Coding RNA Signatures of Genomic Instability in Renal Cell Carcinoma.

BackgroundRenal cell carcinoma (RCC) is a malignant tumor with high morbidity and mortality. It is characterized by a large number of somatic mutations and genomic instability. Long non-coding RNAs (lncRNAs) are widely involved in the expression of genomic instability in renal cell carcinoma. But no studies have identified the genome instability-related lncRNAs (GInLncRNAs) and their clinical significances in RCC.MethodsClinical data, gene expression data and mutation data of 943 RCC patients were downloaded from The Cancer Genome Atlas (TCGA) database. Based on the mutation data and lncRNA expression data, GInLncRNAs were screened out. Co-expression analysis, Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were conducted to explore their potential functions and related signaling pathways. A prognosis model was further constructed based on genome instability-related lncRNAs signature (GInLncSig). And the efficiency of the model was verified by receiver operating characteristic (ROC) curve. The relationships between the model and clinical information, prognosis, mutation number and gene expression were analyzed using correlation prognostic analysis. Finally, the prognostic model was verified in clinical stratification according to TCGA dataset.ResultsA total of 45 GInLncRNAs were screened out. Functional analysis showed that the functional genes of these GInLncRNAs were mainly enriched in chromosome and nucleoplasmic components, DNA binding in molecular function, transcription and complex anabolism in biological processes. Univariate and Multivariate Cox analyses further screened out 11 GInLncSig to construct a prognostic model (AL031123.1, AC114803.1, AC103563.7, AL031710.1, LINC00460, AC156455.1, AC015977.2, ‘PRDM16-dt’, AL139351.1, AL035661.1 and LINC01606), and the coefficient of each GInLncSig in the model was calculated. The area under the curve (AUC) value of the ROC curve was 0.770. Independent analysis of the model showed that the GInLncSig model was significantly correlated with the RCC patients’ overall survival. Furthermore, the GInLncSig model still had prognostic value in different subgroups of RCC patients.ConclusionOur study preliminarily explored the relationship between genomic instability, lncRNA and clinical characteristics of RCC patients, and constructed a GInLncSig model consisted of 11 GInLncSig to predict the prognosis of patients with RCC. At the same time, our study provided theoretical support for the exploration of the formation and development of RCC.

Abstract 2050: Comprehensive genomic instability characteristics analysis in Chinese patients with osteosarcoma

Abstract Background: Previous study indicated that &amp;gt;80% of osteosarcomas (OSs) have genomic instability characteristics similar to BRCA1/2-deficient cancers. Such BRCA-like traits could manifest a deficiency in the DNA damage repair (DDR) pathway. Patients with alterations in DDR pathway was reported sensitive to platinum-based chemotherapy and PARP inhibitors. In order to seek more precision therapeutic opportunities, we explored the molecular profile of Chinese OS patients to identify whether the analogous phenomena exists as reported. Material and Methods: 44 archival Chinese OS patients from 3D Medicines and a public dataset including 35 OS patients (download from cBioPortal), were collected and analyzed next-generation sequencing (NGS) results on tumor tissues for gene mutations, copy number alterations (CNAs) and fusions in 33 cancer-associated DDR genes. Simultaneously, 44 Chinese OS patients' tumor analysis generated homologous recombination deficiency (HRD) scores (defined as the unweighted sum of LOH, TAI and LST scores) through the 733 gene panel sequencing. Results: Among 44 Chinese OS patients, 18 (40.9%) of OS had DDR pathway gene somatic alterations, and TP53 (31.8%) were the most frequently mapped gene. Other DDR somatic alterations were identified in BRCA2 (6.8%), BARD1 (4.5%), BRCA1 (2.3%), PMS2 (2.3%), FANCA (2.3%) and CHEK1 (2.3%). It was worth mention that all gene alterations were copy number reduction except TP53. Collectively, the frequencies of HRR and mismatch repair (MMR) alterations were 15.9% and 2.3%, respectively. HRD score were available in 18 patients, the median was 40.5 (range, 0-81). As to public dataset, 6 (17.1%) patients harbored somatic alterations in DDR genes. The variations were BRCA2 (5.7%), ATR (2.9%), BARD1 (2.9%), FANCC (2.9%), MLH1 (2.9%), MSH6 (2.9%) and PMS2 (2.9%). The HRR mutations frequency was 11% and MMR was 8.6%. Conclusions: This is the first large-scale comprehensive genomic instability characteristics analysis for Chinese patients with OS. Chinese patients and public dataset patients covers many common features. The DDR gene alteration frequency and HRD score are higher in OS patients, the association of variations in DDR genes and HRD score could be considered substitutable in predicting platinum-based therapy and PARP inhibitor response in clinical practice. Citation Format: Yonggang Wang, Hongtao Li, Jing Xia, Shiqing Chen, Yuezong Bai. Comprehensive genomic instability characteristics analysis in Chinese patients with osteosarcoma [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 2050.

Viral Vectors as Gene Therapy Agents for Treatment of Glioblastoma.

Simple SummaryGlioblastoma is the most malignant cancer of the brain and current therapeutic strategies are clearly inadequate. In addition to surgical intervention, conventional drugs and ratio-therapy, scientists are looking at approaches based on gene therapy with genetically modified viruses. In this review we give a snapshot of the current state of play in this field of research and the available information about the clinical trials. We make some suggestions as to what opportunities could be explored further and hope that this review will stimulate discussion and conception of new life saving strategies.In this review, we scrutinize the idea of using viral vectors either as cytotoxic agents or gene delivery tools for treatment of glioblastoma multiforme (GBM) in light of the experience that our laboratory has accumulated over ~20 years when using similar vectors in experimental neuroscience. We review molecular strategies and current clinical trials and argue that approaches which are based on targeting a specific biochemical pathway or a characteristic mutation are inherently prone to failure because of the high genomic instability and clonal selection characteristics of GBM. For the same reasons, attempts to develop a viral system which selectively transduces only GBM cells are also unlikely to be universally successful. One of the common gene therapy approaches is to use cytotoxic viruses which replicate and cause preferential lysis of the GBM cells. This strategy, in addition to its reliance on the specific biochemical makeup of the GBM cells, bears a risk of necrotic cell death accompanied by release of large quantities of pro-inflammatory molecules. On the other hand, engaging the immune system in the anti-GBM response seems to be a potential avenue to explore further. We suggest that a plausible strategy is to focus on viral vectors which efficiently transduce brain cells via a non-selective, ubiquitous mechanism and which target (ideally irreversibly) processes that are critical only for dividing tumor cells and are dispensable for quiescent brain cells.

Macrophages Promote DNA Repair of Double Strand Break in Multiple Myeloma Cells By Non-Homologous End Joining(NHEJ), Nevertheless Decrease Its Accuracy

Multiple myeloma (MM) is a hematological malignancy of B cells, characterized by clonal proliferation of malignant plasma cells. DNA damage and genomic instability play an important role in the pathogenesis of MM. Based on the characteristics of high heterogeneity and genomic instability of MM, and the protective effect of MΦs on MM cells (MMCs), our study intended to further clarify whether MΦs affect MMCs DNA damage response (DDR) and DNA repair, and the relationship between MΦs and genomic instability of MMCs. We found that the content of MΦs in bone marrow biopsy of MM patients was related to the results of cytogenetics (tested by FISH). The higher the content of MΦs, the more complicated the cytogenetic abnormalities of patients, especially in the IgH translocation, D13S319 locus deletion and RB1 deletion. In our study, MΦs were harvested from peripheral blood monocytes (PBMCs) , which were incubated for 7 days with M-CSF. Flow cytometry was used to detect M-CSF induced macrophages (MΦs) in vitro, and CD163 and CD206 were highly expressed in MΦs which implied that MΦs tended to be M2 type. The incubated MΦs were used in the following experiments. Our study showed that MΦs reduced the baseline γH2AX of MMCs, and contributed to MMCs surviving in the case of genomic instability detected by Western blot and immunofluorescence. We also confirmed that MΦs contribute to repairing the DNA damage in myeloma cells with the methods of comet assay. In the case of severe injury of MMCs' DNA, MΦs promoted the DDR and DNA damage repair. We examined the effects of macrophages on HR and NHEJ using U2OS cells. HR repair was measured in U2OS-HR cells loaded with SCR reporter (HR reporter) while NHEJ repair was measured in U2OS-NHEJ cells loaded with vGEJ reporter (NHEJ Reporter). We found that macrophages increased NHEJ but had no sense on total HR. In order to detect NHEJ level in endogenous genes, we adopted paired gRNA-CRISPR/Cas9 system. AAVS1 and HBB were used as detection genes, and the sequence of about 250 bp near the NHEJ interface was sequenced by NGS. The results proclaimed that the MΦs co-culture group significantly increased the efficiency of NHEJ, and decreased proportion of accurate NHEJ repair. In addition, analysis of the length of base sequence loss showed that the probability of base loss &gt;3 bp in the MΦs co-culture group was higher than that MMCs in the group cultured alone. In the HBB site, MΦs also prolonged the average length of base loss in NHEJ. Furthermore, we used gRNA-CRISPR/Cas9 technology to cause fixed-point cleavage in AAVS1 and HBB respectively, and detected translocation by PCR and NGS with comparing the translocation reads between different groups. The results showed that MΦs promoted the probability of chromosomal translocation, which was of great importance in MM's occurrence and progression. Disclosures No relevant conflicts of interest to declare.

Macrophages promote DNA repair of double strand break in multiple myeloma cells by non-homologous end joining(NHEJ), nevertheless sacrifice the accuracy of NHEJ

Multiple myeloma (MM) is a hematological malignancy of B cells, characterized by clonal proliferation of malignant plasma cells. DNA damage and genomic instability play an important role in the pathogenesis of MM. Based on the characteristics of high heterogeneity and genomic instability of MM, and the protective effect of MΦs on MM cells (MMCs), our study intended to further clarify whether MΦs affect MMCs DNA damage response and DNA repair, and the relationship between MΦs and genomic instability of MMCs.

Imaging features of genomic instability in circulating tumor cells (CTCs) from metastatic triple-negative breast cancer (metTNBC) patients (pts) who received PIKTOR.

e14527 Background: A subset of TNBCs have homologous recombination deficiency (HRD) with upregulation of compensatory DNA repair pathways. A combination of TAK-228 and TAK-117 (PIKTOR), investigational oral TORC1/2 and PI3Kα selective inhibitors, respectively, is hypothesized to increase genomic instability (GI) and decrease double strand DNA repair. CTCs from metTNBC pts are being characterized for imaging features that may correlate GI in blood obtained at baseline (BL) prior to beginning PIKTOR, and at disease progression (PD) on PIKTOR. Pts are treated with a cisplatin-based regimen after PD on PIKTOR. Methods: Following IRB-approved informed consent, metTNBC pts received 4 mg PO TAK-228 and 200 mg PO TAK-117 QDx3d QW until PD. Blood samples collected at BL and at PD were sent to Epic Sciences for CTC analyses including enumeration, cell morphology, phenotypic heterogeneity, and GI analysis via a previously developed GI prediction algorithm based on cell phenotypes (Jendrisak et al AACR 2018). Results: The 9 pts treated with PIKTOR to date had mets in lymph nodes (n = 8), lung (n = 5), chest wall (n = 2), and bone (n = 2). Median number of prior regimens for metTNBC was 2; median duration of PIKTOR treatment was 8 wks (range, 5-15). CTCs were detected in 62.5% (5/8) BL and 100% (9/9) PD samples. 2 pts had high phenotypic CTC heterogeneity at both BL and PD (pt#36: 0.69 to 0.64; pt#80: 0.93 to 1.41), 1 pt had increased heterogeneity (pt#75: 0 to 1.68) and 1 pt had decreased heterogeneity (pt#67: 1.26 to 0) post-PIKTOR. 1 pt had predicted phenotypic GI (pGI+) CTCs detected at both BL and PD (pt#80), 1 pt had pGI+ CTCs at BL only (pt#36) and 1 pt at PD only (pt#75). Higher expression of the epithelial marker, cytokeratin (CK), and larger cell size were observed in the PD samples. Conclusions: Following treatment with and PD on PIKTOR, higher CK expression, higher phenotypic heterogeneity, and more pGI+ CTCs were observed in a subset of pts, suggesting that these TN CTCs may have developed epithelial transition and greater GI with PIKTOR treatment. Accrual continues and CTC GI biomarkers will be correlated with pts’ response to subsequent cisplatin-based therapy.

Association of BRCA1- and BRCA2-deficiency with mutation burden, expression of PD-L1/PD-1, immune infiltrates, and T cell-inflamed signature in breast cancer.

Immune checkpoint inhibitors have demonstrated effective anti-tumour response in cancer types with high mutation burden (e.g. melanoma) and in subset of cancers with features of genomic instability (e.g. mismatch-repair deficiency). One possible explanation for this effect is the increased expression of immune checkpoint molecules and pre-existing adaptive immune response in these cancers. Given that BRCA1 and BRCA2 are integral in maintaining genomic integrity, we hypothesise that the inactivation of these genes may give rise to breast cancers with such immunogenic phenotype. Therefore, using two large series of publicly available breast cancer datasets, namely that from The Cancer Genome Atlas and Wellcome Trust Institute, we sought to investigate the association between BRCA1- and BRCA2-deficiency with features of genomic instability, expression of PD-L1 and PD-1, landscape of inferred tumour-infiltrating immune cells, and T-cell inflamed signature in breast cancers. Here, we report that BRCA1 and BRCA2-deficient breast cancers were associated with features of genomic instability including increased mutation burden. Interestingly, BRCA1-, but not BRCA2-, deficient breast cancers were associated with increased expression of PD-L1 and PD-1, higher abundance of tumour-infiltrating immune cells, and enrichment of T cell-inflamed signature. The differences in immunophenotype between BRCA1- and BRCA2-deficient breast cancers can be attributed, in part, to PTEN gene mutation. Therefore, features of genomic instability such as that mediated by BRCA1- and BRCA2- deficiency in breast cancer were necessary, but not always sufficient, for yielding T cell-inflamed tumour microenvironment, and by extension, predicting clinical benefit from immunotherapy.

17 Quantifying microenvironmental signatures of oesophageal carcinogenesis

IntroductionThe tumour-immune crosstalk has been successfully exploited in recent immunotherapy trials, with clear impact in several highly mutated late stage cancers. To what extent it may be exploited at an earlier stage in the cancer’s history is currently unclear. Oesophageal adenocarcinoma is a cancer with dismal survival and early onset of metastases. We have previously described three subtypes of this cancer with distinct therapeutic relevance based on signatures of mutational processes observed during cancer development. Secrier, Li et al, Nat Genet 2016 In particular, an acid reflux-linked signature defines a subgroup of patients with enhanced immune activity that may benefit from immunotherapy approaches. In this work we question whether similar features of tumour-immune crosstalk may precipitate cancer progression in the oesophagus.Material and methodsAiming to explore multiple aetiology backgrounds, we employed a cohort of oesophageal tumours from the Cancer Genome Atlas (n=184) incorporating both adenocarcinomas and squamous cell carcinomas. We used extensively curated signatures of immune function and in-house computational methods to score immune activity from bulk RNA-seq data. Matched whole-exome sequencing data was used to quantify signatures of selected mutational processes and features of genomic instability.Results and discussionsThe activity of cancer associated fibroblasts was significantly associated with defined markers of cancer progression, both in adenocarcinomas as well as squamous cell carcinomas. Signals of rapid cancer progression were stronger in the highly immunogenic subtype, and were accompanied by elevated patterns of macrophage activity, defined on a spectrum of inhibition to stimulation signals. These immune determinants of progression showed similar trends across tissues. Subsequent integration of genomic instability characteristics and specific mutational triggers suggested that complex tumour-immune interactions may be contributing to oesophageal cancer progression in a subtype-specific manner.ConclusionWe have previously demonstrated that historic mutational processes in oesophageal adenocarcinoma unveil distinct trajectories of cancer development, with differentiated impact on immune composition. This work shows a strong microenvironmental contribution to rapid cancer progression, and highlights the importance of stromal cell dynamics in this process. We propose quantifiable features of tumour-immune crosstalk in oesophageal cancer subtypes with potential predictive value.

DNA Damage and Repair Biomarkers of Immunotherapy Response.

DNA-damaging agents are widely used in clinical oncology and exploit deficiencies in tumor DNA repair. Given the expanding role of immune checkpoint blockade as a therapeutic strategy, the interaction of tumor DNA damage with the immune system has recently come into focus, and it is now clear that the tumor DNA repair landscape has an important role in driving response to immune checkpoint blockade. Here, we summarize the mechanisms by which DNA damage and genomic instability have been found to shape the antitumor immune response and describe clinical efforts to use DNA repair biomarkers to guide use of immune-directed therapies.Significance: Only a subset of patients respond to immune checkpoint blockade, and reliable predictive biomarkers of response are needed to guide therapy decisions. DNA repair deficiency is common among tumors, and emerging experimental and clinical evidence suggests that features of genomic instability are associated with response to immune-directed therapies. Cancer Discov; 7(7); 675-93. ©2017 AACR.

Abstract IA10: Biomarker selected therapy for pancreas ductal adenocarcinoma: an emerging reality?

Abstract Pancreas adenocarcinoma remains a major challenge hallmarked by several notables, including a rising incidence, modest effectiveness of current state-of-the-art therapies and a dearth of validated biomarkers for clinical decision making. With regard to germline genomics, about 5-8% of pancreas adenocarcinoma patients will harbor a mutation in BRCA2, BRCA1 and PALB2. These mutations amongst others lead to impaired DNA repair and are identified to be important biomarkers of genomic instability as well as of response to DNA damaging agents. Genomic instability, a result of deleterious mutations of genes functioning in homologous repair mediated DNA repair pathways, yields an exceptional vulnerability in cancer cells named synthetic lethality. Recent studies have elucidated that platinum agents and Poly-ADP-ribose polymerase (PARP) inhibitors may be effective to induce tumor regression in solid tumors bearing an unstable genome including pancreatic cancer. The impact of somatic BRCA mutations on response to DNA targeting agents is currently being investigated. A recent whole genome sequencing study by Waddell et al reported overlapping features in both somatic and germline mutations such as unstable genome along with possible DNA targeting agent sensitivity in both groups. This suggests that gained mutation of DNA repair genes throughout the oncogenesis process may also create synthetic lethality and a larger subset of patients may benefit from these evolving treatment approaches. The characteristics of genomic instability in pancreatic cancer along with its clinical implications and the utility of DNA targeting agents particularly PARP inhibitors as novel treatment approaches will be discussed. Citation Format: Eileen M. O’Reilly.{Authors}. Biomarker selected therapy for pancreas ductal adenocarcinoma: an emerging reality? [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr IA10.

Characterization of copy number alterations in a mouse model of fibrosis-associated hepatocellular carcinoma reveals concordance with human disease.

Hepatocellular carcinoma (HCC) is a prevalent human cancer with rising incidence worldwide. Human HCC is frequently associated with chronic liver inflammation and cirrhosis, pathophysiological processes that are a consequence of chronic viral infection, disturbances in metabolism, or exposure to chemical toxicants. To better characterize the pathogenesis of HCC, we used a human disease‐relevant mouse model of fibrosis‐associated hepatocarcinogenesis. In this model, marked liver tumor response caused by the promutagenic chemical N‐nitrosodiethylamine in the presence of liver fibrosis was associated with epigenetic events indicative of genomic instability. Therefore, we hypothesized that DNA copy number alterations (CNAs), a feature of genomic instability and a common characteristic of cancer, are concordant between human HCC and mouse models of fibrosis‐associated hepatocarcinogenesis. We evaluated DNA CNAs and changes in gene expression in the mouse liver (normal, tumor, and nontumor fibrotic tissues). Additionally, we compared our findings to DNA CNAs in human HCC cases (tumor and nontumor cirrhotic/fibrotic tissues) using publicly available data from The Cancer Genome Atlas (TCGA). We observed that while fibrotic liver tissue is largely devoid of DNA CNAs, highly frequently occurring DNA CNAs are found in mouse tumors, which is indicative of a profound increase in chromosomal instability in HCC. The cross‐species gene‐level comparison of CNAs identified shared regions of CNAs between human fibrosis‐ and cirrhosis‐associated liver tumors and mouse fibrosis‐associated HCC. Our results suggest that CNAs most commonly arise in neoplastic tissue rather than in fibrotic or cirrhotic liver, and demonstrate the utility of this mouse model in replicating the molecular features of human HCC.

Xq28 Duplications and Epilepsy: Influence of the Combinatory Duplication of MECP2 and GDI1

Xq28 duplications including the MECP2 (Methyl-CpG-binding protein 2) cause an X-linked recessive neurodegenerative disorder that presents symptoms such as early developmental delay, progressive deterioration, and intractable epilepsy. Submicroscopic interstitial duplication in the MECP2 region is one of the most frequently observed submicroscopic chromosomal aberrations in patients with intellectual disability. This high prevalence is derived from the genomic instability characteristic of this region due to segmental duplications that are densely located in this region, which are prone to cause nonallelic homologous recombination during meiosis. Patients with MECP2 duplications generally begin to show epilepsy after 6 years of age. Therefore, incidence of epilepsy is high in adolescents. Although many types of seizures have been reported in patients, the most frequently observed seizure types were absence seizure and generalized tonic–clonic seizures. Because drop attacks are frequently seen, such epileptic attacks are sometimes life-threatening. We encountered a patient who showed epileptic spasms during infancy with a relatively larger duplication that encompasses MECP2 and GDI1 (GDP dissociation inhibitor-1). This led us to speculate that the combinatory duplication that includes MECP2 and GDI1 may cause severe epileptic features; however, most of the previously reported patients with combined duplications of MECP2 and GDI1 did not showed early occurrence of epilepsy. Therefore, further information would be required to confirm if the combinatory duplication of MECP2 and GDI1 exerts any influence on the severity of epilepsy.