Genomic Loss Research Articles

Abstract 4499: Genomic editing of EWS-FLI1 and its targets, and its therapeutic potential in treatment of Ewing sarcoma

Abstract Ewing sarcoma (ES) is an aggressive bone and soft tissue tumor. These tumors have a low mutational burden and are mainly driven by chimeric transcription factor, EWS-FLI1 or equivalent fusion gene. Tumor dependency on EWS-FLI1 makes it an ideal therapeutic target but targeting this disordered protein has proven to be a challenge. Here we show that CRISPR-Cas9-mediated genomic editing can a) be employed to effectively target EWS-FLI1 and its target genes, both coding and non-coding, and b) be safely delivered to tumors in mice, thus highlighting its potential as a therapeutic agent. All CRISPR-Cas9 reagents (ThermoFisher Scientific) including Cas9 protein, single guide RNA (sgRNA) directed against various gene targets and controls, and LentiArrayTM lentiviruses were obtained and optimized for in vitro and in vivo use. Cas9-sgRNA ribonucleoprotein (RNP) complexes were transfected using CRISPRMAXTM or delivered to ES cells by CD99-targeted nanoparticles. We were able to achieve about 70% genomic cleavage when we targeted the fusion gene, EWS-FLI1, and its coding target, NR0B1, using two different sgRNA delivered as RNP complexes. Loss of EWS-FLI1 slowed down cell growth, but increased cell adhesion, and cell invasion. For EWS-FLI1 target, long non-coding RNA, FEZF1-AS1, we used paired sgRNA targeting two different regions of the RNA to achieve effective genomic editing (70%) and loss of transcript expression. We further used a lentiviral-generated stable Cas9 expressing A673 cell line to effectively knockout FEZF1-AS1 using lentiviruses expressing paired sgRNA. Both the RNP and lentiviral mediated FEZF1-AS1 knockdown did not affect cell growth but significantly decreased cell invasion. Dual knockdown of EWS-FLI1 and FEZF1-AS1 decreased cell growth and cell invasion. Mice with ES tumors in their flanks were treated with intravenous injections of CD99-targeted nanoparticles carrying Cas9-sgRNA RNP. Mice injected with Cas9-scrambled gRNA RNP had regular tumor growth. Cas9-EWSR1 sgRNA RNP-treated mice had nearly completely suppressed tumor growth for the duration of treatment but failed to totally eliminate the tumors. In conclusion, we show that CRISPR-Cas9 mediated genomic editing can effectively target EWS-FLI1 and disrupt its function in ES pathogenesis. We further demonstrate that modified CRISPR strategy can effectively knockout non-coding RNA and that the combined targeting of EWS-FLI1 and its lncRNA target had an added therapeutic response in controlling tumor growth and invasion. The pilot animal studies (more detailed studies underway) emphasize the importance of developing CRISPR-Cas9 as a therapeutic tool provided we can safely deliver it in vivo using vehicles like targeted nanoparticles to help reduce toxicity caused by untoward targeting effects. Citation Format: Sheetal A. Mitra, Namritha Ravinder, Veronica Magnon, Jon Nagy, Timothy J. Triche. Genomic editing of EWS-FLI1 and its targets, and its therapeutic potential in treatment of Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4499.

Abstract CT179: ATLAS: A Phase II, open-label study of rucaparib in patients with locally advanced or metastatic urothelial carcinoma

Abstract Background: For patients with metastatic urothelial carcinoma (mUC) that has progressed on or after platinum-based chemotherapy (cisplatin or carboplatin) and/or immune checkpoint inhibitors, there are currently no standard treatments, emphasizing the urgent unmet need for additional treatment options. Based on analysis of The Cancer Genome Atlas bladder cancer dataset, ≈60% of muscle invasive bladder tumors have homologous recombination (HR) deficiency (HRD), as defined by high genomic loss of heterozygosity (LOH) or a deleterious mutation in a DNA repair pathway gene. Poly(ADP-ribose) polymerase (PARP) inhibitors such as rucaparib exert their antitumor activity through synthetic lethality in cancer cells with HRD. Tumors with HRD have been shown to be sensitive to PARP inhibitors in breast, ovarian, and prostate cancers, and PARP inhibitors are approved for the treatment of patients with breast or ovarian cancer. Although there are little data on their activity in mUC, we hypothesize that PARP inhibition may have antitumor activity in this tumor type. The ATLAS (NCT03397394) trial is evaluating the efficacy and safety of the PARP inhibitor rucaparib as monotherapy for patients with locally advanced (unresectable) urothelial carcinoma or mUC previously treated with 1-2 anticancer treatments for advanced/metastatic disease. Methods: Eligible patients must have received 1-2 prior treatments (eg, platinum-based chemotherapy, immune checkpoint inhibitor, and/or clinical trial agent) and have radiographic progression, measurable disease (RECIST version 1.1), and adequate organ function. Tumor HRD is not required as rucaparib may potentially benefit patients with HR-proficient or HR-deficient tumors; however, fresh tumor or recently obtained archival tissue is mandatory for central HRD profiling. Prior PARP inhibitor treatment is an exclusion criterion. All patients will receive rucaparib monotherapy (600 mg BID) until disease progression or other reason for discontinuation. The primary endpoint is confirmed objective response rate (investigator assessed per RECIST version 1.1) in the intent-to-treat and HRD-positive (signature based on tumor genomic LOH) populations. Secondary endpoints include response duration, progression-free survival, overall survival, safety, and pharmacokinetics. Exploratory endpoints include evaluating plasma and tumor samples for biomarkers associated with response and resistance to rucaparib. The study has >90% power to reject the null hypothesis (P=0.10) at a one-sided 5% significance level if the true response rate for rucaparib is 20%. ATLAS is currently enrolling at ≈65 sites in 6 countries (France, Germany, Italy, Spain, United Kingdom, and United States), with a target enrollment of 200 patients. Citation Format: Petros Grivas, Min Yuen Teo, Nicholas Vogelzang, Ajjai Alva, Yousef Zakharia, Nabil Adra, Alexandra Drakaki, Arif Hussain, Rafael Morales-Barrera, Andrea Necchi, Alejo Rodriguez-Vida, Susan Feyerabend, Sumati Gupta, Debra H. Josephs, Yohann Loriot, Sandy Srinivas, Kenton Wride, Daleen Thomas, Rachel Dusek, Andrew D. Simmons, Dale Nepert, Simon Chowdhury. ATLAS: A Phase II, open-label study of rucaparib in patients with locally advanced or metastatic urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT179.

PF514 LOSS OF 18Q22.1‐Q23 IS SIGNIFICANTLY ASSOCIATED WITH GCB‐DLBCL AND FAILURE TO ACHIEVE EFS24

Background:Recent large next generation sequencing studies have redefined the genomic landscape of diffuse large B‐cell lymphoma (DLBCL). While many of the more common genetic variants, primarily mutations, have been biologically characterized, little is known about newly emerging genetic aberrations. In particular, somatic copy number alterations remain poorly studied due to the large genomic regions they cover and the number of genes involved. This, in combination with lack of remission or early relapse in a portion of DLBCL, opens new opportunities to explore yet unidentified genetic contributions to lymphomagenesis.Aims:The goal of this study was to identify variants associated with failure to achieve event free survival at 24 months (EFS24) and define their biologic significance in DLBCL.Methods:Newly diagnosed DLBCL treated with standard immunochemotherapy were included in this study (n = 246). Copy number data was generated by using the molecular inversion probe OncoScan™ FFPE Assay Kit (Affymetrix, Santa Clara, CA, USA). OncoScan OSCHP files were analyzed using Nexus Copy Number 9.0 software (Biodiscovery, El Segundo, USA). Data interpretation and copy number calling was done using the human reference genome GRCh37/hg19. Files were analyzed using the Nexus FASST2‐Segmentation algorithm, which is based on a Hidden Markov Model approach for calling genetic events. For functional analysis, SOCS6 was overexpressed and studied in GCB‐DLBCL cell lines.Results:Upon review of genomic copy number data from DLBCL cases (n = 246), loss of 18q22.1‐q23 was a significant event in our cohort (p = 0.018), is found in 11% (n = 27) of DLBCL, and is enriched in germinal center DLBCL (GCB‐DLBCL) patients (18/107, 17%, p < 0.05) compared to activated B‐cell (ABC)/non‐GCB DLBCL patients (1/93, 1%). Furthermore, GCB‐DLBCL patients with a loss of 18q22.1‐q23 were significantly more likely to fail EFS24 (50%, p < 0.05) compared to GCB‐DLBCL patients without the loss (29%). Genes located at 18q22.1‐q23 include, TMX3, DOK6 and SOCS6. SOCS family proteins have been shown to play an important role in tumor biology, although little is known about SOCS6. SOCS6 gene expression is downregulated in a variety of solid tumors and is hypermethylated in some hematological malignancies. RNASeq data from DLBCL suggest variable expression across tumors. Using a panel of GCB cell lines we found that SOCS6 expression was low to negative, and publically available cell line data suggests this region is hypermethylated. Expression of SOCS6 was higher in a panel of mantle cell lymphoma and ABC‐DLBCL cell lines and this again correlated with their cell line methylation data, where no hypermethylation was found in those cell lines. Together, this data suggests that downregulation of SOCS6 through genomic loss or hypermethylation may have biologic importance. To explore the role of SOCS6, we restored expression in GCB cell lines and immuno‐fluorescence staining suggest localization to the nucleus and cytoplasm, as previously reported in other cell types. Studies to explore the role of SOCS6 in cytokine signaling and STAT family protein regulation are ongoing.Summary/Conclusion:Using genome copy number data from 246 DLBCL, we have found that loss of 18q22.1‐q23 (p = 0.018) is associated with GCB‐DLBCL and failure to achieve EFS24. Furthermore, we have identified that SOCS6 may have clinical and biologic significance in GCB‐DLBCL.

The role of apoptosis defects in malignant mesothelioma pathogenesis with an impact on prognosis and treatment.

Malignant mesothelioma (MM) is a highly aggressive tumor that is strongly related to asbestos fiber exposure. The tumorigenesis procedure in MM is complex, and many pathogenetic mechanisms including chronic inflammation, deregulation of cell death, and the genomic copy-number losses and gains may contribute to carcinogenesis. MM cells are resistant to TRAIL-mediated apoptosis due to defects in extrinsic apoptotic pathway. CAPS, a regulator of cell cycle and death, may contribute to the MM development as well. BAP1 is the most frequently inactivated gene in MPM; BAP1 deficiency triggers malignant transformation via disruption of DNA repair, transcription regulation, cell metabolism, apoptosis, and ferroptosis. In addition, bcl-2 family proteins as well as abnormal activation of PI3K/Akt/mTOR pathway and deregulation of the Wnt signaling pathway may result in MM tumorigenesis. Finally, the Hippo pathway plays a critical role in MPM development. Mutations of NF2 and LATS lead to YAP activation in MPM. Thus, inhibition of YAP activity by YAP inhibitors could be a potentially promising treatment option for MM. In conclusion, extensive genetic alterations exist in mesotheliomas associated with the signaling of apoptotic HM cells death. The comprehension of these pathways may contribute to enhancing survival via developing new effective therapeutic strategies.

TAKTIC: A prospective, multicenter, uncontrolled, phase IB/II study of LY2780301 (LY) in combination with weekly paclitaxel (wP) in HER2-negative locally advanced (LA) or metastatic breast cancer (MBC) patients.

1091 Background: Phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR)-pathway is frequently activated in HER2-negative breast cancer and may play a role in taxane-resistance. LY is a dual inhibitor of p70 S6 kinase and AKT. TAKTIC study aimed to determine the recommended phase II dose (RP2D) of the combination of LY with wP (phase Ib) and to estimate overall response rate (ORR) of this regimen (phase II) in HER2-negative LA or MBC patients, both in the overall patient population and in patients with activation of PI3K/AKT pathway (PI3KAKT+). Methods: HER2-negative inoperable LA or MBC patients (pts), with (phase Ib) or without (phase II) previous cytotoxic treatment for advanced disease were eligible. Oral LY (400 or 500mg) was administered daily in combination with intravenous wP (70 or 80mg/m2). A modified CRM using an adaptive Bayesian model guided the dose escalation of both agents. PI3KAKT+ was defined as activating mutation of PIK3CA and/or AKT by targeted NGS or homozygous loss of PTEN by array comparative genomic hybridization (aCGH) or loss of PTEN by immunohistochemistry, as evaluated on available fresh tumor tissue. Results: A total of 12 and 35 patients (pts) were included in the phase Ib and II, respectively. In phase Ib, only 1 dose-limiting toxicity (confusion) was observed at the last dose level (LY, 500 + wP, 80), which was determined as RP2D. Main drug-related adverse events (AE) in phase Ib were skin toxicity (92% of pts, G3-4 in 33%), and paresthesia (50% of pts, G3-4 in 8%). In the phase II study, ORR was 62.9 % [44.9,78.5] including 1 CR and 21 PR in the overall population and 55.6 % [30.8,78.5] in PI3KAKT+ pts (10 PR in 18 pts). Median progression-free survival was 12.4 months [7.9,17.9] and 6-month clinical benefit rate was 82.9% [66.4,93.4]. AEs in phase II were similar to phase I part, except that 17% of pts experienced pneumonia (G3-4 in 9%). Conclusions: Combining LY and wP in HER2-negative LA or MBC was feasible with preliminary evidences of efficacy, independently of PI3K/AKT activation. Clinical trial information: NCT01980277.

Differential copy number alteration profiles among gynecological malignancies.

e14744 Background: Copy number alterations (CNA) constitutes a genomic hallmark that defines specific outcomes in gynecological neoplasms, as evidenced by the integrated analysis of the high-grade serous ovarian carcinoma (HGSOC) and endometrial carcinomas (EC) performed by the TCGA Consortium. The purpose of this study is to set up an NGS-based technology to obtain CNA profiles on formalin-fixed paraffin-embedded (FFPE) samples with clinical utility. Methods: FFPE blocks from 14 EC and 12 HGSOC were analyzed using a SureSelectXT custom panel plus OneSeq backbone, interrogating 147.000 SNPs (Agilent). CNA groups were defined in EC with a RF algorithm based on genotyping data as previously reported (Romero, ASCO 2018) and in HGSOC Homologous recombination deficiency (HRD) was determined by sequencing a serie of 35 HR genes. saasCNV pipeline was used to perform CNA calling. Results: Ten (38.50 %) of our cases were classified as CN Low (CNL), 8 as CN high (CNH)-RF (30.75 %) and 8 CNH-HRD (30.75 %). CNL profiles from EC and OC are homogenous in terms of genomic stability. CNH-RF and CNH-HRD cases harbor the highest percentage of genomic instability (p = 0.012) with distinct patterns of CNA. CNH-HRD is characterized by a higher ratio of genomic losses (p = 0.023). However, CNH-RF is mainly defined by gains (p = 0.011) (Table). Conclusions: Our NGS-based approach represents a FFPE clinically available procedure to define CNA alterations in gynecological cancer with distinct CNH patterns with prognostic potential.[Table: see text]

Profiling of 3,634 cholangiocarcinomas (CCA) to identify genomic alterations (GA), tumor mutational burden (TMB), and genomic loss of heterozygosity (gLOH).

4087 Background: The management of CCA has evolved as targeted and immune checkpoint inhibitor (ICPI) therapies have emerged. We used comprehensive genomic profiling (CGP) to characterize the genomic alterations (GA) that have potential to personalize therapy for CCA. Methods: 3634 CCA underwent hybrid capture based CGP on 0.8-1.1 Mb of the coding genome to identify GAs in exons and select introns in up to 404 genes, TMB, microsatellite status (MSI) and % monoallelic genome (gLOH). PD-L1 expression was determined by IHC (Dako 22C3). Results: 52% of CCA were female with a median age of 62 years (range 16 - > 89). The most common biopsy sites were liver (74%), lymph node (4%), bile duct (3.3%), and lung (2%). MSI-high was rare (1%), 118 and 47 cases had TMB > 10 and > 20 mut/mb respectively. Of the latter, 51% (24/47) were MSI-H. PD-L1 amplification (AMP) was present in 0.27%. Of 490 CCA tested, 43 (9%) were positive for PD-L1 expression. 11% of cases had gLOH > 16%, only 2 cases had both TMB > 20 and gLOH > 16%. GA were most common in TP53 (31%), CDKN2A (29%), KRAS (20%) and ARID1A (17%). Potentially targetable GAs included FGFR2 (11%, 85% fusions), BRAF (5%, 50% V600E), ERBB2 (5%, 72% AMP), MET (2%, 90% AMP), EGFR (0.52%) and rarer ( < 0.5%) FGFR3, RET, FGFR1, ALK, and ROS1 fusions. The FGFR2 fusions had 128 unique 3’ partner genes including BICC1 (26%), CCDC6 (3.2%), AHCYL1 (2.6%) and KIAA1217 (2.6%). FGFR2 fusions occurred in a mutually exclusive fashion from high gLOH (p < 0.002), but not high TMB. GA in IDH1 (15%) were mutually exclusive of FGFR2 fusions (p < 1e-13), but co-occurred with PBRM1 GA (23%, p < 1e-21), ARID1A (26% p < 1e-10). IDH1 GA had gLOH similar to the overall CCA population but were enriched for low TMB (p < 1e-3). Conclusions: Nearly 20% of CCA cases harbor targetable kinase GA, half of which were FGFR2 fusions. Independently, an additional 10% (gLOH) and 1% (high TMB, MSI and/or PD-L1 AMP) may benefit from PARP inhibitors and ICPI respectively. Independently, co-mutation of IDH1 and PBRM1/ARID1A defines a class of CCA that warrants further investigation for sensitivity to PARP inhibitors and may serve as a paradigm for other tumors (ie. gliomas) with a similar co-occurrence landscape.

ATLAS: A phase II open-label study of rucaparib in patients with locally advanced or metastatic urothelial carcinoma (mUC).

TPS496 Background: There are currently no standard treatment options for patients with mUC that has progressed on or after platinum (cisplatin/carboplatin)–based chemotherapy (PBC) and/or immune checkpoint inhibitors (ICIs), emphasizing the need for new therapies. Analysis of The Cancer Genome Atlas bladder cancer dataset suggests that approximately 60% of bladder cancer tumors have homologous recombination deficiency (HRD), as defined by high genomic loss of heterozygosity (LOH) or a deleterious mutation in a homologous recombination pathway gene. The poly(ADP-ribose) polymerase inhibitor (PARPi) rucaparib has demonstrated activity in tumors with HRD and in homologous recombination proficient tumors in the recurrent ovarian cancer setting. The ATLAS (NCT03397394) trial will evaluate the efficacy and safety of rucaparib in patients with locally advanced/unresectable UC or mUC treated with 1–2 prior anticancer treatments. Methods: Eligible patients must have measurable disease per RECIST v1.1, adequate organ function, and radiographic progression after 1–2 prior regimens (eg, PBC and/or ICI). Confirmation of HRD status before enrollment is not required, but fresh tumor tissue or recently obtained archival tissue is mandatory for HRD profiling. Prior PARPi treatment is exclusionary. All patients will receive oral rucaparib 600 mg BID until disease progression or other reason for discontinuation. The coprimary endpoints are confirmed objective response rate (investigator-assessed per RECIST v1.1) in the intent-to-treat and HRD-positive (signature based on tumor genomic LOH) populations. Secondary endpoints include response duration, progression-free survival, overall survival, safety, and pharmacokinetics. Exploratory endpoints include evaluation of molecular biomarkers associated with response and resistance to rucaparib, including changes in plasma and tumor samples, and circulating tumor DNA. Patients are being enrolled in 6 countries (France, Germany, Italy, Spain, UK, and USA), with a target enrollment of 200 patients. The study has > 90% power to reject the null hypothesis (P = 0.10) at a 5% significance level if the true response rate for rucaparib is 20%. Clinical trial information: NCT03397394.

Abstract P3-01-09: Tracing clonal evolution of circulating tumor cells and cell-free DNA in a metastatic breast cancer patient

Abstract Background: Copy number variation analysis has been shown as a valuable tool to infer clonal evolution of tumor cells. While bulk genomic analysis of FFPE tissue can provide information about the characteristics of a primary tumor or a metastatic nodule, these biopsies are rarely repeated due to their invasive nature. Liquid biopsies on the other hand, provide minimally invasive access to tumor derived cells and DNA and can therefore be used to monitor genomic changes of the tumor longitudinally and in real time. Here we evaluate the kinetics of liquid biopsy biomarkers and monitor genomic changes in single circulating tumor cells (CTCs) and cell-free DNA (cfDNA) from 17 longitudinal blood draws collected from a metastatic breast cancer patient over seven treatment regimes. FFPE tissue from the primary tumor and two metastatic sites in liver and bone are available for comparative analysis. Methods: We used the high definition – single cell assay (HD-SCA) platform to detect and enumerate CTCs of the blood of one metastatic breast cancer patient followed for 4 years. CTCs were characterized morphometrically and single CTCs of each draw were sequenced for copy number variation (CNV). cfDNA was extracted from plasma and tissue samples of the primary breast, bone and liver metastasis were micro-dissected. Extracted DNA from tissue samples and plasma underwent CNV analysis and were compared with CNV data of CTCs. Results: CTCs were identified in 14/17 draws and varied from 0 to >3000 cells/ml, spiking at two different times concordant with clinical progression. Changes in cell free tumor DNA fraction (ctDNA) correlated strongly with CTC count. Comparative CNV analysis of CTCs, cfDNA and FFPE tissue confirmed the tumorous origin of both cells and cfDNA. Single cell CNV analysis found four genomically related sub-clones in the patient's blood, three of which were present at time of enrollment, while the fourth clone emerged 3.5 years later concordant with the decline in the patient's health. Genomic copy number losses included tumor suppressor genes such as ARID1A, FOXP1, ATM, BRCA2, RB1, CDH11 and CDH1. Genomic gains were limited but included the proto-oncogenes BCL9, ABL2 and MDM4. It is striking that the sub-clonal structure persisted nearly unchanged for 3.5 years throughout various hormone and cytotoxic treatments, indicating high genomic stability. Despite their genomic differences, cells from the different clones are indistinguishable by immunofluorescence or morphometric analysis. CNV profiles of cfDNA reflect the most abundant CTC clone at each time-point. Conclusion: Using the HD-SCA assay cellular (CTC), and soluble (cfDNA) fractions of liquid biopsies can be compared at the molecular level over a long time course, and verified by comparison with tissue biopsy. This work confirms that liquid biopsies can be used to access the molecular state of a patient's cancer in near real-time and provide insight into the response to treatment years after the original characterization of the primary tissue. Here we show in a single patient example the power of single CTC analysis and highlight how CNV analysis of liquid biopsies could serve as minimally invasive tool to monitor tumor evolution longitudinally. Citation Format: Welter L, Xu L, McKinley D, Dago AE, Restrepo-Vassalli S, Rodriguez Lee M, Kolatkar A, Nieva J, Hicks J, Kuhn P. Tracing clonal evolution of circulating tumor cells and cell-free DNA in a metastatic breast cancer patient [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-09.

Loss of Chromosomes Monitored in Live Cells

Cancer cells exhibit genomic instability, with gains or losses of entire chromosomes and chromosome segments that cause massive changes in gene expression. This aneuploidy occurs early in cancer development and is particularly prominent, ongoing, and variable in solid tumors – stiff tumors in particular. Here we develop live cell fluorescence monitoring for loss or gain of two chromosomes (Ch3 (green) and Ch5 (red)) in a lung cancer-derived cell line in order to quantify factors that affect aneuploidy. In standard culture, ∼10% of mitotic cancer cells show chromosome mis-segregation, but mis-segregation increases several-fold with mechanical pressing during mitosis or drug inhibition of the spindle assembly checkpoint. Because multiple DNA repair factors mis-localize transiently from nucleus to cytoplasm when cells exhibit nuclear envelope rupture, transient knockdown of such physically-regulated factors was also studied, with knockdown cells showing more DNA damage and more chromosome mis-segregation. After such perturbations, more red-fluorescent-null cells are observed indicating a loss of Ch5. The aneuploid changes are confirmed by single cell karyotyping as well as SNP arrays and marker-specific PCR on stable clones. Single cell transcriptomics of these red-null cells further reveal gene expression on Ch5 is significantly downregulated in >80% of cells. In addition, ∼10% total genes on average are mis-regulated more than 2-fold in response to such Ch5 loss. Normal cells are being similarly studied, but viability seems much lower with chromosome loss. Such system thus lend insight into factors affecting genome variation, gene expression, and cell viability.

A phase III, double-blind, randomized study of pamiparib versus placebo as maintenance therapy in patients with inoperable, locally advanced, or metastatic gastric cancer (GC) that responded to platinum-based first-line chemotherapy.

TPS173 Background: Gastric cancer is the fifth most common cancer, and is the third leading cause of cancer deaths worldwide. In patients with locally advanced or metastatic GC, fluoropyrimidine- and platinum-based combination chemotherapy is first-line standard of care. Despite refinement in chemotherapy regimens, outcomes are poor and survival after first-line treatment remains low. A subset of GCs exhibit platinum sensitivity and genomic instability that is characteristic of homologous recombination deficiency (HRD). Poly (ADP-ribose) polymerase proteins 1 and 2 (PARP1/2) are involved in DNA damage repair, and their inhibition is cytotoxic for cells with HRD. Pamiparib is a selective PARP1/2 inhibitor that crosses the blood-brain barrier, has shown potent DNA–PARP trapping, and has demonstrated antitumor activity in preclinical models. In early phase clinical studies (NCT02361723; NCT03333915), pamiparib was generally well tolerated and showed preliminary antitumor activity; 60 mg oral twice daily (BID) was established as the recommended dose. Methods: This ongoing, global, double-blind, placebo-controlled, randomized, multicenter phase III study (NCT03427814) is designed to compare the efficacy, safety, and tolerability of pamiparib vs placebo as maintenance therapy in ~540 patients with advanced GC who have responded to first-line, platinum-based chemotherapy. Patients who are ≤ 8 weeks after their last dose of first-line platinum based chemotherapy will be randomized 1:1 to receive either pamiparib 60 mg BID or placebo in 28-day cycles. Patient randomization will be stratified by genomic loss of heterozygosity status (ie, high vs low), region, and ECOG status. Radiologic assessments will be centrally evaluated per RECIST every 8 weeks after first dose. The primary endpoint is progression-free survival; key secondary endpoints include safety/tolerability, overall survival, objective response rates, time and duration of response, and time to second subsequent treatment. Correlative biomarker analyses in tumor tissues and blood will be performed. Clinical trial information: NCT03427814.

Inter- and intra-tumor heterogeneity of SMAD4 loss in head and neck squamous cell carcinomas.

Reports regarding the frequency of SMAD4 loss in human head and neck squamous cell carcinoma (HNSCC) vary significantly. We have shown that SMAD4 deletion contributes to HNSCC initiation and progression. Therefore, accurately detecting genetic SMAD4 loss is critical to determine prognosis and therapeutic interventions in personalized medicine. We developed a SMAD4 fluorescence in situ hybridization (FISH) assay to identify chromosomal SMAD4 loss at the single cell level of primary HNSCC specimens and patient derived xenograft (PDX) tumors derived from HNSCCs. SMAD4 heterozygous loss was detected in 35% of primary HNSCCs and 41.3% of PDX tumors. Additionally, 4.3% of PDX tumors had SMAD4 homozygous loss. These frequencies of SMAD4 loss were similar to those in The Cancer Genome Atlas (TCGA). However, we identified significant heterogeneities of SMAD4 loss (partial or complete) among cells within each tumor. We also found that aneuploidy (monosomy and polysomy) contributed greatly to how to define chromosomal SMAD4 deletion. Furthermore, in cultured PDX tumors, SMAD4 mutant cells outcompeted SMAD4 wildtype cells, resulting in establishing homogenous SMAD4 mutant HNSCC cell lines with partial or complete genomic SMAD4 loss, suggesting a survival advantage of SMAD4 mutant cells. Taken together, our study reveals inter- and intra-tumor heterogeneities of SMAD4 chromosomal loss in HNSCCs. Further, SMAD4 FISH assay provides a platform for future clinical diagnosis of SMAD4 chromosomal loss that potentially serves as a molecular marker for prognosis and therapeutic intervention in cancer patients.

PPARγ Interaction with UBR5/ATMIN Promotes DNA Repair to Maintain Endothelial Homeostasis

Using proteomic approaches, we uncovered a DNA damage response (DDR) function for peroxisome proliferator activated receptor γ (PPARγ) through its interaction with the DNA damage sensor MRE11-RAD50-NBS1 (MRN) and the E3 ubiquitin ligase UBR5. We show that PPARγ promotes ATM signaling and is essential for UBR5 activity targeting ATM interactor (ATMIN). PPARγ depletion increases ATMIN protein independent of transcription and suppresses DDR-induced ATM signaling. Blocking ATMIN in this context restores ATM activation and DNA repair. We illustrate the physiological relevance of PPARγ DDR functions by using pulmonary arterial hypertension (PAH) as a model that has impaired PPARγ signaling related to endothelial cell (EC) dysfunction and unresolved DNA damage. In pulmonary arterial ECs (PAECs) from PAH patients, we observed disrupted PPARγ-UBR5 interaction, heightened ATMIN expression, and DNA lesions. Blocking ATMIN in PAH PAEC restores ATM activation. Thus, impaired PPARγ DDR functions may explain the genomic instability and loss of endothelial homeostasis in PAH.

Next-Generation Sequencing–Based Assessment of JAK2, PD-L1, and PD-L2 Copy Number Alterations at 9p24.1 in Breast Cancer: Potential Implications for Clinical Management

Genomic amplification at 9p24.1, including the loci for JAK2, PD-L1, and PD-L2, has recently been described as a mechanism of resistance in postchemotherapy, triple-negative breast cancer. This genomic signature holds significant promise as a prognostic biomarker and has implications for targeted therapy with JAK2 inhibitors, as well as with immunotherapy. To guide future screening strategies, the frequency of these alterations was determined. A total of 5399 cases were included in the study. This encompassed 2890 institutional cases tested by the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets assay and 2509 cases from The Cancer Genome Atlas (TCGA). The combined incidence of 9p24.1 amplifications in both the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets and TCGA cohorts was 1.0% (56/5399 cases) and showed a >10-fold higher incidence in triple-negative breast cancer (triple-negative: 5.1%; non-triple-negative: 0.5%). Tumor mutation burden and stromal tumor infiltrating lymphocytes, parameters used to assess response to immunotherapy, were not significantly higher for these cases. The significance of genomic losses at 9p24.1 is unclear, and further studies are needed. Herein, we studied the spectrum of copy number alterations in breast cancer cases within our institutional clinical sequencing cohort and those profiled by TCGA to determine the frequency of genomic alterations thatmay predict response or resistance to JAK2 inhibitors and/or immunotherapy.

Genomic loss of HLA alleles may affect the clinical outcome in low-risk myelodysplastic syndrome patients

The Revised International Prognostic Score and some somatic mutations in myelodysplastic syndrome (MDS) are independently associated with transformation to acute myeloid leukemia (AML). Immunity has also been implicated in the pathogenesis of MDS, although the underlying mechanism remains unclear. We performed a SNP array on chromosome 6 in CD34+ purified blasts from 19 patients diagnosed with advanced MDS and 8 patients with other myeloid malignancies to evaluate the presence of loss of heterozygosity (LOH) in HLA and its impact on disease progression. Three patients had acquired copy-neutral LOH (CN-LOH) on 6p arms, which may disrupt antigen presentation and act as a mechanism for immune system evasion. Interestingly, these patients had previously been classified at low risk of AML progression, and the poor outcome cannot be explained by the acquisition of adverse mutations. LOH HLA was not detected in the remaining 24 patients, who all had adverse risk factors. In summary, the clinical outcome of patients with advanced MDS might be influenced by HLA allelic loss, wich allows subclonal expansions to evade cytotoxic-T and NK cell attack. CN-LOH HLA may therefore be a factor favoring MDS progression to AML independently of the somatic tumor mutation load.

Genomic loss of heterozygosity and survival in the REAL3 trial.

BackgroundHomologous recombination deficiency (HRD) measured using a genomic signature for loss of heterozygosity (LOH) predicts benefit from rucaparib in ovarian cancer. We hypothesized that some oesophagogastric cancers will have high-LOH which would be prognostic in patients treated with platinum chemotherapy.MethodsDiagnostic biopsy DNA from patients treated in the REAL3 trial was sequenced using the Foundation Medicine T5 next-generation sequencing (NGS) assay. An algorithm quantified the percentage of interrogable genome with LOH. Multidimensional optimization was performed to identify a cut-off dichotomizing the population into LOH-high and low groups associated with differential survival outcomes.ResultsOf 158 available samples, 117 were successfully sequenced; LOH was derived for 74 of these. A cut-off of 21% genomic LOH defined an LOH-high subgroup (n=10, 14% of population) who had median overall survival (OS) of 18.3 months (m) versus 11m for the LOH-low group (HR 0.55 95% CI 0.19-0.97, p= 0.10). Progression free survival (PFS) for LOH-high and LOH-low groups was 10.7m and 7.3m (HR 0.61 (95% CI 0.21 – 1.09, p=0.09). Sensitivity analysis censoring operated patients (n=4), demonstrated OS of 18.3m vs. 10.2m (HR 0.43, 95% CI (0.20-0.92), p=0.02; PFS was 10.5m vs. 7.2m (HR 0.55, (95% CI 0.26-1.17), p=0.09 for LOH-high and LOH-low.ConclusionHRD assessment using an algorithmically derived LOH signature on a standard NGS panel identifies oesophagogastric cancer patients with high LOH who have prolonged survival when treated with platinum chemotherapy. Validation work will determine the signature's predictive value in patients treated with a PARP inhibitor and with platinum chemotherapy.

Accounting for the Unique Molecular Landscape of Pediatric Malignancies Improves Target-Agent Pair Identification for Pediatric Precision Oncology

Precision medicine has significant potential to improve therapy options for patients who have exhausted treatment options for their relapsed or refractory cancers. Next-generation sequencing has enabled the detection of genetic abnormalities in individual tumours that bestow sensitivity to selective agents targeting these dysregulated cellular pathways. Several programs have recently launched that utilize sequencing technology to identify patient mutations and match the patients to clinical trials for agents that target the dysregulted pathway. The Pediatric MATCH trial initiated by the National Cancer Institute (NCI) and Children's Oncology Group (COG) exemplifies this approach, but also emphasizes the numerous obstacles involved in molecularly-guided therapiesThe target and agent prioritization strategy for the Pediatric MATCH trial was recently outlined. It employs an amplicon-based targeted sequencing panel (OCAV3) that was originally developed to capture informative variants for adult tumours, with the current version 3 including a set of pediatric-specific variants. However, the landscape of genomic alterations across pediatric malignancies differs from adult malignancies, which translate to distinct oncogenic drivers. The fundamental differences in the genomic landscapes observed in adult and pediatric malignancies must therefore be considered in the optimization of target identification for precision oncology.We hypothesized that a pediatric-focused targeted sequencing panel (OCCRA) will be more informative than an adult-focused panel (OCAV3) for pediatric malignancies. To compare target-agent pairs identified by OCAV3 and OCCRA, we performed a retrospective analysis of 28 childhood tumour samples, 10 B-ALL, 4 T-ALL, 6 neuroblastomas, and 8 additional solid tumours. For 12 of these tumours, whole genome sequencing (WGS) was performed on matched samples and all sequencing results were filtered for pediatric cancer driver genes.For the 12 samples tested by all three sequencing modalities, we found that the greatest discordance occurred in samples with low (<25%) tumour content. In these samples, however, all three modalities gave highly concordant detection of pediatric driver mutations, indicating the robustness of amplicon-based sequencing. Following the prioritization strategies outlined by the Pediatric MATCH committee, at least one target-agent pair was detected for 16 of 28 samples sequenced by the OCAV3 panel with 23 total agents identified due to multiple variants per sample. Using the OCCRA panel, however, we detected at least one target-agent pair for 19 of 28 samples with 25 total agents. Importantly, a fusion was detected by OCCRA in 3 samples, whereas OCAV3 was unable to detect these fusions due to panel content. Of particular note was the detection of homozygous loss in CDKN2A, which was the most commonly identified target, assigned seven times for OCAV3 and 11 times for OCCRA. Although genomic loss in CDKN2A or CDKN2B is not validated by the manufacturer, orthologous verification through clinical reports and WGS indicates that the OCCRA panel called nine of nine verifiable losses in these genes across the 28 samples. Variants were also detected within targetable pathways currently not included in the Pediatric MATCH trial, such as the JAK/STAT or tyrosine kinase pathways; inclusion of inhibitors targeting these pathways increased the total number of agents to 25 for OCAV3 and 29 for OCCRA. In addition, use of the OCCRA panel enabled detection of potentially targetable cancer driver mutations, including gains in RUNX1, ABL2, or ERBB2 and a SMARCA4 SNV, in several samples for which no established target-agent pairing was assigned.Overall, amplicon-based sequencing was highly reproducible across independent panels, robust to low tumour content, and enabled cost-effective target-agent pairing with short 2-3 day turnaround times. Furthermore the pediatric cancer-specific OCCRA panel improved the identification rate of target-agent pairs and resulted in more frequent matching with multiple agents. DisclosuresNo relevant conflicts of interest to declare.

Incidence of HLA Loss in a Global Multicentric Cohort of Post-Transplantation Relapses: Results from the Hlaloss Collaborative Study

Introduction. Genomic loss of an HLA haplotype encoding incompatible alleles (“HLA loss”) has been described in previous single-center studies as a mechanism by which leukemic cells evade the graft-versus-leukemia effect mediated by alloreactive donor T cells and outgrow into a clinically evident relapse. HLA loss accounts for up to 30% of relapses after HLA-haploidentical transplants (Crucitti, Leukemia 2015), but the actual frequency and clinical relevance of this phenomenon in unrelated donor HSCTs, including cord blood transplants, are largely unknown. Here we present the first global collaborative study to investigate the incidence of HLA loss across different transplant settings.Methods. Twenty transplant centers from Europe (n=16), North America (n=3) and Asia (n=1) joined to form the HLALOSS consortium. To date, we collected a total of 619 cases of hematologic relapse from adult patients with acute myeloid leukemia (78.5%), acute lymphoblastic leukemia (13.9%), myelodysplastic syndromes (4%) or myeloproliferative neoplasms (1.1%) after allogeneic HSCT from HLA-haploidentical relatives (31.7%), HLA-mismatched unrelated donors (MMUD, 21.3%), 10/10-matched unrelated donors (MUD, 37.2%), or unrelated cord blood units (UCB, 9.8%). Where available, the donor and patient germlines and the patient pre-transplant disease were collected in parallel. Until today, 476 cases were analyzed using conventional HLA typing of sorted leukemic blasts, the recently developed HLA-KMR assay (Ahci and Toffalori, Blood, 2017) or a novel Next-Generation Sequencing (NGS) method. The latter was developed adapting the HLA typing strategy in use at the DKMS (Lange, BMC Genomics 2013) to the study of chimeric samples, and allowing to cover all possible HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 alleles and to analyze at least 48 different cases in a single run.Results. Out of the 476 relapses analyzed to date, 396 (83.2%) were informative for the study of HLA loss. Of these, 155 occurred after haploidentical HSCT, 101 after MMUD HSCT, 93 after 10/10-matched, HLA-DPB1 mismatched MUD, and 47 after UCB HSCTs. Three-hundred-two (76.2%) of cases were analyzed using the NGS platform. This method resulted particularly robust, reliable and sensitive in analyzing large sample series: the mean coverage across the 6 sequenced loci was over 8500x, up to 0.5% of the HLA allele of interest could be detected in artificial chimerism curves, and relapse samples tested in parallel via the sequencing platform and HLA-KMR (n=10) showed remarkable concordance between the two methods (R2=0.86, p<0.0001). In total, we detected 51 HLA loss post-transplantation relapses out of the 396 cases analyzed (12.8%). Of these, 35 occurred after haploidentical HSCT (22.6% of relapses in this setting), 12 after MMUD HSCT (11.9%), 4 after 10/10 MUD HSCT (4.3%) and, notably, none after UCB HSCT.Conclusions. The present data, obtained from the largest collaborative study on the immunobiology of relapse to date, confirm the clinical relevance of HLA loss as a major mechanism of immune evasion and post-transplantation relapse after allogeneic HSCT, with an incidence which is proportional to the number of donor-recipient HLA mismatches. The only exception is represented by UCB HSCT which, despite being often performed across multiple major HLA incompatibilities, does not appear to be associated with this relapse modality. This finding might reflect the fact that in UCB HSCT, multiple HLA mismatches are often not encoded in cis on the same chromosome, thereby reducing the selective advantage for leukemic cells that undergo an HLA haplotype loss. This phenomenon might in turn contribute to the lower incidence of relapse reported for UCB HSCT compared to other stem cell sources. DisclosuresVago:Moderna TX: Research Funding; GENDX: Research Funding. Stoelzel:Neovii: Speakers Bureau. Gojo:Novartis: Membership on an entity's Board of Directors or advisory committees; Merck inc: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees. Busca:Novartis: Speakers Bureau; Jazz Pharmaceuticals: Honoraria; Pfizer Pharmaceuticals: Honoraria, Speakers Bureau; Merk: Honoraria, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Luznik:WIndMIL Therapeutics: Equity Ownership, Patents & Royalties. Kobbe:Amgen: Honoraria, Research Funding; Celgene: Honoraria, Other: Travel Support, Research Funding; Roche: Honoraria, Research Funding. Kroeger:Novartis: Honoraria, Research Funding; Sanofi: Honoraria; Riemser: Honoraria, Research Funding; Neovii: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; JAZZ: Honoraria. Finke:Neovii: Consultancy, Honoraria, Other: travel grants, Research Funding; Medac: Consultancy, Honoraria, Other: travel grants, Research Funding; Riemser: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Other: travel grants, Research Funding. Mohty:Takeda: Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria; Servier: Consultancy; MaaT Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; Molmed: Consultancy; Jazz Pharmaceuticals: Honoraria, Research Funding, Speakers Bureau; Bristol Myers: Consultancy, Research Funding; Janssen: Honoraria, Research Funding, Speakers Bureau. Beelen:Medac: Consultancy, Other: Travel Support. Fleischhauer:GENDX: Research Funding.

Genomic Copy-Number Loss Is Rescued by Self-Limiting Production of DNA Circles

Copy-number changes generate phenotypic variability in health and disease. Whether organisms protect against copy-number changes is largely unknown. Here, we show that Saccharomyces cerevisiae monitors the copy number of its ribosomal DNA (rDNA) and rapidly responds to copy-number loss with the clonal amplification of extrachromosomal rDNA circles (ERCs) from chromosomal repeats. ERC formation is replicative, separable from repeat loss, and reaches a dynamic steady state that responds to the addition of exogenous rDNA copies. ERC levels are also modulated by RNAPI activity and diet, suggesting that rDNA copy number is calibrated against the cellular demand for rRNA. Last, we show that ERCs reinsert into the genome in a dosage-dependent manner, indicating that they provide a reservoir for ultimately increasing rDNA array length. Our results reveal a DNA-based mechanism for rapidly restoring copy number in response to catastrophic gene loss that shares fundamental features with unscheduled copy-number amplifications in cancer cells.

Molecular Mechanisms of Hepatocarcinogenesis Following Sustained Virological Response in Patients with Chronic Hepatitis C Virus Infection.

Despite the success of direct-acting antiviral (DAA) agents in treating chronic hepatitis C virus (HCV) infection, the number of cases of HCV-related hepatocellular carcinoma (HCC) is expected to increase over the next five years. HCC develops over the span of decades and is closely associated with fibrosis stage. HCV both directly and indirectly establishes a pro-inflammatory environment favorable for viral replication. Repeated cycles of cell death and regeneration lead to genomic instability and loss of cell cycle control. DAA therapy offers >90% sustained virological response (SVR) rates with fewer side effects and restrictions than interferon. While elimination of HCV helps to restore liver function and reverse mild fibrosis, post-SVR patients remain at elevated risk of HCC. A series of studies reporting higher than expected rates of HCC development among DAA-treated patients ignited debate over whether use of DAAs elevates HCC risk compared to interferon. However, recent prospective and retrospective studies based on larger patient cohorts have found no significant difference in risk between DAA and interferon therapy once other factors are taken into account. Although many mechanisms and pathways involved in hepatocarcinogenesis have been elucidated, our understanding of drivers specific to post-SVR hepatocarcinogenesis is still limited, and lack of suitable in vivo and in vitro experimental systems has hampered efforts to examine etiology-specific mechanisms that might serve to answer this question more thoroughly. Further research is needed to identify risk factors and biomarkers for post-SVR HCC and to develop targeted therapies based on more complete understanding of the molecules and pathways implicated in hepatocarcinogenesis.