Focal Copy Number Alterations Research Articles

Deciphering the correlation between Chr3q26.2 amplification through homologous recombination deficiency and improved survival in ovarian cancer under platinum-based adjuvant therapy.

e17537 Background: Discriminant copy number alterations (CNAs) have been linked to aberrant DNA repair mechanisms. This study aimed to identify CNA-based biomarkers, alongside homologous recombination deficiency (HRD) scores for ovarian cancer patients receiving platinum (Pt)-based chemotherapy, especially in BRCA1/2 wild-type or HRD-low patients. Methods: Fourteen most prevalent and significant focal CNA regions (seven amplification and seven deletion) were examined to investigate their correlations with HRD scores via SHAP values of an XGBoost model and the effectiveness of Pt-based adjuvant chemotherapy in a cohort of high-grade serous ovarian cancer (HGSC) patients (TCGA cohort, N=576), and the results were validated in a separate Chinese ovarian cancer cohort (test cohort, N=457). An in-house developed next-generation sequencing HRD pipeline (GeneseeqPrime HRD) was used to determine HRD scores. Histological subtypes, molecular features, and potential molecular mechanisms associated with the identified biomarkers were further explored. Results: Of 14 high-level CNA regions identified by GISTIC in the TCGA cohort, Chromosome 3q26.2 amplification [ Chr3(q26.2) Amp, a prevalence of 30.7%] was associated with increased HRD scores in both BRCA1/2-altered ( p=0.01) and wild-type HGSCs ( p=0.03). Consistently, in the test cohort, higher HRD scores ( p<0.01) and elevated chromosome instability scores ( p<0.01) were observed in Chr3(q26.2) amplified samples. Among 406 patients administrated with Pt-based adjuvant chemotherapy in the TCGA cohort, those with Chr3(q26.2) Amp exhibited a relatively high sensitivity rate ( p=0.09) and significantly superior disease-free survival after adjusting for HRD score, patient age, tumor stage, and residual disease [hazard ratio (HR): 0.65, 95% confidence interval (CI): 0.48–0.88] when compared to those without, representing Chr3(q26.2) Amp as an independent predictive biomarker. Similar results were obtained in 78 patients using Pt-based adjuvant chemotherapy in the test cohort (HR: 0.68, 95% CI: 0.40–1.15). Moreover, Chr3(q26.2) Amp was more prevalent in HGSCs harboring BRCA1/2 or TP53 aberrations than other histological subtypes driven by PIK3CA, PTEN or KRAS mutations ( p<0.01). In comparison to HGSCs without Chr3(q26.2) Amp, Chr3(q26.2) amplified HGSCs exhibited higher Ragnum hypoxia scores ( p<0.01) and a total of four overexpressed proteins, including p62 ( p<0.01), PIK3CA ( p<0.01), CCNB1 ( p<0.01) and TSC1 ( p<0.01), indicative of suppressed homologous recombination repair pathway and cycle cell arrest. Conclusions: Chr3(q26.2) Amp emerges as a promising biomarker for Pt-based adjuvant chemotherapy in ovarian cancer, potentially reflecting a hypoxia-induced etiology through suppressed homologous recombination repair pathway.

Abstract 1769: Pan-cancer genomic characterization of human papillomavirus associated tumors reveals patterns of somatic alteration that associate with virus status and anatomic site

Abstract Human papilloma virus (HPV) infection causes over 600,000 human cancers yearly and accounts for nearly all cervical cancers, increasing rates of head and neck squamous cell carcinomas (HNSCC), and many anogenital cancers - all with varying clinical outcomes due to a lack of personalized care. While recent integrative genomic studies have described molecular features of individual cancer types, few studies have compared genomic changes between HPV(+) and HPV(-) cancers across anatomic sites. Here, we conducted the first pan-cancer genomic analysis of HPV-associated tumors across multiple anatomic tumor types using whole exome and transcriptome data from The Cancer Genome Atlas (TCGA) cohorts of cervical (n=254) and HNSCCs (n=514), and targeted exome sequencing of 800 cancer genes plus full length HPV16/18 genomes in a clinical cohort of squamous tumors from the head and neck (n=458), cervix (n=78), vulva (n=23), anal canal (n=5), and vagina (n=2). Somatic variant calling and filtering, followed by an integrative pathway analysis of commonly altered targets, defined the catalog of somatic mutations and copy number alterations (CNAs) that drive HPV(+) and HPV(-) tumorigenesis. Sequencing reads from viral RNA or DNA determined HPV status, and HPV type, genome structure, integration events, and viral load were characterized in a subset of samples via de novo assembly of viral aligned reads followed by copy number analysis, breakpoint identification, and the calling of structural variants. Overall HPV positivity was 50% (668 out of 1334 total), with HPV16 accounting for 96%, 89%, and 60% of all HPV(+) anogenital, head and neck, and cervical tumors respectively. Significant differences in somatic mutation frequency between HPV(+) and HPV(-) tumors were observed in the full cohort, as well as in analyses stratified by anatomic site. Interestingly, we noticed recurrently mutated “hotspots” attributable to increased APOBEC-mutagenesis in HPV(+) samples across anatomic sites (PIK3CA:E545K, FGFR3:S249C, EP300:D1399N), while hotspot mutations likely caused by tobacco smoking predominate HPV(-) HNSCC (PIK3CA:H1047R/L, CDKN2A:R80*, TP53:R282W). Focal and arm level CNAs were distinctive, including gains of 11q22 in HPV(+) cervical and HPV(-) HNSCC, and losses of 11q22 in HPV(+) HNSCC. Biological pathways commonly altered include epithelial differentiation, cell death, innate immunity, growth factor/kinase signaling, and cell cycle control. In summary, pan-cancer genomic analysis revealed distinct patterns of somatic alteration of conserved biological pathways that associate with HPV status and anatomic site. These findings improve our understanding of tumor biology unique to HPV-associated cancers and may lead to novel treatment and classification strategies to improve patient outcomes in the clinical setting. Citation Format: Jeremiah Ray Holt, Paul Little, Heejoon Jo, Xiaobei Zhao, Hyo Young Choi, Vonn Walter, Benjamin Wahle, Jose P. Zevallos, Angela Mazul, Katherine A. Hoadley, Michele Hayward, David N. Hayes. Pan-cancer genomic characterization of human papillomavirus associated tumors reveals patterns of somatic alteration that associate with virus status and anatomic site [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 1769.

Abstract IA002: Management of newly diagnosed endometrial cancers and molecular classification

Abstract In the past 50 years, the treatment of this cancer has evolved from a regimen of preoperative radiation therapy followed by hysterectomy, to individualized management consisting of surgical treatment with or without additional therapy depending on whether various risk factors are identified. After the diagnosis of endometrial carcinoma has been histologically confirmed, uterine and extrauterine risk factors affect the probability of retroperitoneal lymph node involvement and subsequent survival. Surgical staging is a critical component for the proper diagnosis and management of all endometrial carcinomas. FIGO stage is the most powerful predictor of prognosis. Surgical staging is feasible by minimally invasive techniques. Patients with significant medical comorbidities who are not acceptable candidates for surgery may be managed by alternative means. Women with EC are diagnosed under the age of 40 may be eligibile for nonoperative management if there is no clear evidence of myometrial invasion. As the contemporary management of EC continues to evolve, and the indications for lymph node dissection remain controversial. Minimally invasive management of EC has increasingly been integrated into standard practice. In 2006, The Cancer Genome Atlas (TCGA) began with studies in glioblastoma and high-grade serous ovarian cancer. The National Institutes of Health committed major resources to TCGA to collect and characterize the genomic landscape of more than 20 cancer types. In 2013, TCGA published findings for endometrial cancers. Only serous and endometrioid subtypes were accrued in the endometrial cancer project. TCGA has reclassified endometrioid and serous endometrial cancers in four broad categories using comprehensive genomic approaches. One-third of the cancers is characterized by microsatellite instability (MSI) and associated hyper-mutation. The cancers without MSI can be further separated into two groups. Serous cases with extensive DNA copy number alterations and genomic instability comprise one group, and endometrioid cases with few or only focal copy number alterations comprise the other. A smaller fourth sub-group contains samples with very high mutation rates and is characterized by mutations in POLE. Molecular features can be distinctly overlaid onto these four sub-classifications, and each have important clinical implications that are currently being used in both clinical practice and clinical trial design. In this educational presentation, we will discuss the management of newly diagnosed endometrial cancers, review the molecular classification of endometrial cancers, and identify approaches that incorporate these findings into clinical care and clinical trial design. Citation Format: Douglas A. Levine. Management of newly diagnosed endometrial cancers and molecular classification [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr IA002.

Development of a rapid and comprehensive genomic profiling test supporting diagnosis and research for gliomas.

A prompt and reliable molecular diagnosis for brain tumors has become crucial in precision medicine. While Comprehensive Genomic Profiling (CGP) has become feasible, there remains room for enhancement in brain tumor diagnosis due to the partial lack of essential genes and limitations in broad copy number analysis. In addition, the long turnaround time of commercially available CGPs poses an additional obstacle to the timely implementation of results in clinics. To address these challenges, we developed a CGP encompassing 113 genes, genome-wide copy number changes, and MGMT promoter methylation. Our CGP incorporates not only diagnostic genes but also supplementary genes valuable for research. Our CGP enables us to simultaneous identification of mutations, gene fusions, focal and broad copy number alterations, and MGMT promoter methylation status, with results delivered within a minimum of 4days. Validation of our CGP, through comparisons with whole-genome sequencing, RNA sequencing, and pyrosequencing, has certified its accuracy and reliability. We applied our CGP for 23 consecutive cases of intracranial mass lesions, which demonstrated its efficacy in aiding diagnosis and prognostication. Our CGP offers a comprehensive and rapid molecular profiling for gliomas, which could potentially apply to clinical practices and research primarily in the field of brain tumors.

Cancer Genomic Profiling and Minimal Residual Disease Monitoring By Cell-Free DNA Sequencing in Pediatric Leukemia

Introduction: Cell-free DNA (cfDNA) sequencing has been reported as a promising non-invasive tool for detecting cancer and monitoring treatment response. We sought to evaluate the potential of a cfDNA-based strategy for detecting diverse classes of somatic mutations relevant to pediatric leukemia and cancers. Methods: Plasma samples collected at diagnosis from 135 acute lymphoblastic leukemia (ALL) and 34 acute myeloid leukemia (AML) patients were evaluated, including 114 B-ALL and 21 T-ALL. We also studied diagnostic plasma samples from 38 solid tumor and 19 brain tumor patients, and plasma collected from 12 patients with B-ALL at days 15, 22 and end of induction chemotherapy (EOI). Plasma cfDNA extraction and library preparation were performed with the NucleoSnap cfDNA kit and ThruPLEX Tag-Seq HV kit, respectively. Next-generation sequencing was performed using a capture panel covering 355 pediatric cancer genes (including noncoding regions covering hotspots of structural variation) and 8,103 heterozygous population polymorphisms. We developed analytic approaches for de novo detection of all types of somatic mutations, including focal copy number alterations, rearrangements, and large chromosomal abnormalities. Whole-genome sequencing (WGS) was also performed for 10 cfDNA samples from leukemia. Data were compared with corresponding whole-genome, exome and transcriptome tumor cell sequencing data at diagnosis. Results: At diagnosis, plasma cfDNA yield was higher for leukemias than for solid and brain tumors (median, 142 ng/ml, 7.16 ng/ml, and 7.07 ng/ml, respectively. The average coverages of leukemia cfDNA sequencing were 790X by capture panel post error suppression and 69X by WGS. We found that 100% cases of leukemia at diagnosis had detectable circulating tumor DNA (ctDNA) in plasma cfDNA, and the median ctDNA purity was 77%, comparable to primary tumor purity from diagnostic sequencing. Overall, our panel covered 86% somatic variants present in the tumor samples. Of those covered events, capture-based cfDNA sequencing detected 98% SNVs/indels, 95% focal copy gains/losses, 100% internal tandem duplications, 95% chromosomal translocations, and 99% aneuploidy. In addition, capture-based sequencing identified cfDNA-specific variants such as NRAS/KRAS SNVs and a IKZF1 deletion that were subclonal in plasma cfDNA. Of those uncovered events, 85% were structural variants with their breakpoints in intronic/noncoding regions, and cfDNA WGS identified all somatic events present in the 10 sequenced tumor samples. To detect minimal residual disease (MRD) in B-ALL patients during induction, we increased cfDNA sequencing depth for low VAF events, and the detection limit was 0.4% for SNV/indel and structural variations. Compared to the MRD results from flow cytometry, somatic mutations were detected in 7/8 MRD-positive and 1/4 MRD-negative patients on day 15, 3/4 MRD-positive patients on day 22, and 0/2 MRD-positive patients at EOI. We also sequenced cfDNA from peripheral blood and bone marrow plasma for 2 B-ALL patients on day 0, day 15, and EOI, with comparable ctDNA purity and dynamics observed between the two compartments. In a patient with concurrent ALL and neuroblastoma (NBL) who experienced NBL relapse four months after the primary diagnosis, cfDNA genomic profiling identified ctDNA derived from ALL (52% of total plasma cfDNA), primary NBL (20%), and relapsed NBL (10%), suggesting that genetic variations of multiple tumors could be detected in a single cfDNA sample. Conclusion: Our study has demonstrated the utility of cfDNA sequencing to identify both sequence and structural somatic mutations in childhood acute leukemia. This approach will be useful to monitor disease, as well as providing a non-invasive diagnostic approach.

Comprehensive Characterisation of the Copy Number Landscape in Acute Lymphoblastic Leukemia Using Digitalmlpa

Introduction: Acute lymphoblastic leukemia (ALL) is a heterogeneous disease affecting various signaling pathways including cell differentiation and cell cycle control. ALL subtypes are defined by their genetic abnormalities, encompassing gross chromosomal aberrations, ploidy changes, translocations or rearrangements, regional or focal (intragenic) copy number alterations (CNAs), gene fusions and point mutations. Novel biomarkers for risk stratification are still being discovered, further mapping the diverse genetic ALL landscape. Testing for these newly identified genetic biomarkers and characterising the copy number (CN) landscape in ALL samples can provide useful new insights for risk stratification and patient-tailored treatment regimens. Methods: For multiplex detection of CNAs in ALL samples, a new test version of SALSA® digitalMLPA™ Probemix D007 Acute Lymphoblastic Leukemia was developed. Hereafter, this new test version will be referred to as D007 Acute Lymphoblastic Leukemia. This assay contains probes for CNA detection of 70 key genes and regions frequently altered in B-cell ALL and T-cell ALL ( Figure 1). In addition, this probemix contains digital karyotyping probes covering each chromosomal arm for the detection of gross chromosomal aberrations (except the p-arm of acrocentric chromosomes). A selection of these digital karyotyping probes also serve as reference probes. Finally, a set of single nucleotide polymorphism (SNP) probe pairs, targeting both alleles of each of the SNPs, was included for sample identification and guiding the correct CN baseline determination, facilitating optimal data normalisation in samples with complex karyotypes. To test the performance of this new test version of the probemix, DNA samples from 29 ALL cell lines were analysed. digitalMLPA-derived PCR amplicons were sequenced on a NextSeq 1000 Sequencing System (Illumina). FastQ files were analysed using Coffalyser digitalMLPA™ (MRC Holland) and a custom-built algorithm was used for copy number determination using SNP probe pairs. Results: digitalMLPA data of all 29 cell lines was analysed and compared to Cancer Cell Line Encyclopedia (CCLE) copy number data (Depmap, Copy Number Public 23Q2 download). CNAs were detected with D007 Acute Lymphoblastic Leukemia, including clinically relevant biomarkers such as the IKZF1 plus profile, PAX5alt, TCF3 fusions, focal 22q11.22 deletion (incl. VPREB1), intrachromosomal amplification of chromosome 21 (iAMP21), hypo- and hyperdiploidy. Other CNAs of emerging significance in ALL risk stratification included 6q15-q16 deletions, DNMT3B, CDKN1B and KRAS alterations. SNP probe pairs were effectively used to facilitate correct CN baseline determination, and results were confirmed using CCLE data (Depmap, CCLE_ABSOLUTE_combined 2019 download). Correct CN baseline detection is particularly important for samples with complex karyotypes, such as the P12-ICHIKAWA cell line. In this cell line, our data revealed that most chromosomal regions targeted by the SNP probe pairs had a copy number ≥3 ( Figure 2), consistent with the hypotetraploid karyotype previously described for this cell line. Conclusions: Using this new test version of SALSA® digitalMLPA™ D007 Acute Lymphoblastic Leukemia, numerous CNAs can be detected in a single reaction. Results obtained with this probemix showed a high correlation with CN data from the CCLE database in a panel of 29 ALL cell lines. digitalMLPA provides valuable insights into the CN of key target sequences relative to reference sequences in the genome. Moreover, our novel approach using pairs of probes that target both SNP alleles provides an additional layer of information, guiding CN and ploidy status determination in highly complex karyotypes. The SNP probe pair based approach for detection of CN levels in complex samples shows promising results and will be further validated in an independent cohort of 140 ALL patient samples. These results will be presented at the ASH meeting.

PPM1D activity promotes the replication stress caused by cyclin E1 overexpression.

Oncogene-induced replication stress has been recognized as a major cause of genome instability in cancer cells. Increased expression of cyclin E1 caused by amplification of the CCNE1 gene is a common cause of replication stress in various cancers. Protein phosphatase magnesium-dependent 1 delta (PPM1D) is a negative regulator of p53 and has been implicated in termination of the cell cycle checkpoint. Amplification of the PPM1D gene or frameshift mutations in its final exon promote tumorigenesis. Here, we show that PPM1D activity further increases the replication stress caused by overexpression of cyclin E1. In particular, we demonstrate that cells expressing a truncated mutant of PPM1D progress faster from G1 to S phase and fail to complete licensing of the replication origins. In addition, we show that transcription-replication collisions and replication fork slowing caused by CCNE1 overexpression are exaggerated in cells expressing the truncated PPM1D. Finally, replication speed and accumulation of focal DNA copy number alterations caused by induction of CCNE1 expression was rescued by pharmacological inhibition of PPM1D. We propose that increased activity of PPM1D suppresses the checkpoint function of p53 and thus promotes genome instability in cells expressing the CCNE1 oncogene.

The copy number and mutational landscape of recurrent ovarian high-grade serous carcinoma

The drivers of recurrence and resistance in ovarian high grade serous carcinoma remain unclear. We investigate the acquisition of resistance by collecting tumour biopsies from a cohort of 276 women with relapsed ovarian high grade serous carcinoma in the BriTROC-1 study. Panel sequencing shows close concordance between diagnosis and relapse, with only four discordant cases. There is also very strong concordance in copy number between diagnosis and relapse, with no significant difference in purity, ploidy or focal somatic copy number alterations, even when stratified by platinum sensitivity or prior chemotherapy lines. Copy number signatures are strongly correlated with immune cell infiltration, whilst diagnosis samples from patients with primary platinum resistance have increased rates of CCNE1 and KRAS amplification and copy number signature 1 exposure. Our data show that the ovarian high grade serous carcinoma genome is remarkably stable between diagnosis and relapse and acquired chemotherapy resistance does not select for common copy number drivers.

PAQR8 promotes breast cancer recurrence and confers resistance to multiple therapies

BackgroundBreast cancer mortality is principally due to recurrent disease that becomes resistant to therapy. We recently identified copy number (CN) gain of the putative membrane progesterone receptor PAQR8 as one of four focal CN alterations that preferentially occurred in recurrent metastatic tumors compared to primary tumors in breast cancer patients. Whether PAQR8 plays a functional role in cancer is unknown. Notably, PAQR8 CN gain in recurrent tumors was mutually exclusive with activating ESR1 mutations in patients treated with anti-estrogen therapies and occurred in > 50% of both patients treated with anti-estrogen therapies and those treated with chemotherapy or anti-Her2 agents.MethodsWe used orthotopic mouse models to determine whether PAQR8 overexpression or deletion alters breast cancer dormancy or recurrence following therapy. In vitro studies, including assays for colony formation, cell viability, and relative cell fitness, were employed to identify effects of PAQR8 in the context of therapy. Cell survival and proliferation were quantified by immunofluorescence staining for markers of apoptosis and proliferation. Sphingolipids were quantified by liquid chromatography-high resolution mass spectrometry.ResultsWe show that PAQR8 is necessary and sufficient for efficient mammary tumor recurrence in mice, spontaneously upregulated and CN gained in recurrent tumors that arise following therapy in multiple mouse models, and associated with poor survival following recurrence as well as poor overall survival in breast cancer patients. PAQR8 promoted resistance to therapy by enhancing tumor cell survival following estrogen receptor pathway inhibition by fulvestrant or estrogen deprivation, Her2 pathway blockade by lapatinib or Her2 downregulation, and treatment with chemotherapeutic agents. Pro-survival effects of PAQR8 were mediated by a Gi protein-dependent reduction in cAMP levels, did not require progesterone, and involved a PAQR8-dependent decrease in ceramide levels and increase in sphingosine-1-phosphate levels, suggesting that PAQR8 may possess ceramidase activity.ConclusionsOur data provide in vivo evidence that PAQR8 plays a functional role in cancer, implicate PAQR8, cAMP, and ceramide metabolism in breast cancer recurrence, and identify a novel mechanism that may commonly contribute to the acquisition of treatment resistance in breast cancer patients.

TRAF7-mutated Fibromyxoid Spindle Cell Tumors Are Associated With an Aggressive Clinical Course and Harbor an Undifferentiated Sarcoma Methylation Signature: A Molecular and Clinicopathologic Study of 3 Cases.

TRAF7 somatic mutations are rare and have been reported in meningiomas, intraneural perineuriomas, and mesotheliomas. Triggered by an index case of an unclassified low-grade mesenchymal tumor with TRAF7 mutation as the only genetic alteration, we searched our files and identified 2 additional cases with similar features. The tumors arose in 2 females and 1 male, aged 63 to 75 years old (median: 67y). They were infiltrative deep soft tissue masses involving the shoulder, chest wall, and thigh, measuring 7.0 to 9.1cm in greatest dimensions. One tumor was locally aggressive, and 2 were associated with lung and bone metastases. The tumors displayed alternating fibrous and myxoid stroma with mild to moderate cellularity and consisted of uniform spindle cells with open chromatin, inconspicuous nucleoli and scant cytoplasm. Significant mitotic activity or necrosis were not present. However, the metastatic tumor of 1 case showed an epithelioid morphology and brisk mitotic activity. Immunohistochemically, the tumors showed nonspecific and focal smooth muscle actin or CD34 expression. By DNA sequencing, all 3 cases harbored TRAF7 missense mutations involving the C-terminal WD40 domains as the only somatic mutations, showed nonrecurrent focal copy number alterations, and were negative for gene fusions by targeted RNA sequencing. On methylation profiling, the tumors clustered with the undifferentiated sarcoma and myxofibrosarcoma methylation classes and were distinct from morphologic mimics. On follow-up (5 to 36mo), 2 patients died of disease following aggressive chemotherapeutic regimens. We describe a novel TRAF7- mutated mesenchymal tumor characterized by aggressive clinical behavior despite the histologic appearance of a low-grade fibromyxoid spindle cell tumor.

Replication stress generates distinctive landscapes of DNA copy number alterations and chromosome scale losses

BackgroundA major driver of cancer chromosomal instability is replication stress, the slowing or stalling of DNA replication. How replication stress and genomic instability are connected is not known. Aphidicolin-induced replication stress induces breakages at common fragile sites, but the exact causes of fragility are debated, and acute genomic consequences of replication stress are not fully explored.ResultsWe characterize DNA copy number alterations (CNAs) in single, diploid non-transformed cells, caused by one cell cycle in the presence of either aphidicolin or hydroxyurea. Multiple types of CNAs are generated, associated with different genomic regions and features, and observed copy number landscapes are distinct between aphidicolin and hydroxyurea-induced replication stress. Coupling cell type-specific analysis of CNAs to gene expression and single-cell replication timing analyses pinpointed the causative large genes of the most recurrent chromosome-scale CNAs in aphidicolin. These are clustered on chromosome 7 in RPE1 epithelial cells but chromosome 1 in BJ fibroblasts. Chromosome arm level CNAs also generate acentric lagging chromatin and micronuclei containing these chromosomes.ConclusionsChromosomal instability driven by replication stress occurs via focal CNAs and chromosome arm scale changes, with the latter confined to a very small subset of chromosome regions, potentially heavily skewing cancer genome evolution. Different inducers of replication stress lead to distinctive CNA landscapes providing the opportunity to derive copy number signatures of specific replication stress mechanisms. Single-cell CNA analysis thus reveals the impact of replication stress on the genome, providing insights into the molecular mechanisms which fuel chromosomal instability in cancer.

Estrogen Receptor Alpha Gene Amplification Is an Independent Predictor of Long-Term Outcome in Postmenopausal Patients with Endocrine-Responsive Early Breast Cancer.

Estrogen receptor (ER) expression is a prognostic parameter in breast cancer, and a prerequisite for the use of endocrine therapy. In ER+ early breast cancer, however, no receptor-associated biomarker exists that identifies patients with a particularly favorable outcome. We have investigated the value of ESR1 amplification in predicting the long-term clinical outcome in tamoxifen-treated postmenopausal women with endocrine-responsive breast cancer. 394 patients who had been randomized into the tamoxifen-only arm of the prospective randomized ABCSG-06 trial of adjuvant endocrine therapy with available formalin-fixed, paraffin-embedded tumor tissue were included in this analysis. IHC ERα expression was evaluated both locally and in a central lab using the Allred score, while ESR1 gene amplification was evaluated by FISH analysis using the ESR1/CEP6 ratio indicating focal copy number alterations. Focal ESR1 copy-number elevations (amplifications) were detected in 187 of 394 (47%) tumor specimens, and were associated with a favorable outcome: After a median follow-up of 10 years, women with intratumoral focal ESR1 amplification had a significantly longer distant recurrence-free survival [adjusted HR, 0.48; 95% confidence interval (CI), 0.26-0.91; P = 0.02] and breast cancer-specific survival (adjusted HR 0.47; 95% CI, 0.27-0.80; P = 0.01) as compared with women without ESR1 amplification. IHC ERα protein expression, evaluated by Allred score, correlated significantly with focal ESR1 amplification (P < 0.0001; χ2 test), but was not prognostic by itself. Focal ESR1 amplification is an independent and powerful predictor for long-term distant recurrence-free and breast cancer-specific survival in postmenopausal women with endocrine-responsive early-stage breast cancer who received tamoxifen for 5 years.

Risk assessment with low-pass whole-genome sequencing of cell-free DNA before CD19 CAR T-cell therapy for large B-cell lymphoma

AbstractPatients with relapsed or refractory large B-cell lymphomas (rrLBCL) can achieve long-term remission after CD19 chimeric antigen receptor T-cell therapy (CART19). However, more than half of recipients will experience treatment failure. Thus, approaches are needed to identify high-risk patients who may benefit from alternative or consolidative therapy. We evaluated low-pass whole-genome sequencing (lpWGS) of cell-free DNA (cfDNA) before CART19 as a new approach for risk stratification. We performed lpWGS on pretreatment plasma samples from 122 patients at time of leukapheresis who received standard-of-care CART19 for rrLBCL to define DNA copy number alterations (CNAs). In multivariable selection, high focal CNA score (FCS) denoting genomic instability was the most significant pretreatment variable associated with inferior 3-month complete response rates (28% vs 56%, P = .0029), progression-free survival (PFS; P = .0007; hazard ratio, 2.11), and overall survival (OS; P = .0026; hazard ratio, 2.10). We identified 34 unique focal CNAs in 108 (89%) patients; of these, deletion 10q23.3 leading to loss of FAS death receptor was the most highly associated with poor outcomes, leading to inferior PFS (P < .0001; hazard ratio, 3.49) and OS (P = .0027; hazard ratio, 2.68). By combining FCS with traditional markers of increased tumor bulk (elevated lactate dehydrogenase and >1 extranodal site), we built a simple risk model that could reliably risk stratify patients. Thus, lpWGS of cfDNA is a minimally invasive assay that could rapidly identify high-risk patients and may guide patient selection for and targeted therapies to evaluate in future clinical trials.

A Risk Score Incorporating Low Pass Whole Genome Sequencing of Cell Free DNA from Patients Receiving CD19 CAR T-Cell Therapy for Large B-Cell Lymphoma

A Risk Score Incorporating Low Pass Whole Genome Sequencing of Cell Free DNA from Patients Receiving CD19 CAR T-Cell Therapy for Large B-Cell Lymphoma

Tumor heterogeneity assessed by sequencing and fluorescence in situ hybridization (FISH) data.

Computational reconstruction of clonal evolution in cancers has become a crucial tool for understanding how tumors initiate and progress and how this process varies across patients. The field still struggles, however, with special challenges of applying phylogenetic methods to cancers, such as the prevalence and importance of copy number alteration (CNA) and structural variation events in tumor evolution, which are difficult to profile accurately by prevailing sequencing methods in such a way that subsequent reconstruction by phylogenetic inference algorithms is accurate. In this work, we develop computational methods to combine sequencing with multiplex interphase fluorescence in situ hybridization to exploit the complementary advantages of each technology in inferring accurate models of clonal CNA evolution accounting for both focal changes and aneuploidy at whole-genome scales. By integrating such information in an integer linear programming framework, we demonstrate on simulated data that incorporation of FISH data substantially improves accurate inference of focal CNA and ploidy changes in clonal evolution from deconvolving bulk sequence data. Analysis of real glioblastoma data for which FISH, bulk sequence and single cell sequence are all available confirms the power of FISH to enhance accurate reconstruction of clonal copy number evolution in conjunction with bulk and optionally single-cell sequence data. Source code is available on Github at https://github.com/CMUSchwartzLab/FISH_deconvolution. Supplementary data are available at Bioinformatics online.

Genetically-Defined Diffuse Large B-Cell Lymphoma Subsets Arise By Distinct Pathogenetic Mechanisms and Predict Outcome

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease that largely arises from antigen-exposed B-cells that transit through the germinal center (GC). DLBCL is further classified into transcriptional subtypes - activated B-cell (ABC) and GC B-cell (GCB). ABC DLBCLs exhibit genetic alterations in NF-kB modifiers and proximal components of the B-cell receptor (BCR) pathway and perturbed terminal B-cell differentiation. GCB DLBCLs have reported alterations in chromatin-modifying enzymes, PI3K signaling and Gα-migration pathway components and frequent translocations of BCL2 .Despite the recognized molecular heterogeneity in DLBCL, previously published genomic analyses have largely focused on single types of alterations - mutations, copy number alterations (CNAs) or structural variants (SVs) - in smaller data sets with more limited clinical annotation. An unbiased comprehensive analysis of all three alteration types is needed to define discrete, clinically annotated subtypes of DLBCL.We performed whole exome sequencing (WES) of 304 newly diagnosed DLBCLs, using an expanded bait set that captures recurrent SVs; 85% of study patients were uniformly treated with R-CHOP and had long-term follow-up. Somatic alterations (mutations, CNAs and SVs) and their clonality were determined with analytical pipelines developed at the Broad Institute. Notably, half of our DLBCL cohort lacked patient-matched normal samples, prompting the successful development of new methods to analyze tumor-only WES data. Significantly mutated genes (SMGs) were identified with MutSig2CV andrecurrent CNAs were defined with GISTIC2.0 .With increased sample size and improved methodology, we identified 158 SMGs, CNAs and SVs; the 98 SMGs included ones previously unreported in DLBCL but described in other lymphoid malignancies or cancer. Additional insights into the putative biological function of newly identified alterations were obtained by overlaying the predicted protein changes onto their 3-dimensional protein structures.We analyzed the mutational signatures in these DLBCLs and found that the majority of exome mutations were caused by spontaneous deamination at CpGs, a clock-like genetic signature associated with aging; only a minority of mutations were attributed to activation-induced cytidine deaminase (AID), an enzyme required for physiologic immunoglobulin receptor editing and aberrant somatic hypermutation.IGH, BCL2, BCL6 and MYC were the most frequently rearranged genes (40%, 21%, 19% and 8%, respectively). There were 18 arm-level and 18 focal regions of copy gain and 2 arm-level and 32 focal regions of copy loss with frequencies ranging between 5-32%.SMGs were significantly more likely to reside within focal CNAs (p=1x10-44), suggesting that these driver genes were perturbed by multiple mechanisms. Individual DLBCLs had a median of 17 genetic drivers, highlighting the need for a more comprehensive analysis. Therefore, we applied an integrated clustering approach to the recurrent mutations, CNAs and SVs and delineated 5 genetically distinct DLBCL subtypes and determined the likely temporal order of alterations within each cluster.This unbiased approach identified a previously unappreciated favorable risk ABC subset with genetic features of an extrafollicular, possibly marginal zone origin; a more tightly defined poor risk group of ABC DLBCLs with frequent BCL2 gain and concordant MYD88L265P / CD79B mutations; a distinct poor risk subset of GCB tumors with BCL2 SVs and alterations of PTEN and epigenetic enzymes and a discrete group of good risk GCB DLBCLs with specific alterations in BCR/PI3K, JAK/STAT and BRAF pathway components and multiple histones; and a Cell-of-Origin-independent subset with biallelic inactivation of TP53, 9p21.3/CDKN2A and associated genomic instability. These findings likely explain the variability in outcome predictions with the binary ABC- vs. GCB-DLBCL transcriptional classification and the challenges of therapeutically targeting less well defined DLBCLs.The genetically distinct DLBCL subsets provide a framework for assessing previously unrecognized heterogeneity in this disease, characterizing combinations of genetic alterations that drive DLBCL biology and guiding the development of rational single-agent and combination therapies in patients with the greatest need. DisclosuresCerhan:Janssen: Other: Scientific Advisory Board (REMICADELYM4001); Janssen: Other: Multiple Myeloma Registry Steering. Rodig:Bristol-Myers Squibb: Honoraria, Research Funding. Neuberg:Synta Pharmaceuticals: Other: Stock shares. Shipp:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Other: Scientific Advisory Board; Cell Signaling: Honoraria; Bayer: Research Funding; Takeda: Other: Scientific Advisory Board; Merck: Other: Scientific Advisory Board; AstraZeneca: Honoraria.

Clinical utility of whole genome sequencing in hematological neoplasms.

7028 Background: Hematological neoplasms are often characterized by acute onset and rapid disease progression. Cytogenetics, FISH, SNP arrays, targeted DNA and RNA sequencing are performed to inform diagnosis, risk stratification and guide treatment decisions. Whole genome sequencing (WGS) offers the opportunity to comprehensively characterize all putative biomarkers in a single assay. However, a limitation in current WGS implementation is the requirement for a germline sample, as sources of control tissue are frequently contaminated with leukemic cells resulting in false negative calls. Methods: To evaluate the clinical utility and feasibility of WGS in the diagnostic work up of leukemias, we analyzed 57 B-cell acute lymphoblastic leukemia (B-ALL) from the UKALL14 trial (NCT01085617) with no informative biomarkers at diagnosis. WGS analysis was performed on the leukemic sample and a matching control sample (with minimal residual disease level of &lt;1%). Using this dataset, we trained the development of an unmatched (uWGS) analytical workflow (Isabl) for a tumor only WGS study. This workflow was validated across 20 hematologic neoplasms (12 B-ALL, 6 AML and 2 T-ALL). Results: Among the 57 cases, 5 failed QC owing to low tumor content (&lt;25%). Of the remaining 52, putative biomarkers of clinical relevance were identified by WGS in 69% (36/52). These included delineation of aberrant karyotypes where conventional chromosome banding failed (4/52), the detection of newly described fusion genes (such as IGH-DUX4 and EP300-ZNF384 in 21/52) and recurrent gene mutations (i.e. PAX5 P80R, ZEB2 H1038R in 11/52). uWGS workflow in our training dataset captured 86% of biomarkers identified in the matched analysis (3/3 ploidy, 21/22 fusion and 7/11 coding). Concordance between the matched and uWGS workflow for arm-level and focal copy number alterations (CNAs), structural variants (SVs) and annotated hotspot mutations were 94%, 84%, 83% and 100% respectively. Independent validation of the uWGS workflow across 20 myeloid and lymphoid neoplasms, recapitulated all clinically reported biomarkers (14/15 CNAs, 16/16 SVs) as well as captured two novel findings not previously detected in two B-ALL patients, to include a focal deletion in BTG1 and the fusion gene P2RY8-CRLF2, as well as a NOTCH1 translocation in T-ALL. Conclusions: Our findings demonstrate that comprehensive WGS allows for the detection of the same biomarkers as a range of clinical assays using a single test, as well as the opportunity to discover novel clinical and research findings to support future correlative research and biomarker development. Additionally, we developed and validated an uWGS workflow that allows WGS analysis of hematopoietic neoplasms at diagnosis, enabling detection and reporting of clinically relevant biomarkers.

Analytic validation and clinical utilization of the comprehensive genomic profiling test, GEM ExTra®.

We developed and analytically validated a comprehensive genomic profiling (CGP) assay, GEM ExTra, for patients with advanced solid tumors that uses Next Generation Sequencing (NGS) to characterize whole exomes employing a paired tumor-normal subtraction methodology. The assay detects single nucleotide variants (SNV), indels, focal copy number alterations (CNA), TERT promoter region, as well as tumor mutation burden (TMB) and microsatellite instability (MSI) status. Additionally, the assay incorporates whole transcriptome sequencing of the tumor sample that allows for the detection of gene fusions and select special transcripts, including AR-V7, EGFR vIII, EGFRvIV, and MET exon 14 skipping events. The assay has a mean target coverage of 180X for the normal (germline) and 400X for tumor DNA including enhanced probe design to facilitate the sequencing of difficult regions. Proprietary bioinformatics, paired with comprehensive clinical curation results in reporting that defines clinically actionable, FDA-approved, and clinical trial drug options for the management of the patient’s cancer. GEM ExTra demonstrated analytic specificity (PPV) of > 99.9% and analytic sensitivity of 98.8%. Application of GEM ExTra to 1,435 patient samples revealed clinically actionable alterations in 83.9% of reports, including 31 (2.5%) where therapeutic recommendations were based on RNA fusion findings only.

Evolution of late-stage metastatic melanoma is dominated by aneuploidy and whole genome doubling

Although melanoma is initiated by acquisition of point mutations and limited focal copy number alterations in melanocytes-of-origin, the nature of genetic changes that characterise lethal metastatic disease is poorly understood. Here, we analyze the evolution of human melanoma progressing from early to late disease in 13 patients by sampling their tumours at multiple sites and times. Whole exome and genome sequencing data from 88 tumour samples reveals only limited gain of point mutations generally, with net mutational loss in some metastases. In contrast, melanoma evolution is dominated by whole genome doubling and large-scale aneuploidy, in which widespread loss of heterozygosity sculpts the burden of point mutations, neoantigens and structural variants even in treatment-naïve and primary cutaneous melanomas in some patients. These results imply that dysregulation of genomic integrity is a key driver of selective clonal advantage during melanoma progression.

MBRS-59. SINGLE-CELL WHOLE-GENOME SEQUENCING DISSECTS INTRA-TUMOURAL GENOMIC HETEROGENEITY AND CLONAL EVOLUTION IN CHILDHOOD MEDULLOBLASTOMA

Medulloblastomas harbor clinically-significant intra-tumoral heterogeneity for key biomarkers (e.g. MYC/MYCN, β-catenin). Recent studies have characterized transcriptional heterogeneity at the single-cell level, however the underlying genomic copy number and mutational architecture remains to be resolved. We therefore sought to establish the intra-tumoural genomic heterogeneity of medulloblastoma at single-cell resolution. Copy number patterns were dissected by whole-genome sequencing in 1024 single cells isolated from multiple distinct tumour regions within 16 snap-frozen medulloblastomas, representing the major molecular subgroups (WNT, SHH, Group3, Group4) and genotypes (i.e. MYC amplification, TP53 mutation). Common copy number driver and subclonal events were identified, providing clear evidence of copy number evolution in medulloblastoma development. Moreover, subclonal whole-arm and focal copy number alterations covering important genomic loci (e.g. on chr10 of SHH patients) were detected in single tumour cells, yet undetectable at the bulk-tumor level. Spatial copy number heterogeneity was also common, with differences between clonal and subclonal events detected in distinct regions of individual tumours. Mutational analysis of the cells allowed dissection of spatial and clonal heterogeneity patterns for key medulloblastoma mutations (e.g. CTNNB1, TP53, SMARCA4, PTCH1) within our cohort. Integrated copy number and mutational analysis is underway to establish their inter-relationships and relative contributions to clonal evolution during tumourigenesis. In summary, single-cell analysis has enabled the resolution of common mutational and copy number drivers, alongside sub-clonal events and distinct patterns of clonal and spatial evolution, in medulloblastoma development. We anticipate these findings will provide a critical foundation for future improved biomarker selection, and the development of targeted therapies.