Genomic Copy Number Aberrations Research Articles

Reciprocal chromosome translocation t(3;4)(q27;q31.2) with deletion of 3q27 and reduced FBXW7 expression in a patient with developmental delay, hypotonia, and seizures.

Reciprocal chromosomal translocation is one of genomic variations. When cytogenetically de novo reciprocal translocations are identified in patients with some clinical manifestations, the genes in the breakpoints are considered to be related to the clinical features. In this study, we encountered a patient with severe developmental delay, intractable epilepsy, growth failure, distinctive features, and skeletal manifestations. Conventional karyotyping revealed a de novo translocation described as 46,XY,t(3;4)(q27;q31.2). Chromosomal microarray testing detected a 1.25-Mb microdeletion at 3q27.3q28. Although the skeletal manifestations may have been affected by this deletion, the neurological features of this patient were severe and could not be fully explained by this deletion. Since no genomic copy number aberration was detected on chromosome 4, long-read whole-genome sequencing analysis was performed and a precise breakpoint was confirmed. A 460-bp deletion was detected between the two breakpoints; however, no gene was disrupted. FBXW7, the gene responsible for developmental delay, hypotonia, and impaired language, is in the 0.5-Mb telomeric region. Most of the patient's clinical features were considered consistent with symptoms of FBXW7-related disorders, but were more severe. FBXW7 expression in the immortalized lymphoblasts of the patient was reduced compared to that in controls. Based on these findings, we suspect that FBXW7 is affected by downstream position effects of chromosomal translocations. The severe neurological features of the patient may have been affected not only by the 3q27-q28 deletion but also by impaired expression of FBXW7 derived from the breakage of chromosome 4.

Mixed responses to targeted therapy driven by chromosomal instability through p53 dysfunction and genome doubling

The phenomenon of mixed/heterogenous treatment responses to cancer therapies within an individual patient presents a challenging clinical scenario. Furthermore, the molecular basis of mixed intra-patient tumor responses remains unclear. Here, we show that patients with metastatic lung adenocarcinoma harbouring co-mutations of EGFR and TP53, are more likely to have mixed intra-patient tumor responses to EGFR tyrosine kinase inhibition (TKI), compared to those with an EGFR mutation alone. The combined presence of whole genome doubling (WGD) and TP53 co-mutations leads to increased genome instability and genomic copy number aberrations in genes implicated in EGFR TKI resistance. Using mouse models and an in vitro isogenic p53-mutant model system, we provide evidence that WGD provides diverse routes to drug resistance by increasing the probability of acquiring copy-number gains or losses relative to non-WGD cells. These data provide a molecular basis for mixed tumor responses to targeted therapy, within an individual patient, with implications for therapeutic strategies.

Abstract B102: Mutational and copy number-based ctDNA profiles mirror high-grade serous cancer tumors and enable detection of genetic changes appearing at recurrence

Abstract Circulating tumor DNA (ctDNA) is the cancer-derived fraction of extracellular DNA fragments present in the bloodstream as cell-free DNA (cfDNA). Consequently, ctDNA harbors the distinct genomic information specific to the cancer from which it derives. Therefore, ctDNA from liquid biopsies provides a non-invasive, universally available alternative to traditional tumor biopsies. ctDNA amount may provide insight into tumor burden, as its concentration is typically highest during diagnosis and relapse. The non-invasive nature of this method facilitates frequent monitoring, enabling longitudinal tracking of the disease's trajectory and response to treatments. By focusing on the key genomic changes, either short mutations or copy number aberrations (CNA), liquid biopsy allows the investigation of cancer progression and evolution over time, providing invaluable insight into the behavior and adaptability of the cancer. We collected 152 longitudinal plasma samples and 92 fresh tissue samples from 29 patients enrolled in the DECIDER cohort (NCT04846933), after a diagnosis of high-grade serous carcinoma (HGSC), the most common and deadliest histological subtype of ovarian cancer. With ultra-deep targeted sequencing of a gene panel of more than 700 cancer-related genes we were able to comprehensively evaluate the cancer-related genomic changes. The concordance rate between mutations in plasma compared to tumor tissue was 83 % at diagnosis and 90 % at relapse, confirming the reliability of exploiting ctDNA in monitoring genomic alterations. Furthermore, by comparing plasma- and tissue-derived mutations, we demonstrated that comparable levels of ctDNA are released from adnexa, intra-abdominal lesions, and ascites, proposing that a liquid biopsy represents mutations from various locations. Analysis of CNA showed that our approach successfully detected concordant profiles in most plasma samples, even with tumor content as low as 3%. Additionally, highly amplified regions were identified in samples with approximately 1% tumor content. Longitudinal analysis of both mutations and CNA revealed genomic changes in plasma samples at relapse. Twenty-four new exonic mutations were identified in eight patients at relapse; two of these alterations were related to HRD status, supporting PARP-inhibitors as a valid treatment option. Furthermore, mutations in FGFR3, JAK2, FLT3 and CDKN2A could be investigated as target indications for extended use of currently available therapies. Eleven patients had high tumor content in their plasma samples at the time of relapse. We detected altered CNA profiles in seven of these eleven patients, so that five patients harbored only a few novel focal amplifications or losses, while two patients showed significant changes in their copy-number profile during the therapy. In conclusion, our study identified recurrence-specific genomic aberrations, proposing that ctDNA more accurately represents the metastatic tumor and provides possibilities for personalized therapy options. Citation Format: Giovanni Marchi, Mai T.N. Nguyen, Anna Rajavuori, Taru Muranen, Kaisa Huhtinen, Sinikka Oksa, Sakari Hietanen, Johanna Hynninen, Jaana Oikkonen, Sampsa Hautaniemi. Mutational and copy number-based ctDNA profiles mirror high-grade serous cancer tumors and enable detection of genetic changes appearing at recurrence [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B102.

Multiomics and Multidimensional Testing for Efficacy Monitoring of Patients with Lymphoma

To explore the role of a new blood-based, multiomics and multidimensional method for evaluating the efficacy of patients with lymphoma. 10 ml peripheral blood was extracted from each patient, and the genomic copy number aberrations (CNA) and fragment size (FS) were evaluated by low-depth whole genome sequencing of cfDNA, and the level of a group of plasma tumor marker (PTM) were detected at the same time. The cancer efficacy score (CES) was obtained by standardized transformation of the value of above three numerical indexes, and the changes of CES before and after treatment were compared to evaluate the patient's response to the treatment regimen. A total of 35 patients' baseline data were collected, of which 23 cases (65.7%) had elevated CES values. 18 patients underwent the first time test. The results showed that the CES value of 9 patients with positive baseline CES decreased significantly at the first test, and the efficacy evaluation was PR, which was highly consistent with the imaging evaluation results of the same period. At the same time, the CNA variation spectrum of all patients were evaluated and it was found that 23 patients had partial amplification or deletion of chromosome fragments. The most common amplification site was 8q24.21, which contains important oncogenes such as MYC. The most common deletion sites were 1p36.32, 4q21.23, 6q21, 6q27, 14q32.33, and tumor suppressor-related genes such as PRDM1, ATG5, AIM1, FOXO3 and HACE1 were expressed in the above regions, so these deletions may be related to the occurrence and development of lymphoma. With the advantages of more convenience, sensitivity and non-invasive, this multiomics and multidimensional efficacy detection method can evaluate the tumor load of patients with lymphoma at the molecular level, and make more accurate efficacy evaluation, which is expected to serve the clinic better.

Association of ctDNA tumor fraction with survival in advanced non-small cell lung cancer (NSCLC) treated with maintenance durvalumab in the UNICANCER SAFIR02-Lung/IFCT1301 trial.

2516 Background: ctDNA shed is a promising biomarker for cancer immunotherapy, with patients (pts) having higher concentration of plasma ctDNA being reported to have poorer outcome. Its role in the maintenance setting with immune checkpoint inhibitors remains unexplored. In SAFIR02-Lung, advanced NSCLC pts were enrolled to receive a platinum-based chemotherapy. After 4 cycles, if stable or in response, pts with no targetable alterations were oriented to the immunotherapy sub-study and randomized between maintenance durvalumab or Standard of Care (SoC) (PMID 35802649). Durvalumab prolonged survival only in the PD-L1 ≥ 1% subgroup. Here we explored Tumor Fraction (TF) as determined by Low-Pass Whole Genome Sequencing as a biomarker for immunotherapy irrespective of PD-L1. Methods: SAFIR02-Lung (NCT02117167) is a multicentric, randomized phase 2 clinical trial. Plasma samples were obtained at randomization, just before maintenance treatment initiation. DNA was extracted using the Maxwell RSC ccfDNA Plasma Kit (Promega, AS1840). Sequencing libraries were prepared using the Low Input SureSelectXT protocol (Agilent, G9916A). Pooled libraries were sequenced on a NovaSeq 6000 platform (Illumina) as 2 × 150 bp paired-end reads (NovaSeq 6000 SP Reagent Kit v1.5). Genomic copy number aberrations and TF were investigated using the ichorCNA (V0.2.0) tool. TF ≥ 2% was considered positive. For the survival analyses, a landmark approach was performed, by considering the randomization date as the landmark time. Progression Free Survival (PFS) was the primary endpoint, defined as the time from randomization until the date of objective radiological disease progression, clinical progression or death. The secondary endpoint was Overall Survival (OS). Results: 50 out of 121 plasma samples from pts randomized to durvalumab were analyzed for the feasibility part. 34 pts (68%) were < 65 years, 33 (66%) were male. PD-L1 expression was available for 18 pts and 39% of them had PD-L1 ≥ 1%. Median TF was 1.1%, 8 pts (16%) had a TF ≥ 2%, ranging from 2.2% to 23.3%. TF at randomization was marginally correlated with the presence of liver metastases (p 0.063) and with the sum of target lesions (p 0.081). No significant correlation was observed with PD-L1 positivity. Pts with TF ≥ 2% had lower median PFS 1.7months (m) (95% CI 1.2 – 4.2) vs 5.8m (95% CI 2.9 – 7.5) for those with TF < 2%, p 0.0003. Similarly, median OS was lower for pts with TF > 2% 10.4m (95% CI 4.2 – 18.7) vs 25.9m (95% CI 18.2 – NR) for those with TF < 2%, p 0.0002. Data will be presented on the whole cohort, including control group receiving SoC, to discriminate between a prognostic or a predictive value of TF. Conclusions: TF was positive in 16% of pts randomized to durvalumab in the SAFIR02-Lung trial. This population seems to have a limited benefit from maintenance durvalumab after induction chemotherapy. Clinical trial information: NCT02117167 .

A molecular study of pediatric pilomyxoid and pilocytic astrocytomas: Genome-wide copy number screening, retrospective analysis of clinicopathological features and long-term clinical outcome.

Pilocytic Astrocytoma (PA) is the most common pediatric brain tumors. PAs are slow-growing tumors with high survival rates. However, a distinct subgroup of tumors defined as pilomyxoid astrocytoma (PMA) presents unique histological characteristics and have more aggressive clinical course. The studies on genetics of PMA are scarce. In this study, we report one of the largest cohort of pediatric patients with pilomyxoid (PMA) and pilocytic astrocytomas (PA) in Saudi population providing a comprehensive clinical picture, retrospective analysis with long-term follow-up, genome-wide copy number changes, and clinical outcome of these pediatric tumors. We examined and compared genome-wide copy number aberrations (CNAs) and the clinical outcome of the patients with PA and PMA. The median progression free survival for the whole cohort was 156 months and it was 111 months for the PMA, however, not statistically significantly different between the groups (log-rank test, P = 0.726). We have identified 41 CNAs (34 gains and 7 losses) in all tested patients. Our study yielded the previously reported KIAA1549-BRAF Fusion gene in over 88% of the tested patients (89% and 80% in PMA and PA, respectively). Besides the fusion gene, twelve patients had additional genomic CNAs. Furthermore, pathway and gene network analyses of genes in the fusion region revealed alterations in retinoic acid mediated apoptosis and MAPK signaling pathways and key hub genes that may potentially be involved in tumor growth and progression, including BRAF, LUC7L2, MKRN1, RICTOR, TP53, HIPK2, HNF4A, POU5F, and SOX4. Our study is the first report of a large cohort of patients with PMA and PA in the Saudi population that provides detailed clinical features, genomic copy number changes, and outcome of these pediatric tumors and may help better diagnosis and characterization of PMA.

Long-read sequence analysis for clustered genomic copy number aberrations revealed architectures of intricately intertwined rearrangements.

Most chromosomal aberrations revealed by chromosomal microarray testing (CMA) are simple; however, very complex chromosomal structural rearrangements can also be found. Although the mechanism of structural rearrangements has been gradually revealed, not all mechanisms have been elucidated. We analyzed the breakpoint-junctions (BJs) of two or more clustered copy number variations (CNVs) in the same chromosome arms to understand their conformation and the mechanism of complex structural rearrangements. Combining CMA with long-read whole-genome sequencing (WGS) analysis, we successfully determined all BJs for the clustered CNVs identified in four patients. Multiple CNVs were intricately intertwined with each other, and clustered CNVs in four patients were involved in global complex chromosomal rearrangements. The BJs of two clustered deletions identified in two patients showed microhomologies, and their characteristics were explained by chromothripsis. In contrast, the BJs in the other two patients, who showed clustered deletions and duplications, consisted of blunt-end and nontemplated insertions. These findings could be explained only by alternative nonhomologous end-joining, a mechanism related to polymerase theta. All the patients had at least one inverted segment. Three patients showed cryptic aberrations involving a disruption and a deletion/duplication, which were not detected by CMA but were first identified by WGS. This result suggested that complex rearrangements should be considered if clustered CNVs are observed in the same chromosome arms. Because CMA has potential limitations in genotype-phenotype correlation analysis, a more detailed analysis by whole genome examination is recommended in cases of suspected complex structural aberrations.

Abstract 5150: Multimodal analysis of plasma cell-free DNA methylome for sensitive multi-cancer detection

Abstract Introduction: Genomic scale copy number, methylation, and fragmentation aberrations are proven genetic and epigenetic biomarkers of circulating tumor DNA (ctDNA). We developed a novel cell-free DNA (cfDNA) methylome sequencing assay that allows for integrative analysis of these genomic features for sensitive detection of multiple types of cancer. Methods: Whole methylome sequencing (WMS) libraries were generated from enzymatically converted cfDNA. Low-pass (~2X) paired-end NGS sequencing was performed on WMS libraries and paired whole genome sequencing (WGS) libraries of unconverted cfDNA for technical comparison and analytical validation. For development of cancer detection models, we profiled the genome-wide methylation density (MD), fragment size index (FSI), fragment end motif (motif) and chromosome instability (CIN) based on WMS data from a discovery cohort of 352 healthy controls and 559 newly diagnosed cancer patients (45 breast, 105 colorectal, 44 esophageal, 79 gastric, 79 liver, 110 lung, 83 pancreatic, and 14 others), 34.5% of which were at stage I or II. Machine learning models, including KNN, SVM, LR, GBDT, and random forest were trained and tested for individual biomarker types, with a final ensemble classifier to integrate all biomarkers. Performance of the predictive model was confirmed on an independent validation cohort consisting of 145 healthy controls and 236 cancer patients (21 breast, 45 colorectal, 18 esophageal, 35 gastric, 34 liver, 47 lung, and 36 pancreatic), among which 31.8% were at early stages (I or II). Results: WMS and WGS data from 512 cfDNA samples showed high concordance in CIN (R=0.988, 95% CI: 0.986-0.990) and FSI (R=0.961, 0.954-0.967) profiles. On the independent validation cohort, the optimal model selected for each of individual genomic features achieved following area under the ROC curve (AUC) values for cancer detection: MD-KNN, 0.830 (0.789-0.870); FSI-SVM, 0.904 (0.874-0.933); motif-SVM, 0.943 (0.920-0.966); and CIN-PAscore, 0.812 (0.770-0.854). The ensemble classifier based on linear SVM outperformed individual biomarkers, with an AUC value of 0.952 (0.934-0.971), which translated to, at 95% specificity, detection sensitivity of 66.7% for breast, 77.8% for colorectal, 83.3% for esophageal, 62.9% for gastric, 82.4% for liver, 66.0% for lung, and 77.8% for pancreatic cancers. Noteworthily, the overall sensitivity on early-stage cancer was 74.7%. Conclusions: These results demonstrate the first proof of principle on the feasibility of integrating multiple genomic cancer markers on the same WMS technical platform. Low-pass WMS on plasma cfDNA from 10ml of blood with integrative multimodal analysis of methylation, fragmentation, and CNV profiles yields in satisfactory sensitivity and specificity for detection of multiple types of cancer, warranting a forthcoming prospective study to further assess its clinical performance in a larger cohort. Citation Format: Yulong Li, Fenglong Bie, Fengwei Tan, Tiancheng Han, Shunli Yang, Fang Lv, Peiyao Nie, Qi Zhang, Yuanyuan Hong, Zhijie Wang, Ji He, Weizhi Chen, Liang Zhao, Shugeng Gao. Multimodal analysis of plasma cell-free DNA methylome for sensitive multi-cancer detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5150.

Abstract 5263: Whole-genome sequencing as an alternative to analyze copy number abnormalities in acute myeloid leukemia and myelodysplastic syndrome

Abstract Genomic alternations especially copy number aberrations (CNAs) are particularly imperative for diagnostic classification and risk stratification in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). Although conventional cytogenetic analysis (CCA) has been mandatory in clinical routine to detect recurrent genomic abnormalities in AML/MDS, it presents a few obvious shortcomings, such as time-consuming in vitro cell-culturing procedure, low resolution (no smaller than 10 Mbp), and subjective bias introduced from the visual interpretation by cytogeneticists. With the technological advances of next generation sequencing (NGS), the current CCA test and the molecular tests are likely to be challenged by whole-genome sequencing (WGS), which is a more unbiased method to detect all types of genomic aberrations not only those well-characterized clinically actionable mutations but also cryptic alternations yet to be defined and annotated. In this study a shallow whole-genome sequencing (sWGS) based assay, LeukoPrint, was utilized to depict genomic CNA profiles from the bone marrow cells of 107 newly diagnosed patients with AML (n=63, 58.9%) or MDS (n=44, 41.1%). Bone marrow was collected and mononuclear cells were separated to harvest genomic DNA which was sequenced without capture at approximately 1x coverage depth. Genome-wide CNAs was analyzed for each sample using an automated pipeline. European Leukemia Net (ELN) or Revised International Prognostic Scoring System (IPSS-R)-defining CNAs were selected and used to assign patients to a genetic risk group through the same classification systems that are used for CCA. It demonstrated 98.1% concordance of CNA profiles at individual patient level with cytogenetics and/or FISH. It is advantageous in detecting CNAs of short segments (1 Mb) and from samples with low leukemic cell content, more accurate for describing complex karyotypes and less confounded by subjective bias. LeukoPrint improved the overall diagnostic yield by redefining the risk categories for 16 patients (15%) by presenting new information. Nearly identical CNA profiles from the paired plasma and bone marrow suggest that peripheral blood may serve as a substitute to spare bone marrow aspiration. In summary, LeukoPrint provided an automated, convenient and cost-effective approach to describe genomic CNA profiles in AML/MDS. It brought greater diagnostic yield and risk stratification information by incorporating into the routine cytogenetics based on the standard ELN/IPSS-R guidelines. Citation Format: Xiaodong Lyu, Tao Li, Dandan Zhu, Mengna Zhang, Yuexin Cheng, Yan Chen, Zhenling Li, Shiyong Li, Wei Wu, Shuaipeng Geng, Jingshuai Li, Xiangxiang He, Yangwei Li, Yinyin Chang, Zunmin Zhu, Mao Mao, Yongping Song. Whole-genome sequencing as an alternative to analyze copy number abnormalities in acute myeloid leukemia and myelodysplastic syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5263.

Whole-genome sequencing as an alternative to analyze copy number abnormalities in acute myeloid leukemia and myelodysplastic syndrome

Copy number aberrations (CNA) are the core determinants for diagnosis, risk stratification and prognosis in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). In this study, a shallow whole-genome sequencing-based assay, LeukoPrint, was utilized to depict genomic CNA profiles from the bone marrow of 137 newly diagnosed AML/MDS patients. It demonstrated 98.1% concordance of CNA profiles with cytogenetics and/or fluorescence in situ hybridization (FISH). It is advantageous in detecting CNAs of short segments (1 Mb) and from samples with low leukemic cell content, more accurate for describing complex karyotypes and less confounded by subjective bias. LeukoPrint improved the overall diagnostic yield by redefining the risk categories for 16 patients by presenting new information. In summary, LeukoPrint provided an automated, convenient, and cost-effective approach to describe genomic CNA profiles. It brought greater diagnostic yield and risk stratification information by incorporating into the routine cytogenetics based on the CNA-related criteria of standard ELN/IPSS-R guidelines.

The Progenetix oncogenomic resource in 2021.

In cancer, copy number aberrations (CNAs) represent a type of nearly ubiquitous and frequently extensive structural genome variations. To disentangle the molecular mechanisms underlying tumorigenesis as well as identify and characterize molecular subtypes, the comparative and meta-analysis of large genomic variant collections can be of immense importance. Over the last decades, cancer genomic profiling projects have resulted in a large amount of somatic genome variation profiles, however segregated in a multitude of individual studies and datasets. The Progenetix project, initiated in 2001, curates individual cancer CNA profiles and associated metadata from published oncogenomic studies and data repositories with the aim to empower integrative analyses spanning all different cancer biologies. During the last few years, the fields of genomics and cancer research have seen significant advancement in terms of molecular genetics technology, disease concepts, data standard harmonization as well as data availability, in an increasingly structured and systematic manner. For the Progenetix resource, continuous data integration, curation and maintenance have resulted in the most comprehensive representation of cancer genome CNA profiling data with 138 663 (including 115 357 tumor) copy number variation (CNV) profiles. In this article, we report a 4.5-fold increase in sample number since 2013, improvements in data quality, ontology representation with a CNV landscape summary over 51 distinctive National Cancer Institute Thesaurus cancer terms as well as updates in database schemas, and data access including new web front-end and programmatic data access. Database URL: progenetix.org

The landscape of copy number variations in classical Hodgkin lymphoma: a joint KU Leuven and LYSA study on cell-free DNA

The low abundance of Hodgkin/Reed-Sternberg (HRS) cells in lymph node biopsies in classical Hodgkin lymphoma (cHL) complicates the analysis of somatic genetic alterations in HRS cells. As circulating cell-free DNA (cfDNA) contains circulating tumor DNA (ctDNA) from HRS cells, we prospectively collected cfDNA from 177 patients with newly diagnosed, mostly early-stage cHL in a monocentric study at Leuven, Belgium (n = 59) and the multicentric BREACH study by Lymphoma Study Association (n = 118). To catalog the patterns and frequencies of genomic copy number aberrations (CNAs), cfDNA was sequenced at low coverage (0.26×), and data were analyzed with ichorCNA to yield read depth-based copy number profiles and estimated clonal fractions in cfDNA. At diagnosis, the cfDNA concentration, estimated clonal fraction, and ctDNA concentration were significantly higher in cHL cases than controls. More than 90% of patients exhibited CNAs in cfDNA. The most frequent gains encompassed 2p16 (69%), 5p14 (50%), 12q13 (50%), 9p24 (50%), 5q (44%), 17q (43%), 2q (41%). Losses mostly affected 13q (57%), 6q25-q27 (55%), 4q35 (50%), 11q23 (44%), 8p21 (43%). In addition, we identified loss of 3p13-p26 and of 12q21-q24 and gain of 15q21-q26 as novel recurrent CNAs in cHL. At diagnosis, ctDNA concentration was associated with advanced disease, male sex, extensive nodal disease, elevated erythrocyte sedimentation rate, metabolic tumor volume, and HRS cell burden. CNAs and ctDNA rapidly diminished upon treatment initiation, and persistence of CNAs was associated with increased probability of relapse. This study endorses the development of ctDNA as gateway to the HRS genome and substrate for early disease response evaluation.

Lymphovascular invasion in breast cancer is associated with gene expression signatures of cell proliferation but not lymphangiogenesis or immune response.

While the prognostic relevance of lymphovascular invasion (LVI) in breast cancer is well known, its molecular biology is poorly understood. We hypothesized that pathologically determined LVI reflects molecular features of tumors and can be discerned from their genomic and transcriptomic profiles. LVI status and Nottingham histological scores of primary breast tumors of The Cancer Genome Atlas (TCGA) project were assessed from pathology reports; other clinical and molecular data were obtained from TCGA data portals and publications. Two independent datasets (GSE5460 and GSE7849) were combined and used for validation. LVI status was determinable for 639 and 196 cases of the TCGA and validation cohorts, among whom LVI incidence was 37.8% and 37.2%, respectively. LVI was associated with high tumor Ki67 expression, advanced pathologic stage, and high Nottingham scores. LVI-positive cases had worse overall and progression-free survival regardless of cancer subtype. Surprisingly, in both cohorts, LVI was not associated with lymphangiogenesis or lymphatic vessel density as estimated from tumor expression of lymphatic endothelium-associated genes. LVI-positive tumors had higher genome copy number aberrations, aneuploidy, and homologous recombination defects, but not single-nucleotide variations or intra-tumor genome heterogeneity. Tumor immune cell composition and cytolytic activity was not associated with LVI status. On the other hand, expression of cell proliferation-related genes was significantly increased in LVI-positive tumors. Our study suggests that breast cancer with LVI is a highly proliferative cancer, and it does not correlate with gene expression markers for lymphangiogenesis or immune response.

Multiparametric magnetic resonance imaging of multifocal prostate cancer to reveal intra-prostatic genomic heterogeneity and novel radio-genomic correlates: Results of the Smarter Prostate Interventions and Therapeutics (SPIRIT) study.

20 Background: Multi-focality and heterogeneity in prostate cancer can confound the selection of appropriate clinical management. Our study aimed to explore radio-genomic correlations using multiparametric magnetic resonance imaging (mpMRI) against a histopathologic reference standard. Methods: Eight men with prostate cancer who underwent mpMRI followed by prostatectomy were selected for this pilot. Whole-mount histopathology was digitized and co-registered to corresponding MRI slices using a validated high-fidelity methodology.(1) Foci, including central/transitional and peripheral zone lesions were identified by a pathologist, and contoured on digitized histopathology specimens and these digitized maps were used to guide macrodissection of the individual foci for genomic copy-number aberration (CNA) analysis. Correlation of radiomics signatures with the histologic findings and genomic analysis was performed. Results: We found a broad range of CNAs revealing inter-patient and intra-prostatic heterogeneity. Recurrently-altered loci ( e.g., 8p21) containing genes of known significance ( e.g., NKX3-1) were observed. Only radiomic features derived from apparent diffusion coefficient (ADC) independently correlated with both Gleason grade (Rho=-0.62, p=0.003) and median CNA burden (Rho=-0.68, p<0.001). While greater CNA burden expectedly correlated with higher grade, intermediate-grade (Gleason score 3+4 or 4+3) lesions appeared more like either high-grade (Gleason scores ≥4+4) or low-grade (Gleason score 3+3) disease when clustered based on CNA and ADC metrics. Conclusions: These findings suggest ADC derived radiomic metrics may be a useful imaging biomarker across both central and peripheral zone lesion and could aid in further characterization of intra-prostatic biologic heterogeneity. These proof-of-principle data reveal novel radio-genomic correlations that could supplement histologic grading and conventional imaging, thus warranting expanded study and validation. 1) Int J Rad Oncol Biol Phys. 2016; 96(1):188-96.

Negative Selective Pressure Exerted By Maintenance Therapy Promotes the Extinction of Sub-Clones Carrying High-Risk Lesions in Multiple Myeloma

Negative Selective Pressure Exerted By Maintenance Therapy Promotes the Extinction of Sub-Clones Carrying High-Risk Lesions in Multiple Myeloma

42P - Genome wide copy number analysis of circulating tumour cells in breast cancer liver metastasis

BackgroundCirculating tumor cells (CTCs) enter peripheral blood from primary breast cancer tumors and seed liver metastasis (BCLM). The genome-wide sequencing of CTCs offers noninvasive diagnostic factor for survival in BCLM patients, which but has been impeded by low single-cell genome coverage of scarce CTCs. MethodsWe conducted a prospective clinical investigation to confirm the diagnostic value of genome-wide sequencing in BCLM. We have developed the genome-wide pattern differences in copy number variations (CNVs) and to improve a CNV signature as an adjunct test for the routine histopathologic classification of BCLM. We compared the consistency and efficiency of genome-wide sequencing of CTCs with other comment detection methods. Furthermore, functional prediction of candidate gene biomarkers was confirmed in BCLM patients. ResultsCTCs were isolated from 26 patients with BCLM, 12 of which were successfully profiled for high CTCs (> 15), including matched archival primary tumor from 17 patients. Our results characteristic cancer-metastatic variations and insertions/deletions in exomes of CTCs. Comparison with primary breast tumors in metastatic niches cell revealed similar frequencies of BCLM genomic copy number aberrations. Higher CTCs frequencies (> 35) are correlated with disease severity and metastatic progress, which suggesting effective value of CTCs for variety of staging of BCLM. The CNV pattern of CTC is similar to that of hepatic metastasis, but the mutation pattern of CTC is different from that of primary lesion. Twenty- five genes were identified as a CNV signature of BCLM. Functional pathways analysis shows all these genes might play similar and critical roles in migration and progression of BCLM that could be independent risk factors for BCLM (p < 0.05). ConclusionsOur approach utilized to explore reproducibility of cancer-specific CNVs involved in progression of metastatic breast cancer. Our study contributed a new potential non-invasive biomarker for CTC-based cancer diagnostics of BCLM to monitor therapeutic efficacy of targeted therapies. Editorial acknowledgementThe authorswould like to express their appreciation to appreciate Southwest Medical Universityfor its financial support. We also wish to thank the oncology and pathology technicians of The Affiliated Hospital of Southwest Medical University for their assistance in the work reported here. We also thank the anonymous referee for his/her very helpful comments, which remarkably improved the quality of this research. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Comparable Genomic Copy Number Aberrations Differ across Astrocytoma Malignancy Grades.

A collection of intracranial astrocytomas of different malignancy grades was analyzed for copy number aberrations (CNA) in order to identify regions that are driving cancer pathogenesis. Astrocytomas were analyzed by Array Comparative Genomic Hybridization (aCGH) and bioinformatics utilizing a Bioconductor package, Genomic Identification of Significant Targets in Cancer (GISTIC) 2.0.23 and DAVID software. Altogether, 1438 CNA were found of which losses prevailed. On our total sample, significant deletions affected 14 chromosomal regions, out of which deletions at 17p13.2, 9p21.3, 13q12.11, 22q12.3 remained significant even at 0.05 q-value. When divided into malignancy groups, the regions identified as significantly deleted in high grades were: 9p21.3; 17p13.2; 10q24.2; 14q21.3; 1p36.11 and 13q12.11, while amplified were: 3q28; 12q13.3 and 21q22.3. Low grades comprised significant deletions at 3p14.3; 11p15.4; 15q15.1; 16q22.1; 20q11.22 and 22q12.3 indicating their involvement in early stages of tumorigenesis. Significantly enriched pathways were: PI3K-Akt, Cytokine-cytokine receptor, the nucleotide-binding oligomerization domain (NOD)–like receptor, Jak-STAT, retinoic acid-inducible gene (RIG)-I-like receptor and Toll-like receptor pathways. HPV and herpex simplex infection and inflammation pathways were also represented. The present study brings new data to astrocytoma research amplifying the wide spectrum of changes that could help us identify the regions critical for tumorigenesis.

Multiplex ligation-dependent probe amplification in oncohematological diagnostics and research

Genetic abnormalities associated with the development, progression and treatment resistance of hematological malignancies are extensively characterized. Rapid, reliable and cost-efficient techniques are needed to screen the clinically relevant aberrations in routine diagnostics. Multiplex ligation-dependent probe amplification is an efficient tool to analyze genomic copy number aberrations at 55-60 different genomic loci. The method allows the profiling of prognostic and predictive markers; thus, it can efficiently be combined with karyotyping and fluorescence in situ hybridization, the most commonly used diagnostic techniques to detect cytogenetic lesions. Furthermore, the method can interrogate methylation status and unravel point mutations at specific sites, providing results in 24 hours. Here, we describe the technical background of multiplex ligation-dependent probe amplification, summarize its advantages and limitations as well as discuss its role in oncohematological diagnostics and research. Finally, future outlook is provided, with emphasis on recent technological advances related to next-generation sequencing. Orv Hetil. 2018; 159(15): 583-592.

DNA aneuploidy relationship with patient age and tobacco smoke in OPMDs/OSCCs.

The aim of this study was to investigate the relationship between tobacco smoke habit, patient age, DNA aneuploidy and genomic DNA copy number aberrations (CNAs) in oral potentially malignant disorder (OPMD) and oral squamous cell carcinoma (OSCC) patients. DNA aneuploidy was detected by high-resolution DNA flow cytometry (hr DNA-FCM) on DAPI stained nuclei obtained from multiple tissue samples from OPMDs/OSCCs in 220 consecutive patients. Nuclear genomic aberrations were determined in a subset of 65 patients by genome-wide array comparative genomic hybridization (aCGH) using DNA extracted from either diploid or aneuploid nuclei suspension sorted by FCM. DNA aneuploidy and mean nuclear genomic aberrations were associated with patients’ age. In particular, DNA aneuploidy strongly associated with age in non-smoker OPMDs/OSCCs patients. OSCCs from smokers showed a lower prevalence of DNA aneuploidy compared to OSCCs from non-smokers. A higher occurrence of DNA aneuploidy (particularly in smokers’ OPMDs) was observed in patients characterized by involvement of a single oral subsite. Our study suggests that: 1) DNA aneuploidy in non-smokers is mainly related to aging; 2) OPMDs/OSCCs involving multiple oral subsites in smokers are less likely to develop DNA aneuploidy compared to non-smokers; 3) OSCC development is characterized by both CIN and CIN-independent mechanisms and that the latter are more relevant in smokers. This study provides evidence that DNA diploid OPMDs may be considered at lower risk of cancerization than DNA aneuploid ones in non-smokers but not in smokers.

Abstract 2733: Comparison of copy number aberrations (CNAs) between plasma cell free DNA (cfDNA) and tissue DNA in metastatic castrate resistant prostate cancer (mCRPC)

Abstract Background: Circulating cell-free DNA (cfDNA) analysis is emerging as a less invasive approach to assessing tumor genomic alterations in cancer patients. Although high concordance has been reported between tumor tissue NGS and cfDNA in studies investigating specific genetic alterations, the fidelity of cfDNA to tumor tissue DNA in the global genomic scale is largely unknown. In a correlative study of a prospective clinical trial (NCT# 01953640) conducted in mCRPC stage patients treated with abiraterone acetate/prednisone (A/P), we evaluated correlation of genomic CNA in tumor DNA obtained from biopsy of metastatic lesions and matched plasma cfDNA. Methods: mCRPC patients (pts) underwent two image-guided core biopsies of metastases prior to initiation of A/P (visit 1, V1) and 12 weeks after treatment (visit 2, V2). At both time points plasma was obtained at the time of the core biopsies and cfDNA was extracted using DNA Blood Mini Kit (Qiagen, Valencia, CA). High coverage (for tumor tissue) and low coverage (for cfDNA) whole genome sequencing reads were first mapped to the human genome hg19. Read counts (RC) from the mapped sequence files were then binned into 1Mb windows. The RC ratio in each genomic bin was calculated by comparing tumor tissue DNA to lymphocyte gDNA derived from the same patient, and was further log2 transformed, corrected for GC content, and normalized by CGHnormaliter. The fully normalized log2 ratios data was subjected to segmentation using DNAcopy algorithm. Results: Between 05/2013 and 09/2015, 92 patients (pts) were enrolled of which tissue and plasma NGS data both visits was available for 18 pts. The correlation of CNAs between tumor tissue and its cfDNA counterpart ranges from 0.013 to 0.83 for V1 samples, and -0.05 to 0.9 for V2 samples. The decreased correlation in some pairs of samples is largely due to low tumor content and heterogeneity in the cfDNA. Although there is a wide range of correlation, commonly shared CNAs were identified in multiple chromosomal regions, including loss in 8p, gain in 8q and chromosome 5 and X. On the other hand, several genomic regions show inconsistent CNAs between tumor tissue and cfDNA among 15 pairs of samples in which cfDNA have large tumor content. These include: loss in 6q, 1p, 2p, 9q, 11q, 13p, 15p, 20p and gain in 8q and 10p in cfDNA but not in tumor tissue; marked loss in chromosome 22 and gain in p arm of X chromosome in tumor tissue but much less evident in cfDNA. Conclusion: High concordance of CNAs between cfDNA and tumor tissue DNA can be achieved given sufficient tumor content of cfDNA. However, more CNAs can be identified in cfDNA than its tumor tissue counterpart. Our result suggests that cfDNA NGS is a useful tool to investigate clonal evolution associated with cancer progression. Citation Format: Chiang-Ching Huang, Meijun Du, Liguo Wang, Yen-chen Lin, Hua Huang, Liewei Wang, Liang Wang, Manish Kohli. Comparison of copy number aberrations (CNAs) between plasma cell free DNA (cfDNA) and tissue DNA in metastatic castrate resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2733. doi:10.1158/1538-7445.AM2017-2733