Copy Number Aberration Profiles Research Articles

Abstract PO-058: Decoding tissue of origin patterns by tumor DNA and plasma tumor proteins

Abstract Background: Compared to standard cancer screening paradigms, blood-based cancer screening test was able to identify asymptomatic cancer patients in a less invasive manner. However, it is an imperative but challenging attribute of a blood-based multi-cancer detection test to accurately localize the tissue of origin (TOO) to direct the diagnostic workup. In this study, we analyzed the somatic copy number aberrations (CNA) and a panel of cancer-associated plasma protein markers via artificial intelligence algorithms to recognize the TOO patterns for specific cancer types. Method: CNA mutational data were downloaded from TCGA SNP6.0 array level 3 dataset. In total, 2600 cancer tissue samples from 13 common cancer types were selected, including bladder, breast, cervical, colorectal, endometrial, gastric, liver, lung, lymphoma, ovarian, prostate, renal and thyroid. Human reference genome hg19 was segmented as individual 5 megabase (M)-length bins. Average log R ratio of each bin was calculated based on SNP6.0 array data. We randomly selected 200 samples from each cancer type as the training set, and the bin log R ratios of each cancer type were used to train the TOO classification algorithm through random forest method. The remaining samples were used as validation cohort. This process was repeated 10 times in order to generate steady results. Average prediction value from 10 repetitions was regarded as final prediction. The same method was applied to analyze seven plasma tumor protein expression data of 1005 cancer patient samples from 8 cancer types, including breast, colorectal, esophageal liver, lung, ovarian, pancreatic, and gastric cancer (Cohen, J. D. et al., Science, 2018). Results: By analyzing the CNA features of different cancer types, the overall accuracy of TOO classification was 69.4%. The accuracy in distinguishing individual cancer types varied with highest in ovarian (92.2%), followed by renal (84.7%), and thyroid (77.6%) cancer, and lowest in bladder (50.7%) and endometrial (42.6%) cancer. On the other hand, when distinctive protein expression traits were exploited, the overall accuracy of TOO classification was 61.7%, with highest in colorectal cancer (79.4%) . Conclusion: Different cancer types acquire distinctive genomic features which may have causal relationship with tumorigenesis and progression. This study indicates that genomic features such as CNA were able to predict cancer types with high accuracy, which warrants further study of this feature in circulating tumor DNA to trace the TOO in a blood-based cancer detection test. We acknowledge that our analysis was based on CNA profiles of tumor tissues, and blood-based CNA detection from cell-free DNA is dependent on fractional concentrations of tumor-derived DNA in blood. Thus it is crucial to estimate the CNA concordance between cancer tissue and blood samples in future studies. Citation Format: Shuaipeng Geng, Wei Wu, Shiyong Li, Mengna Zhang, Yan Chen, Mao Mao. Decoding tissue of origin patterns by tumor DNA and plasma tumor proteins [abstract]. In: Proceedings of the AACR Virtual Special Conference on Artificial Intelligence, Diagnosis, and Imaging; 2021 Jan 13-14. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(5_Suppl):Abstract nr PO-058.

Ultra-low coverage whole genome sequencing of ccfDNA in multiple myeloma: A tool for laboratory routine?

Multiple myeloma (MM), is a heterogeneous disease in which chromosomal abnormalities are important for prognostic risk stratification. Cytogenetic profiling with FISH on plasma cells from bone marrow samples (BM-PCs) is the current gold standard, but variable infiltration of plasma cells or failed aspiration can hamper this process. Ultra-low coverage sequencing (ULCS) of circulating cell-free DNA (ccfDNA) may offer a minimally invasive alternative for the work-up of these cases. We compared ULCS, aCGH and FISH on selected BM-PCs in a routine setting with ULCS of ccfDNA for the detection of somatic copy number aberrations (CNAs) in MM. MethodsPurified CD138+ BM-PCs of 23 MM patients at initiation of their treatment were subjected to aCGH, FISH and ULCS. Paired samples of peripheral blood-ccfDNA obtained at diagnosis were analyzed by ULCS and compared to the results found in BM-PCs. ResultsUsing ULCS of ccfDNA, cytogenetic markers were identified in 18 out of 23 patients; five cases could not be analyzed due to low (≤3%) tumor fraction (TF). High similarity between CNA profiles of BM-PCs and ccfDNA was found. Moreover, 78% of the ccfDNA profiles resulted in the same risk classification as the routine FISH and/or BM-PCs ULCS and aCGH. Chromothripsis was detected in five patients; these had the highest TF values (range 7.1% to 42%) in our series and their profiles showed other high-risk anomalies. ConclusionThis proof-of-principle study indicates that ULCS of ccfDNA can reveal CNAs in MM and should be explored further as a cost-efficient alternative, especially in cases where BM-PC purification fails.

Inferring Copy Number from Triple-Negative Breast Cancer Patient Derived Xenograft scRNAseq Data Using scCNA.

Cancer can develop from an accumulation of alterations, some of which cause a nonmalignant cell to transform to a malignant state exhibiting increased rate of cell growth and evasion of growth suppressive mechanisms, eventually leading to tissue invasion and metastatic disease. Triple-negative breast cancers (TNBC) are heterogeneous and are clinically characterized by the lack of expression of hormone receptors and human epidermal growth factor receptor 2 (HER2), which limits its treatment options. Since tumor evolution is driven by diverse cancer cell populations and their microenvironment, it is imperative to map TNBC at single-cell resolution. Here, we describe an experimental procedure for isolating a single-cell suspension from a TNBC patient-derived xenograft, subjecting it to single-cell RNA sequencing using droplet-based technology from 10× Genomics and analyzing the transcriptomic data at single-cell resolution to obtain inferred copy number aberration profiles, using scCNA. Data obtained using this single-cell RNA sequencing experimental and analytical methodology should enhance our understanding of intratumor heterogeneity which is key for identifying genetic vulnerabilities and developing effective therapies.

Copy number evolution and its relationship with patient outcome\u2014an analysis of 178 matched presentation-relapse tumor pairs from the Myeloma XI trial

Structural chromosomal changes including copy number aberrations (CNAs) are a major feature of multiple myeloma (MM), however their evolution in context of modern biological therapy is not well characterized. To investigate acquisition of CNAs and their prognostic relevance in context of first-line therapy, we profiled tumor diagnosis–relapse pairs from 178 NCRI Myeloma XI (ISRCTN49407852) trial patients using digital multiplex ligation-dependent probe amplification. CNA profiles acquired at relapse differed substantially between MM subtypes: hyperdiploid (HRD) tumors evolved predominantly in branching pattern vs. linear pattern in t(4;14) vs. stable pattern in t(11;14). CNA acquisition also differed between subtypes based on CCND expression, with a marked enrichment of acquired del(17p) in CCND2 over CCND1 tumors. Acquired CNAs were not influenced by high-dose melphalan or lenalidomide maintenance randomization. A branching evolution pattern was significantly associated with inferior overall survival (OS; hazard ratio (HR) 2.61, P = 0.0048). As an individual lesion, acquisition of gain(1q) at relapse was associated with shorter OS, independent of other risk markers or time of relapse (HR = 2.00; P = 0.021). There is an increasing need for rational therapy sequencing in MM. Our data supports the value of repeat molecular profiling to characterize disease evolution and inform management of MM relapse.

Gamma Knife Radiosurgery does not alter the copy number aberration profile in sporadic vestibular schwannoma

IntroductionIonizing radiation is a known etiologic factor in tumorigenesis and its role in inducing malignancy in the treatment of vestibular schwannoma has been debated. The purpose of this study was to identify a copy number aberration (CNA) profile or specific CNAs associated with radiation exposure which could either implicate an increased risk of malignancy or elucidate a mechanism of treatment resistance.Methods55 sporadic VS, including 18 treated with Gamma Knife Radiosurgery (GKRS), were subjected to DNA whole-genome microarray and/or whole-exome sequencing. CNAs were called and statistical tests were performed to identify any association with radiation exposure. Hierarchical clustering was used to identify CNA profiles associated with radiation exposure.ResultsA median of 7 (0–58) CNAs were identified across the 55 VS. Chromosome 22 aberration was the only recurrent event. A median aberrant cell fraction of 0.59 (0.25–0.94) was observed, indicating several genetic clones in VS. No CNA or CNA profile was associated with GKRS.ConclusionGKRS is not associated with an increase in CNAs or alteration of the CNA profile in VS, lending support to its low risk. This also implies that there is no major issue with GKRS treatment failure being due to CNAs. In agreement with previous studies, chromosome 22 aberration is the only recurrent CNA. VS consist of several genetic clones, addressing the need for further studies on the composition of cells in this tumor.

Abstract LB-270: Discovery of genomic correlates and tumor purity as an independent clinical factor of poor outcome in advanced prostate cancer lpWGS CNA data

Abstract Background: We, and others, have shown that responses to therapies in metastatic castration-resistant prostate cancer (mCRPC) can be monitored using plasma cell-free DNA (cfDNA), and that this can be both quantitative and qualitative. In this study, we explored the utility of low pass whole genome sequencing (lpWGS) of cfDNA in mCRPC patients treated on two large prospectively collected Phase III trials of taxane chemotherapy, FIRSTANA (NCT01308567) and PROSELICA (NCT01308580). Methods: Plasma samples collated from the FIRSTANA and PROSELICA trials were evaluated. cfDNA was isolated and analysed with lpWGS using the Illumina NovaSeq 6000. Reads were aligned using BWA-MEM and quality controlled using Picard and FASTQC. Whole-genome copy number aberration (CNA) profiles were generated using ichorCNA, which also estimated tumor purity and ploidy. Log-rank tests were used for univariable survival comparisons, and Cox proportional hazard models were used for multivariable survival analysis. Results: Overall, lp-WGS data was generated from 528 samples acquired at three different timepoints (Baseline [SCR and C1], C4 and EOS), representing 188 unique patients. We observed CNA profiles in line with emergent mCRPC subtypes including highly aberrant, large-scale events which have been linked to homologous-repair deficient tumors. We found that CNAs in this cohort matched other published studies with frequent amplifications of AR (~30%), copy-gains of PI3KCA (~45%) and copy-loss of NKX3-1 (~75%), RB1 (~70%) and PTEN (~50%). We did not observe changes in baseline tumor purity or ploidy between the FIRSTANA and PROSELICA cohorts. Following calculation of a large-scale transition (LST) score (measuring CNA breakpoints), we observed that this was significantly elevated (Wilcox test, p=0.0073) in cases that had prior exposure to second line androgen deprivation (abiraterone or enzalutamide). Baseline tumor purity values were associated with several mCRPC clinical variables including ECOG performance status. High lpWGS tumor purity values were univariably associated with poorer overall survival in both FIRSTANA and PROSELICA cohorts (log-rank test p=0.0021 and 0.0009), and were also associated with significantly poorer RPFS and PSAPFS. Tumor purity was also associated (HR 1.66, p=0.036) with survival when assessed using multivariable models alongside other mCRPC clinical variables, including cell-free DNA concentration. Further, longitudinal changes in tumor purity on treatment can be illustrative of drug responses. Conclusions: In our analysis of this prospectively collected clinical trial data, we show mCRPC CNA profiles in cfDNA, and that these CNAs may be induced by prior aggressive treatments. We also found that tumor purity estimation can serve as a robust, competitive indicator of both overall survival and, assessed longitudinally, shows clear association with drug response. We envision that these techniques will enable improved strategies for monitoring mCRPC patients. Citation Format: George Seed, Semini Sumanasuriya, Claudia Bertan, Harry Parr, Gemma Fowler, Rossitza Christova, Lorna Pope, Jane Goodall, Maryou Lambros, Pasquale Rescigno, Penelope Flohr, Suzanne Carreira, Wei Yuan, Mustapha Chadjaa, Sandrine Mace, Johann De Bono. Discovery of genomic correlates and tumor purity as an independent clinical factor of poor outcome in advanced prostate cancer lpWGS CNA data [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-270.

Abstract 1118: Absence of mouse-specific tumor evolution in patient-derived cancer xenografts

Abstract Patient-Derived Xenografts (PDXs) are preclinical models largely used to study tumor biology and drug response. Recent literature highlighted the possibility that growth of human tumors in a mouse microenvironment imposes a selection driving mouse-specific genetic evolution of PDXs, which may compromise their reliability as human cancer models. Conversely, independent studies observed a conservation of the genomic landscape during PDX engraftment and passaging. We noticed that PDX genetic evolution was particularly evident in studies based on copy number aberration (CNA) inferred from gene expression data, while it was negligible when DNA-based CNA profiles were employed. Therefore, in a joint international effort of the EurOPDX and PDXNet consortia, we assembled a dataset of 37 hepatocellular and 54 gastric carcinoma tumor or PDX samples with matched RNA-based and DNA-based CNA profiles. We found that DNA-based CNA profiles invariably yield higher concordance between patient's tumor and derived PDXs than those inferred from RNA. RNA-based profiles displayed poor concordance with matched DNA-based profiles, and much lower resolution, so that they missed many focal copy number events detected by DNA-based methods. These results revealed that CNA measurements cannot be accurately estimated by expression data and that a systematic reassessment of CNA dynamics in PDXs based on DNA data is required. To this aim, we generated CNA profiles by low-pass whole genome sequencing (WGS) of 87 colorectal and 43 breast cancer triplets, each composed of matched patient's tumor (PT) and PDX at early (PDX-early) and later (PDX-late) passage. In this way, for each tumor type, we generated three perfectly matched PT, PDX-early and PDX-late cohorts and performed CNA recurrence analysis by GISTIC in each cohort. The hypothesis was that if the mouse host induces a selective pressure capable of shaping the CNA landscape during PDX engraftment and propagation, GISTIC analysis would highlight systematic and progressive changes, from the PT to the PDX-early cohort, and then to the PDX-late cohort. Notably instead, the CNA profiles of the PT and PDX-early/late cohorts were virtually indistinguishable, with no progressive accumulation or loss of CNA during PDX passage and only minor changes not functionally related or associated to cancer-driver or actionable genes. These results were not consequence of insufficient capture of the CNA repertoire, since the GISTIC profiles recapitulated those generated by TCGA for colorectal and breast cancer. In summary, our analyses highlighted that while RNA-based CNA inferences have inadequate resolution and accuracy to study genomic evolution in PDXs, DNA-based CNA profiles confirm retention of CNAs in PTs and PDXs, excluding a systematic mouse driven selection via copy number changes. Ultimately, these results support the robustness of PDXs as preclinical models for predicting drug response. Citation Format: Jessica Giordano, Xing Yi Woo, Anuj Srivastava, Zi-Ming Zhao, Michael W. Lloyd, Roebi de Bruijn, Yun-Suhk Suh, Francesco Galimi, Andrea Bertotti, Adam Lafferty, Alice C. O'Farrell, Elodie Modave, Diether Lambrechts, Petra ter Brugge, Violeta Serra, Elisabetta Marangoni, Rania El Botty, Jong-Il Kim, Han-Kwang Yang, Charles Lee, Dennis A. Dean, Brandi Davis-Dusenbery, Yvonne A. Evrard, James H. Doroshow, Alana L. Welm, Bryan E. Welm, Michael T. Lewis, Bingliang Fang, Jack Roth, Funda Meric-Bernstam, Meenhard Herlyn, Michael Davies, Li Ding, Shunqiang Li, Ramaswamy Govindan, Jeffrey A. Moscow, Carol J. Bult, Claudio Isella, Livio Trusolino, Annette T. Byrne, Jos Jonkers, Jeffrey H. Chuang, Enzo Medico, EurOPDX consortium & PDXNET consortium. Absence of mouse-specific tumor evolution in patient-derived cancer xenografts [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1118.

High-Throughput Molecular Cancer Cell Line Characterization Using Digital Multiplex Ligation-Dependent Probe Amplification for Improved Standardization of in Vitro Research

Tumor cell lines are widely used for cancer research, but challenges regarding quality control of cell line identity, cross contamination, and tumor somatic molecular stability remain, demanding novel approaches beyond conventional short tandem repeat profiling. A total of 21 commonly used multiple myeloma cell lines obtained from public repositories were analyzed by digital multiplex ligation-dependent probe amplification (digitalMLPA) to characterize germline single-nucleotide polymorphisms, insertions/deletions, and somatic copy number aberrations (CNAs). Using generated profiles and an in-house developed analytical pipeline, blinded experiments were performed to determine capability of digitalMLPA to predict cell line identity and potential spike-in DNA contamination in 41 anonymized cell line samples. The dominant cell line was correctly identified in all cases, and cross contamination was correctly detected in 33 of 37 samples with spike-in DNA; there were no false-positive predictions. The four samples in which spike in was not detected all carried low levels of contamination (1%), whereas levels of contamination ≥5% were correctly identified in all cases. Unsupervised clustering of CNA profiles identified shared commonalities that correlated with initiating Ig heavy locus translocation events. Longitudinal CNA assessment of nine cell lines revealed changes under standard culturing conditions not detected by insertion/deletion profiling alone. Results suggest that digitalMLPA can be utilized as a high-throughput tool for advanced quality assurance for invitro cancer research.

MP01-12 DETECTION OF UROTHELIAL CARCINOMA BASED ON COPY NUMBER PROFILES OF URINARY CFDNA USING SHALLOW WHOLE-GENOME SEQUENCING

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I (MP01)1 Apr 2020MP01-12 DETECTION OF UROTHELIAL CARCINOMA BASED ON COPY NUMBER PROFILES OF URINARY CFDNA USING SHALLOW WHOLE-GENOME SEQUENCING Yanqing Gong*, Guangzhe Ge, Ding Peng, Bao Guan, Yuanyuan Zhou, Weimin Ci, Xuesong Li, and Liqun Zhou Yanqing Gong*Yanqing Gong* More articles by this author , Guangzhe GeGuangzhe Ge More articles by this author , Ding PengDing Peng More articles by this author , Bao GuanBao Guan More articles by this author , Yuanyuan ZhouYuanyuan Zhou More articles by this author , Weimin CiWeimin Ci More articles by this author , Xuesong LiXuesong Li More articles by this author , and Liqun ZhouLiqun Zhou More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000815.012AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Current non-invasive assays for urothelial cancer (UC) lack sensitivity and specificity. Given the utility of plasma cell-free DNA (cfDNA) biomarkers, the development of urinary cfDNA biomarkers may improve the diagnostic sensitivity. METHODS: We assessed copy number aberrations (CNAs) by shallow genome-wide sequencing of urinary cfDNA in 95 cancer-free individuals and 65 UC, 58 kidney cancer and 45 prostate cancer patients. We used a support vector machine (SVM) to develop a diagnostic classifier based on CNA profiles to detect UC (UCdetector). The model was further validated in an independent cohort (52 cases). Genome sequencing data of tumor specimens from 90 upper tract urothelial cancers (UTUC) and CNA data for 410 urothelial carcinomas of bladder (UCB) from TCGA were used to validate the classifier. Genome sequencing data for urine sediments of 32 UC patients were compared with cfDNA. To monitor the treatment efficacy, we collected cfDNA from 7 post-treatment patients. RESULTS: We showed that urinary cfDNA could be a more sensitive alternative to urinary sediment. The UCdetector can detect UC at a median sensitivity of 86.5% and specificity of 94.7%. The UCdetector performed well in an independent validation dataset. Notably, the CNA features selected by UCdetector were specific markers for both UTUC and UCB. Moreover, the CNA changes in cfDNA were consistent with the treatment effects. Meanwhile, the same strategy could localize genitourinary cancers to tissue-of-origin in 70.1% of patients. CONCLUSIONS: Our findings underscore the potential utility of urinary cfDNA CNA profiles as a basis for non-invasive UC detection and surveillance. Source of Funding: This work was supported by CAS Strategic Priority Research Program (XDA16010102 to W.C.), the National Key R&D Program of China (2018YFC2000100 to W.C.), CAS (QYZDB-SSW-SMC039 to W.C.), the National Natural Science Foundation of China (81672541 to W.C.), and K.C.Wong Education Foundation to W.C. © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e6-e6 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yanqing Gong* More articles by this author Guangzhe Ge More articles by this author Ding Peng More articles by this author Bao Guan More articles by this author Yuanyuan Zhou More articles by this author Weimin Ci More articles by this author Xuesong Li More articles by this author Liqun Zhou More articles by this author Expand All Advertisement PDF downloadLoading ...

Whole genome analysis identifies the association of TP53 genomic deletions with lower survival in Stage III colorectal cancer

DNA copy number aberrations (CNA) are frequently observed in colorectal cancers (CRC). There is an urgent need for CNA-based biomarkers in clinics,. n For Stage III CRC, if combined with imaging or pathologic evidence, these markers promise more precise care. We conducted this Stage III specific biomarker discovery with a cohort of 134 CRCs, and with a newly developed high-efficiency CNA profiling protocol. Specifically, we developed the profiling protocol for tumor-normal matched tissue samples based on low-coverage clinical whole-genome sequencing (WGS). We demonstrated the protocol’s accuracy and robustness by a systematic benchmark with microarray, high-coverage whole-exome and -genome approaches, where the low-coverage WGS-derived CNA segments were highly accordant (PCC >0.95) with those derived from microarray, and they were substantially less variable if compared to exome-derived segments. A lasso-based model and multivariate cox regression analysis identified a chromosome 17p loss, containing the TP53 tumor suppressor gene, that was significantly associated with reduced survival (P = 0.0139, HR = 1.688, 95% CI = [1.112–2.562]), which was validated by an independent cohort of 187 Stage III CRCs. In summary, this low-coverage WGS protocol has high sensitivity, high resolution and low cost and the identified 17p-loss is an effective poor prognosis marker for Stage III patients.

Comprehensive profiling of disease-relevant copy number aberrations for advanced clinical diagnostics of pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia is the most common pediatric cancer characterized by a heterogeneous genomic landscape with copy number aberrations occurring at various stages of pathogenesis, disease progression, and treatment resistance. In this study, disease-relevant copy number aberrations were profiled in bone marrow samples of 91 children with B- or T-cell precursor acute lymphoblastic leukemia using digital multiplex ligation-dependent probe amplification (digitalMLPATM). Whole chromosome gains and losses, subchromosomal copy number aberrations, as well as unbalanced alterations conferring intrachromosomal gene fusions were simultaneously identified with results available within 36 hours. Aberrations were observed in 96% of diagnostic patient samples, and increased numbers of copy number aberrations were detected at the time of relapse as compared with diagnosis. Comparative scrutiny of 24 matching diagnostic and relapse samples from 11 patients revealed three different patterns of clonal relationships with (i) one patient displaying identical copy number aberration profiles at diagnosis and relapse, (ii) six patients showing clonal evolution with all lesions detected at diagnosis being present at relapse, and (iii) four patients displaying conserved as well as lost or gained copy number aberrations at the time of relapse, suggestive of the presence of a common ancestral cell compartment giving rise to clinically manifest leukemia at different time points during the disease course. A newly introduced risk classifier combining cytogenetic data with digitalMLPATM-based copy number aberration profiles allowed for the determination of four genetic subgroups of B-cell precursor acute lymphoblastic leukemia with distinct event-free survival rates. DigitalMLPATM provides fast, robust, and highly optimized copy number aberration profiling for the genomic characterization of acute lymphoblastic leukemia samples, facilitates the decipherment of the clonal origin of relapse and provides highly relevant information for clinical prognosis assessment.

Comprehensive Profiling of Disease-Relevant Copy Number Aberrations Improves Risk Assessment and Unveils the Clonal Origin of Relapse in Pediatric Acute Lymphoblastic Leukemia

Introduction: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy characterized by a heterogeneous genomic landscape. Copy number aberrations (CNA) emerge during the development, progression and treatment resistance of ALL, and can serve as genomic markers for prognostic classification of patients or for scrutinizing clonal evolution associated with relapse. While identification of distinct CNAs with well-characterized prognostic significance has its own value, uncovering the co-segregation of driver aberrations in individual patient samples could allow for a more personalized risk assessment and treatment response prediction. Methods: Disease-relevant CNAs were profiled in children with B- or T-cell precursor ALL using a next-generation sequencing based digital multiplex ligation-dependent probe amplification (digitalMLPA) assay containing 598 probes specific for 54 genes with key relevance in ALL. Besides the diagnostic samples of 91 patients treated according to the BFM protocols, 14 matching samples drawn at the time of first or second relapse were comparatively analyzed. Clonal relationship between B-cell precursor cell populations prevailing at different time points during the disease course was also investigated by screening immunoglobulin heavy-chain gene rearrangements in matching diagnostic and relapse samples using Illumina deep-sequencing with >20,000x coverage. Results: Whole chromosome gains and losses, subchromosomal CNAs as well as alterations conferring intrachromosomal gene fusions were simultaneously identified by digitalMLPA with results available within 36 hours. Aberrations were observed in 96% of diagnostic patient samples and increased numbers of CNAs were detected in individual samples at the time of relapse as compared to diagnosis. DigitalMLPA results were successfully validated by conventional MLPA, FISH and PCR data. Comparative scrutiny of 24 matching diagnostic and relapse samples from 11 patients harboring CNAs revealed three different patterns of clonal relationships with (i) one patient displaying identical CNA profiles at diagnosis and relapse, (ii) six patients showing clonal evolution with all lesions detected at diagnosis being present at relapse and (iii) four patients displaying conserved as well as lost or gained CNAs at the time of relapse, suggestive of the presence of a common ancestral cell compartment giving rise to clinically manifest leukemia at different time points during the disease course. Time between diagnosis and first relapse of T-ALL patients displaying altered CNA profiles suggested a prolonged time requirement of clonal evolution, and of the development of manifest leukemia from an ancestral clone compared to the quick return of an identical clone at the time of relapse. Comparison of the IGH gene rearrangements identified at diagnoses and relapse revealed identical compositions of the most abundant clonotypes in all but one B-ALL patients analyzed; hence, IGH repertoire did not reveal an additional depth of clonal history in our cohort, e.g. by demonstrating the presence of an ancestral clone as the major source of clonal expansion at disease progression in a patient with altered CNA profiles suggesting direct clonal evolution between diagnosis and relapse. Copy number profiles acquired by digitalMLPA were used for determining CNA-based risk groups (Table 1) which were combined with karyotyping and molecular cytogenetic data in order to establish an extended prognostic classifier for patients with B-cell precursor ALL. This novel classifier distinguished four combined genetic risk groups showing significantly different 5-year survival rates (GR: 97%, IR: 84%, IHR: 63% and PR: 13%). Conclusions: DigitalMLPA allows for a rapid, scalable and highly optimized copy number profiling of genomic regions recurrently altered by driver aberrations in pediatric ALL. Based on the comparison of CNA profiles at diagnosis and relapse, clonal evolution and emergence of relapse from an ancestral clone are the predominant driving mechanisms of disease progression. Comprehensive copy number profiling by digitalMLPA identifies distinct prognostic groups for risk assessment in B-cell precursor ALL. Supporting grants : LP95021, K_16 #119950, NVKP_16-1-2016-0004, KH17-126718, BO/00320/18/5, FK_19 #131476, ÚNKP-19-4-SE-77 Disclosures Benard-Slagter: MRC Holland: Employment. de Groot:MRC Holland: Employment. Savola:MRC Holland: Employment.

Clonality analysis of pulmonary tumors by genome-wide copy number profiling.

Multiple tumors in patients are frequently diagnosed, either synchronous or metachronous. The distinction between a second primary and a metastasis is important for treatment. Chromosomal DNA copy number aberrations (CNA) patterns are highly unique to specific tumors. The aim of this study was to assess genome-wide CNA-patterns as method to identify clonally related tumors in a prospective cohort of patients with synchronous or metachronous tumors, with at least one intrapulmonary tumor. In total, 139 tumor pairs from 90 patients were examined: 35 synchronous and 104 metachronous pairs. Results of CNA were compared to histological type, clinicopathological methods (Martini-Melamed-classification (MM) and ACCP-2013-criteria), and, if available, EGFR- and KRAS-mutation analysis. CNA-results were clonal in 74 pairs (53%), non-clonal in 33 pairs (24%), and inconclusive in 32 pairs (23%). Histological similarity was found in 130 pairs (94%). Concordance between histology and conclusive CNA-results was 69% (74 of 107 pairs: 72 clonal and two non-clonal). In 31 of 103 pairs with similar histology, genetics revealed non-clonality. In two out of four pairs with non-matching histology, genetics revealed clonality. The subgroups of synchronous and metachronous pairs showed similar outcome for the comparison of histological versus CNA-results. MM-classification and ACCP-2013-criteria, applicable on 34 pairs, and CNA-results were concordant in 50% and 62% respectively. Concordance between mutation matching and conclusive CNA-results was 89% (8 of 9 pairs: six clonal and two non-clonal). Interestingly, in one patient both tumors had the same KRAS mutation, but the CNA result was non-clonal. In conclusion, although some concordance between histological comparison and CNA profiling is present, arguments exist to prefer extensive molecular testing to determine whether a second tumor is a metastasis or a second primary.

Molecular characterization of colorectal adenomas reveals POFUT1 as a candidate driver of tumor progression.

Removal of colorectal adenomas is an effective strategy to reduce colorectal cancer (CRC) mortality rates. However, as only a minority of adenomas progress to cancer, such strategies may lead to overtreatment. The present study aimed to characterize adenomas by in‐depth molecular profiling, to obtain insights into altered biology associated with the colorectal adenoma‐to‐carcinoma progression. We obtained low‐coverage whole genome sequencing, RNA sequencing and tandem mass spectrometry data for 30 CRCs, 30 adenomas and 18 normal adjacent colon samples. These data were used for DNA copy number aberrations profiling, differential expression, gene set enrichment and gene‐dosage effect analysis. Protein expression was independently validated by immunohistochemistry on tissue microarrays and in patient‐derived colorectal adenoma organoids. Stroma percentage was determined by digital image analysis of tissue sections. Twenty‐four out of 30 adenomas could be unambiguously classified as high risk (n = 9) or low risk (n = 15) of progressing to cancer, based on DNA copy number profiles. Biological processes more prevalent in high‐risk than low‐risk adenomas were related to proliferation, tumor microenvironment and Notch, Wnt, PI3K/AKT/mTOR and Hedgehog signaling, while metabolic processes and protein secretion were enriched in low‐risk adenomas. DNA copy number driven gene‐dosage effect in high‐risk adenomas and cancers was observed for POFUT1, RPRD1B and EIF6. Increased POFUT1 expression in high‐risk adenomas was validated in tissue samples and organoids. High POFUT1 expression was also associated with Notch signaling enrichment and with decreased goblet cells differentiation. In‐depth molecular characterization of colorectal adenomas revealed POFUT1 and Notch signaling as potential drivers of tumor progression.

Frequent clonal relations between metastases and non-index prostate cancer lesions.

Primary prostate cancer lesions are clonally heterogeneous and often arise independently. In contrast, metastases were reported to share a monoclonal background. Because prostate cancer mortality is the consequence of distant metastases, prevention of metastatic outgrowth by primary tumor ablation is the main focus of treatment for localized disease. Focal therapy is targeted ablation of the primary index lesion, but it is unclear whether remaining primary lesions metastasize at a later stage. In this study, we compared copy number aberration profiles of primary prostate cancer lesions with matching pelvic lymph node metastases of 30 patients to establish clonality between a lymph node metastasis and multiple primary lesions within the same patient. Interestingly, in 23.3% of the cases, the regional metastasis was not clonally linked to the index primary lesion. These findings suggest that focal ablation of only the index lesion is potentially an undertreatment of a significant proportion of prostate cancer patients.

Unraveling the Landscape of Copy Number Aberrations in Hodgkin Lymphoma: A Joint KU Leuven and Lysa Study on Circulating Cell Free DNA

Introduction. For decades, the study of genomic alterations in Hodgkin lymphoma (HL) has been hampered by the low abundance of the malignant Hodgkin Reed-Sternberg (HRS) cells in HL lymph node biopsies. Laser microdissection or flow cytometric cell sorting allow the study of purified HRS cells, but the use of these applications is restricted to specialized research centers. We have recently demonstrated proof-of-principle that copy number aberrations (CNA) in HRS cells can be retrieved in circulating cell-free DNA (cfDNA) of HL patients (Vandenberghe et al., Lancet Haematol. 2015). Taking advantage of the presence of HRS cell-derived DNA (ctDNA) in plasma, the aim of this study was to catalogue CNA in HL in a large series of prospectively recruited HL patients.Methods. We analyzed plasma collected from 177 patients (median age 29, range 3-86) with newly diagnosed HL. 60 cases were diagnosed at our institution, including all disease subtypes and stages, with a majority of nodular sclerosis. 118 patients were recruited in the context of the BREACH study, a multicentric Phase 2B study for unfavorable early classical HL (NCT02292979). After cfDNA extraction, samples were low-pass sequenced (0.1x coverage) and analyzed using ichorCNA, an algorithm that produces read depth-based log2 CNA profiles and quantifies the cfDNA tumor content.Results. At diagnosis, 86 % (152/177) of patients showed obvious genomic imbalances in cfDNA, in early-stage (85 % (131/155)) as well as in advanced cases (95 % (21/22)). Among the abnormal profiles, gains encompassing 2p16, 5p15, 9p24, 12q13 and 19q13 were the most frequent aberrations, occurring in 79 %, 53 %, 57 %, 63 % and 80 % respectively. Losses most frequently affected regions 4q34, 6q23, 11q22 and 13q13, in 53 %, 63 %, 49 % and 59 % of abnormal profiles respectively (fig. 1A). All these CNAs have been previously described with varying frequencies in smaller series of 10-53 patients, using arrayCGH or whole exome sequencing on microdissected HRS cells or even HRS cell-derived cell lines as input material. The observed pattern of CNAs is distinct from the pattern we observed in cfDNA obtained from other hematological malignances, e.g. multiple myeloma and diffuse large B-cell lymphoma (fig. 1B, C).Analysis of follow-up cfDNA samples revealed that the majority of patients rapidly clear their profiles during the first two treatment cycles. Of 123 samples analysed at d15 of cycle 1, 89 samples (72 %) showed a normal profile without CNAs.This suggests a relationship between the fraction of ctDNA in cfDNA and the disease burden. However, we could not find a correlation between the ctDNA fraction as calculated by ichorCNA and the metabolic tumor volume as computed from PET/CT scans. This could be due to several factors, e.g. the small fraction of HRS cells in the metabolic tumor volume. We are currently investigating whether a correlation does exist between the HRS content in the lymph node biopsy and the ctDNA load.Conclusions. In this largest study of CNA in HL to date, we provide a comprehensive catalogue of the types of CNAs, as well as their frequencies and patterns in HL. In this series of 177 patients, gains affecting 2p, 12q and 19q and loss of 6q and 13q emerge as the most commonly recurrent CNAs in HL, across all HL subtypes and stages. The data confirm and extend our previous findings that the majority of HL patients, including those with early-stage disease, have detectable CNAs in their cfDNA at diagnosis. We demonstrate a broad spectrum of aberrations, gains and losses, some of which recur at higher frequencies than gains of 9p24, harboring PD-L1. This warrants further studies on how these CNAs are implicated in the pathogenesis of HL. It further endorses the use of ctDNA as an alternative gateway to the genome of HRS cells, and as a substrate for the evaluation of early disease response. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract A051: Liquid biopsy by apheresis: Molecular characterization of circulating tumor cells and their organoid culture reflects intrapatient heterogeneity and clonal evolution

Abstract Liquid biopsy components from blood, such as cell free DNA (cfDNA) and circulating tumor cells (CTCs), are prognostic for overall survival in advanced prostate cancer patients and allow the study of clonal evolution. cfDNA is easily obtained and has been widely used for molecular characterization and reflects pooled genomic profiles in a patient, but has limitations regarding gene copy number calls. CTC single-cell genomic studies generate precise gene copy number calls and elucidate intrapatient intercellular genomic heterogeneity. The main limitation of CTC analyses has been the low CTC count found in many cancer patients. We elected to study whether liquid biopsy by apheresis in advanced prostate cancer patients increases the yield of CTC to study tumor genomics, intrapatient heterogeneity, and ex vivo organotypic 3D models. Advanced metastatic prostate cancer patients being considered for clinical trials were invited to consent to apheresis. Apheresis CTC counts using CellSearchTM (Menarini) were acquired from 16 patients. The contents of the CellSearch cartridges were sorted into pure single cells by fluorescence-activated cell sorting and subsequently assessed by array comparative genomic hybridization (aCGH, Agilent Technology) for copy number aberrations (CNA). Exome and aCGH from tissue biopsies were compared to the single cell aCGH results. We generated patient-derived organoid (PDOs) cultures from apheresis products by preenrichment using density gradient (Lymphoprep) and subsequent CTC enrichment by EpCAM positive selection (EasySep StemCell Technologies). PDOs were characterized by immunofluorescence (IF) as DAPI+/CK+/EpCAM+ and CD45- cells and subsequently by aCGH for CNA. All sixteen patients (median age of 70 years; range 60-77 years) tolerated apheresis without any adverse effects. CTC counts from peripheral blood (PB) prior to apheresis ranged from 13 to 711 (median = 96), and did not significantly change post apheresis. The estimated CTC yield per apheresis ranged from 660-35473 per apheresis product (median = 3351). This constitutes an increase of 102-fold when compared to median CTC capture from 7.5mL of PB. A total of 170 single CTCs from 15 apheresis patients were genomically profiled and the copy number aberration profiles confirmed prostate cancer with multiple genomic hallmarks including CNAs such as AR amplification, chromosome 8q gain (MYC locus), and PTEN, RB1, BRCA2, TP53, CHD1 loss. CNA profiles of PDOs showed similar genomic aberrations to same patient CTCs and also reflected intrapatient heterogeneity detected by single CTC analysis. In conclusion, apheresis from advanced prostate cancer patients is a well-tolerated procedure and in our study increased the CTC yield by 102-fold when compared to PB. CTC and PDOs from apheresis products shared similar CNA profile compared with tissues biopsies and furthermore gave us an insight of the tumor heterogeneity and clonal evolution. Citation Format: Maryou B.K. Lambros, Veronica Gil, Mateus Crespo, Mariane S. Fontes, Alan Mackay, Gemma Folwer, Gemma Folwer, Berni Ebbs, Rui Neves, Penny Flohr, Susana Miranda, Semini Sumanasuriya, Daniel N. Rodrigues, Rita Pereira, Geroge Seed, Wei Yuan, Joanne Hunt, Deirdre Moloney, Dionne Ayanda, Niven Mehra, Jane Goodall, Claudia Bertan, Suzanne Carreira, Nikolas H. Stoecklein, Leon W.M.M. Terstappen, Gunther Boysen, Joahnn S. De Bono. Liquid biopsy by apheresis: Molecular characterization of circulating tumor cells and their organoid culture reflects intrapatient heterogeneity and clonal evolution [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A051.

Abstract GS1-04: Copy number aberration analysis to predict response to neoadjuvant anti-HER2 therapy: Results from the NeoALTTO phase III trial

Abstract Background In the NeoALTTO trial, the combination of trastuzumab and lapatinib for the neoadjuvant treatment of HER2 positive early breast cancer patients (pts) nearly doubled the rate of pathological complete response (pCR) compared with either anti-HER2 treatment alone. In the same trial, pCR was shown to be a surrogate for long-term outcome. Expression of ERBB2, ESR1 and immune signatures were the main drivers of pCR (Fumagalli et al. JAMA Oncol 2016). The main aim of the current study was to investigate the relevance of copy number aberrations (CNAs) for pCR and event-free survival (EFS). Methods For 271 out of the 455 pts enrolled in the study with sufficient tumour content, DNA was hybridized on CytoScan HD arrays. The log2 ratio intensities and B allele frequencies were segmented jointly using the multitrack segmentation algorithm. Integer level estimates of total copy number and major allele were obtained using GAP. Recurrent CNAs were found with GISTIC2. The genome instability index (GII) was defined as the median absolute deviation of the normalized copy number. Tumor infiltrating lymphocytes (TILs) and gene expression were obtained as described previously. The correlations between differents parameters were assessed using Spearman correlations (ρ). The Mann-Whitney test was used to relate binary and numerical features. EFS analysis was performed using the Cox proportional hazard model. Results CNAs estimates were obtained for 185 out of 271 pts. CNA distribution was similar to the ones observed in HER2+ pts from the METABRIC and the TCGA datasets. There were 64% of diploid pts, 26% of triploid and 9% of tetraploid or more. Aneuploidy level was not associated with pCR or EFS. Of interest, there were many significant differences in CNA profiles between ER+ and ER- pts. Those differences mirror those observed between ER+ and ER- among HER2- pts in TCGA (ρ=63%) and METABRIC (ρ=56%). ERBB2 amplification was predictive of high pCR (p=0.0007), albeit less so than ERBB2 expression, and ceased to be significant correcting for ERBB2 expression. The pCR rate increased with the GII (p=0.03), independently of ERBB2 amplification. The effect was stronger in ER+ patients (p=0.01). GISTIC analysis revealed 159 recurrent CNA regions. Amplification of 2 regions on 6q23-24 was significantly associated with higher pCR (p=0.00005 and p=0.00087, FDR=0.006 and 0.05). The most significant segment of 6q23-24 contained 39 genes, some whose expression level also predicted pCR (e.g. MAP3K5, p=10<sup style="font-family: Calibri;">-4). Gene ontology analysis of the genes correlated with this segment highlighted the category 'response to interferon-alpha' (p=4.3x10<sup style="font-family: Calibri;">-7). No amplified region or gene was found to be predictive of EFS, after multiple testing correction. Conclusions The amplification of ERBB2 was shown to be predictive of pCR, however its expression was more predictive. High genomic instability was associated with higher rate of pCR in ER+ subgroup. Of interest, a novel amplified region, involving chromosome 6 was shown to be predictive of pCR, which may warrant further investigation. Citation Format: Sotiriou C, Rothé F, Maetens M, Fumagalli D, Brown DN, Salgado R, Bradbury I, Pusztai L, Harbeck N, Ignatiadis M, Sarp S, de la Pena L, de Azambuja E, Huober J, Nuciforo P, Baselga J, Piccart M, Loi S, Venet D. Copy number aberration analysis to predict response to neoadjuvant anti-HER2 therapy: Results from the NeoALTTO phase III trial [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS1-04.

Abstract P2-07-02: Uncovering TNBC molecular heterogeneity by applying integrative “omics” analyses with potential clinical implications

Abstract Introduction: Triple-negative breast cancer (TNBC), representing about 10-20% of all breast cancers (BC), is a heterogeneous disease characterized by a clinically aggressive course, higher relapse rates and worse overall survival as compared to other BC subtypes. Recent efforts of genome-wide gene expression profiling have improved our understanding of the biological diversity of TNBCs reporting at least 6 different molecular subtypes namely Basal-like 1 (BL1), basal-like 2 (BL2), immunomodulatory (IM), mesenchymal (M), mesenchymal stem-like (MSL) and luminal androgen receptor (LAR). Little is known regarding the potential driving molecular events characterizing each subtype, their difference in survival and response to therapy. Moreover, limited progress has been made so far in the development of effective therapeutic strategies in TNBC partly due to the lack of obvious genetic targets. Further insight into the underlying genomic alterations is therefore needed. Aims: Here, we aimed to study the genomic aberrations that drive each of the TNBC molecular subtypes as defined by Lehmann et al. by applying an integrative analysis combining somatic mutation, copy number aberrations and gene expression profiles of 272 TNBC derived from METABRIC consortium. Methods: In silico analyses were performed using microarray, copy number aberrations profiles (CNA) and mutation profiles data retrieved from 272 TNBC patients from the METABRIC consortium. Somatic mutation profiles were derived from targeted sequencing of 173 cancer genes. CNAs at the gene level were computed using GISTIC 2.0.22. The relationship between TNBC molecular subtypes and genomic aberrations were analysed using Fisher test. Survival analyses were performed using Cox proportional hazard models adjust for standard clinical and pathologic variables. Results: Using a multivariate model, IM and M subtypes were significantly associated with good (HR=0.53; FDR=0.01) and poor (HR=1.8; FDR=0.01) prognosis respectively. BL1 subtype was found to be the most genomically instable subtype with high frequency of TP53 mutations (93%) and copy-number deletions in genes involved in DNA repair mechanisms (BRCA2, MDM2, RB1 and TP53) and MYC overexpression. BL2 subtype was enriched with TP53 (95%) and PIK3CA mutations (52%), whereas LAR subtype was characterized by high frequency of PI3KCA mutations (44%). Of interest, for these two subtypes, mutations in genes in the PI3K signalling pathway were mutually exclusive. IM subtype was enriched in NCOR2 mutations (13%), a co-repressor of the Notch pathway, associated with lower gene expression (p=0.035). M and MSL subtypes were significantly associated with higher signature scores for angiogenesis. In addition, M subtype was associated with higher frequency of NOTCH1 mutations (M p=2.4E-2). Conclusions: This integrative analysis combining somatic mutation, copy number and gene expression profiles shed new light on TNBC molecular heterogeneity, highlighting TNBC subtype-specific genomic aberrations which could potentially open new avenues for the development of effective targeted therapies in each TNBC molecular subtype such as DNA repair, Notch and angiogenesis. Citation Format: Bareche Y, Venet D, Rothe F, Sotiriou C. Uncovering TNBC molecular heterogeneity by applying integrative “omics” analyses with potential clinical implications [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-07-02.

GeneBreak: detection of recurrent DNA copy number aberration-associated chromosomal breakpoints within genes.

Development of cancer is driven by somatic alterations, including numerical and structural chromosomal aberrations. Currently, several computational methods are available and are widely applied to detect numerical copy number aberrations (CNAs) of chromosomal segments in tumor genomes. However, there is lack of computational methods that systematically detect structural chromosomal aberrations by virtue of the genomic location of CNA-associated chromosomal breaks and identify genes that appear non-randomly affected by chromosomal breakpoints across (large) series of tumor samples. 'GeneBreak' is developed to systematically identify genes recurrently affected by the genomic location of chromosomal CNA-associated breaks by a genome-wide approach, which can be applied to DNA copy number data obtained by array-Comparative Genomic Hybridization (CGH) or by (low-pass) whole genome sequencing (WGS). First, 'GeneBreak' collects the genomic locations of chromosomal CNA-associated breaks that were previously pinpointed by the segmentation algorithm that was applied to obtain CNA profiles. Next, a tailored annotation approach for breakpoint-to-gene mapping is implemented. Finally, dedicated cohort-based statistics is incorporated with correction for covariates that influence the probability to be a breakpoint gene. In addition, multiple testing correction is integrated to reveal recurrent breakpoint events. This easy-to-use algorithm, 'GeneBreak', is implemented in R ( www.cran.r-project.org) and is available from Bioconductor ( www.bioconductor.org/packages/release/bioc/html/GeneBreak.html).