Copy Number Alterations In Patients Research Articles

Transcriptomic data analysis coupled with copy number aberrations reveals a blood-based 17-gene signature for diagnosis and prognosis of patients with colorectal cancer.

Background: Colorectal cancer (CRC) is the third most common cancer and third leading cause of cancer-associated deaths worldwide. Diagnosing CRC patients reliably at an early and curable stage is of utmost importance to reduce the risk of mortality. Methods: We identified global differentially expressed genes with copy number alterations in patients with CRC. We then identified genes that are also expressed in blood, which resulted in a blood-based gene signature. We validated the gene signature's diagnostic and prognostic potential using independent datasets of gene expression profiling from over 800 CRC patients with detailed clinical data. Functional enrichment, gene interaction networks and pathway analyses were also performed. Results: The analysis revealed a 17-gene signature that is expressed in blood and demonstrated that it has diagnostic potential. The 17-gene SVM classifier displayed 99 percent accuracy in predicting the patients with CRC. Moreover, we developed a prognostic model and defined a risk-score using 17-gene and validated that high risk score is strongly associated with poor disease outcome. The 17-gene signature predicted disease outcome independent of other clinical factors in the multivariate analysis (HR = 2.7, 95% CI = 1.3-5.3, p = 0.005). In addition, our gene network and pathway analyses revealed alterations in oxidative stress, STAT3, ERK/MAPK, interleukin and cytokine signaling pathways as well as potentially important hub genes, including BCL2, MS4A1, SLC7A11, AURKA, IL6R, TP53, NUPR1, DICER1, DUSP5, SMAD3, and CCND1. Conclusion: Our results revealed alterations in various genes and cancer-related pathways that may be essential for CRC transformation. Moreover, our study highlights diagnostic and prognostic value of our gene signature as well as its potential use as a blood biomarker as a non-invasive diagnostic method. Integrated analysis transcriptomic data coupled with copy number aberrations may provide a reliable method to identify key biological programs associated with CRC and lead to improved diagnosis and therapeutic options.

A Single Next Generation Sequencing Assay for Detection of Driver Mutations, Translocations and Copy Number Alterations in Patients with Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by several genetic abnormalities that impact risk stratification, outcome, and response to therapy; with fluorescence in situ hybridization (FISH) considered the gold-standard assay to detect the presence of chromosomal translocations and copy-number variations (CNV). Here, we evaluate the performance of a single clinically actionable targeted sequencing (NGS) assay for detecting cytogenetic abnormalities in addition to recurrent driver mutations in MM. Patient-derived bone marrow aspirates were obtained and enriched for CD138+ plasma cells. Targeted sequencing was performed using a 2.3 Mb custom capture panel covering 139 genes and regions surrounding MYC, immunoglobulin heavy chain (IgH), and immunoglobulin light chain kappa (IgK) and lambda (IgL) loci. Chromosomal translocations were identified using an in-house algorithm designed specifically for targeted capture DNA sequencing data analyses. Structural variants were called based on discordant read pairs as well as junction split reads, solving the challenges from both the overlapping paired-end reads due to the longer read lengths (150 bp) and the mapping ambiguities in the Ig regions. CNV were assessed using both on and off target reads, using a 200 kb bin size for segmentation covering the entire genome. Clinical annotations and FISH panel reports were abstracted. We sequenced 264 MM patient samples, 94% of which had available FISH information. A median of two somatic mutated genes per sample (range 0-13) was found. The most commonly mutated genes per sample were: KRAS (35%), NRAS (25%), FAM46C (15%), DIS3 (13%), BRAF (13%), ATM (11%), and TP53 (11%). Comparing NGS with FISH, we found a perfect concordance for the identification of t(4;14), t(6;14), t(14;16), and t(14;20); a 100% specificity and 97% sensitivity for t(11;14); and sensitivity >90% and an average specificity of 85% for trisomies of chromosomes 3, 7, 9, 11, and 15. The NGS approach was also able to detect gain(1q) and mono(13) with a sensitivity and specificity >90%, and del(17p) with a 60% sensitivity and 100% specificity. The reduced sensitivity for del(17p) detection was mostly due to focal deletions and low tumor purity. Because the TP53 gene is included in the custom capture panel, we increased the sensitivity by determining six separate regions of coverage using on-target probes, which enabled the detection of focal del(17p), increasing the sensitivity to 78%. The NGS approach also allowed the characterization of 16 (6%) samples with unavailable FISH. Of the 27 samples with del(17p) detected by FISH, NGS was able to identify TP53 mutations (i.e., biallelic inactivation) in 14 samples (52%). Due to the existence of multiple and promiscuous chromosomal partners involved in IgH translocation, the identification of the chromosomal partners is limited to the clinical FISH probe sets; in fact, 19 study samples presented abnormal IgH without a known partner by FISH. Our NGS assay was able to identify these partners, confirming MYC-IGH translocations in seven (37%) of these cases. Additionally, we detected MYC translocations with immunoglobulin light chains: 14 (5%) cases with IGK, and eight (3%) with IGL. Although IgL translocations, particularly IgL-MYC translocations, have been recently associated to poor prognosis, they are not routinely evaluated in FISH assays. The risk stratification and clinical utility of the NGS assay was demonstrated by the shorter overall survival observed in samples classified as high risk (mSMART 3.0; hazard ratio [HR]= 2.81; CI:1.89-4.17; p<0.0001), which was also observed with risk assessment by FISH (HR = 2.48; CI:1.72-3.58; p<0.0001). Therefore, in addition to the detection of clinically relevant somatic mutations, the targeted NGS approach showed robust detection of recurrent MM specific IgH translocations and CNV. Furthermore, the detection of relevant abnormalities not routinely assessed in most clinical labs, such as TP53 bi-allelic inactivation, IgL and MYC rearrangements, supports the use of this small, targeted panel as a clinically relevant, single molecular assay, for the comprehensive profiling of MM.

Clinicopathological Features of FGFR3 - Mutated Upper Tract Urothelial Carcinoma: A Genomic Database Analysis.

Upper tract urothelial carcinomas (UTUCs) arise in the renal pelvis or the ureter, accounting for approximately 5% of all urothelial carcinomas. Recent years have witnessed the publication of several studies aimed at assessing the molecular, biologic, and clinical features of UTUC, reporting that FGFR3 mutations are the most observed genetic aberrations; however, several knowledge gaps persist in the understanding of the genomic landscape of this genitourinary malignancy with few treatment options. In the current study, we aimed to comprehensively analyze clinicopathological features of FGFR3-mutated UTUCs patients in public datasets to increase the current knowledge of the molecular and biologic profile of UTUC. Data regarding clinical outcomes, mutational profiles, and copy number alterations in patients affected by UTUC were downloaded from the cBioPortal for Cancer Genomics Database. UTUC data were available from 4 studies, for a total number of 358 patients; among these, 150 UTUC patients presented FGFR3 mutations. The current database analysis of the mutational profile of 150 FGFR3-mutated UTUCs suggested that FGFR3 mutations may represent a prognostic factor in this disease, with a statistically longer overall survival compared to wild-type patients treated with radical surgery. In addition, FGFR3 mutations were more frequent in low-grade UTUCs with early-stage disease (pT1, pT2, and pT3). Genomic characterization of UTUC is destined to become increasingly important, and more efforts aimed at implementing UTUC genomics analysis are warranted.

Early Breast Cancer Evolution by Autosomal Broad Copy Number Alterations.

The availability of comprehensive genomic datasets across patient populations enables the application of novel methods for reconstructing tumor evolution within individual patients. To this end, we propose studying autosomal broad copy number alterations (CNAs) as a framework to better understand early tumor evolution. We compared the broad CNAs and somatic mutations of patients with 1 to 10 autosomal broad CNAs against the full set of patients, using data from The Cancer Genome Atlas breast cancer project. We reveal here that the frequency of a chromosome arm obtaining a broad CNA and a genome acquiring somatic mutations changes as autosomal broad CNAs accumulate. Therefore, we propose that the number of autosomal broad CNAs is an important characteristic of breast tumors that needs to be taken into consideration when studying breast tumors. To investigate this idea more in-depth, we next studied the frequency that specific chromosome arms acquire broad CNAs in patients with 1 to 10 broad CNAs. With this process, we identified the broad CNAs that exhibit the fastest rates of accumulation across all patients. This finding suggests a likely order of occurrence of these alterations in patients, which is apparent when we consider a subset of patients with few broad CNAs. Here, we lay the foundation for future studies to build upon our findings and use autosomal broad CNAs as a method to monitor breast tumor progression in vivo to further our understanding of how early tumor evolution unfolds.

Genome-wide Sequencing of Cell-free DNA Enables Detection of Copy-number Alterations in Patients with Cancer Where Tissue Biopsy is Not Feasible.

When tissue biopsy is not medically prudent or tissue is insufficient for molecular testing, alternative methods are needed. Because cell-free DNA (cfDNA) has been shown to provide a representative surrogate for tumor tissue, we sought to evaluate its utility in this clinical scenario. cfDNA was isolated from the plasma of patients and assayed with low-coverage (∼0.3×), genome-wide sequencing. Copy-number alterations (CNA) were identified and characterized using analytic methods originally developed for noninvasive prenatal testing (NIPT) and quantified using the genomic instability number (GIN), a metric that reflects the quantity and magnitude of CNAs across the genome. The technical variability of the GIN was first evaluated in an independent cohort comprising genome-wide sequencing results from 27,754 women who consented to have their samples used for research and whose NIPT results yielded no detected CNAs to establish a detection threshold. Subsequently, cfDNA sequencing data from 96 patients with known cancers but for whom a tissue biopsy could not be obtained are presented. An elevated GIN was detected in 35% of patients and detection rates varied by tumor origin. Collectively, CNAs covered 96.6% of all autosomes. Survival was significantly reduced in patients with an elevated GIN relative to those without. Overall, these data provide a proof of concept for the use of low-coverage, genome-wide sequencing of cfDNA from patients with cancer to obtain relevant molecular information in instances where tissue is difficult to access. These data may ultimately serve as an informative complement to other molecular tests.

Abstract 1563: The impact of heterogeneity of AKT1/2 copy number alterations between primary tumors and lymph node metastases in non-small cell lung cancer

Abstract Background: Copy number alterations (CNAs) arise during the evolution of tumors as a consequence of its genomic instability. It is reported that the increase of gene copy number of AKT1/2 correlated with clinicopathological profiles in lung cancer. On the other hand, tumor heterogeneity was also reported to associate with clinical outcomes of the patients in several cancers. However, the clinical significance of AKT1/2 CNAs in patients with non-small cell lung cancer (NSCLC) has been still unclear, especially its contribution to patients’ prognoses. We here explored the relationships between intertumoral heterogeneity of CNAs of AKT1/2 and clinicopathological features using the resected tissue pairs of primary sites and corresponding lymph node metastases (LNM). Method: AKT1/2 CNAs in 130 tissue pairs of NSCLC were assessed by fluorescent in situ hybridization (FISH), which were classified into three categories: amplification, polysomy, and disomy. The associations between CNAs and clinical features were retrospectively analyzed. In addition, we investigated the effect of the increased AKT1/2 copy number and intertumoral heterogeneity of AKT1/2 CNAs between the resected tissue pairs on the patient prognoses. Result: The median age of patients was 66.0 years old (33-84 years old) and 98 (75.4%) patients were male. 100 (76.9%) patients had smoking history and 69 (53.1%) patients had adenocarcinoma. AKT1 CNAs including amplification and polysomy were observed in 27.3% of primary sites (2.3% for amplification and 25% for polysomy), and in 43.8% of LNM (9.4% for amplification and 34.4% for polysomy). AKT2 CNAs were observed in 33.1% of primary sites (5.5% for amplification and 27.6% for polysomy), and in 36.3% of LNM (9.7% for amplification and 26.6% for polysomy). AKT1 CNAs were significantly associated with sex (p=0.004 for primary sites, p=0.034 for LNM) and smoking history (p=0.004 for primary sites, p=0.038 for LNM). EGFR mutations were more frequently observed in the patients with AKT1/2 CNAs in primary sites (p=0.009 for AKT1, and p=0.037 for AKT2). The mean copy number significantly increased in the LNM compared to the primary sites in AKT1 (2.1 for primary sites vs 3.4 for LNM, p&amp;lt;0.001), whereas there were no significant differences but an increasing tendency in AKT2 (2.9 for primary sites vs 3.5 for LNM, p=0.147). There were no significant differences for recurrence-free survival (RFS) according to AKT1/2 CNA categories. However, cases with the increased AKT1/2 copy number in the corresponding LNM showed significantly worse survivals for RFS (Log-rank, p=0.02 for AKT1, and p=0.04 for AKT2). Conclusion: AKT1/2 copy number increased in LNM compared to primary sited, and which also associated with worse prognosis. The intertumoral heterogeneity of AKT1/2 CNAs may possess distinct clinical significance in NSCLC. Citation Format: Kazuo Tsuchiya, Katsuhiro Yoshimura, Yusuke Inoue, Yuji Iwashita, Takashi Yamashita, Akikazu Kawase, Masayuki Tanahashi, Hiroshi Ogawa, Kazuhito Funai, Kazuya Shinmura, Hiroshi Niwa, Takafumi Suda, Haruhiko Sugimura. The impact of heterogeneity of AKT1/2 copy number alterations between primary tumors and lymph node metastases in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1563.

MA13.03 Heterogeneity Analysis of EBUS-TBNA-Derived Specimens for Evaluation of PD-L1 Expression and Copy Number Alterations in Patients with NSCLC

MA13.03 Heterogeneity Analysis of EBUS-TBNA-Derived Specimens for Evaluation of PD-L1 Expression and Copy Number Alterations in Patients with NSCLC

Abstract 5693: Low coverage genome-wide sequencing of cell-free DNA enables detection, therapeutic monitoring, and characterization of copy number alterations in patients with solid tumors

Abstract Circulating tumor DNA (ctDNA) has been detected in the blood of patients with many cancer types and is thought to reflect the combined genetic profile of the primary tumor and metastases. The majority of studies into “liquid biopsies” have focused on detecting focal events including single nucleotide variants, fusions, and specific copy number alterations (CNAs); however, these assays are limited to mutations occurring in targeted regions and require deep sequencing. The objective of this study was to use low coverage, genome-wide sequencing of cell-free DNA (cfDNA) to evaluate the frequency, distribution, and persistence of genome-wide CNAs in the plasma of patients with solid tumors. cfDNA was isolated from a total of 203 plasma aliquots from 72 patients diagnosed with a variety of solid tumors. For almost half of the patients, multiple collections were available (range: 2-13) to enable longitudinal monitoring of treatment response. Greater than 25% of baseline cfDNA samples showed evidence of one or more CNAs and greater than 40% of patients had a CNA detected in at least one sample. Among patients with longitudinal monitoring, greater than 35% of individuals showed evidence of CNAs at baseline and approximately 25% of samples showed similar evidence in subsequent sampling. Collectively, CNAs were detected on all autosomes, suggesting that genome-wide profiling is necessary to fully assess CNA distributions. Additionally, we devised a metric which quantified the level of genomic instability. This metric, termed the genomic instability number (GIN), was associated with disease classification. In patients capable of longitudinal monitoring, GIN values were observed concomitant with therapeutic response (including, interestingly, response to immunotherapy) or disease progression and may provide information earlier than standard imaging-based methods. In addition, total cfDNA amount was evaluated as a predictor. When analysis was performed across all patients, there was a poor correlation between total cfDNA amount and the GIN; however, total cfDNA amount was strongly correlated with the cumulative abundance of CNA events within individual patients, suggesting that the CNAs detected in the cfDNA were derived from the additional genetic material contributed by the tumor. These data demonstrate the feasibility of detecting ctDNA in a significant portion of cancer patients solely on the basis of low coverage CNA profiling. Further, the nature of CNA events observed varied both between patients and across longitudinal samples from individual patients, suggesting potential for monitoring therapy response or disease progression. This study suggests that genome-wide CNA profiling may be useful alone or as a complement to standard variant detection strategies in the development of liquid biopsy technologies. Citation Format: Christopher K. Ellison, Aaron M. Goodman, Sabine Riethdorf, Amin R. Mazloom, Lisa Tran, Prachi Nakashe, Erin McCarthy, Tobias Gorges, Alexander Stein, Julia Quidde, Daniel S. Grosu, Mathias Ehrich, Taylor J. Jensen, Klaus Pantel, Razelle Kurzrock. Low coverage genome-wide sequencing of cell-free DNA enables detection, therapeutic monitoring, and characterization of copy number alterations in patients with solid tumors [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 5693. doi:10.1158/1538-7445.AM2017-5693

Copy Number Alterations in patients with mature B (Burkitt-type) acute lymphoblastic leukaemia treated with specific immunochemotherapy

S174 EBF1 and PAX5 deletions. In the IKZF1 deleted cohort, 13 patients (28%) showed whole gene deletions and Ik6 was the most recurrent short isoform (26%). Multivariate analysis showed that advanced age andEBF1 deletions were negative prognostic factors for achieving Complete Remission (CR) andWBC count and IKZF1 deletions significantly reduced CR duration in both the total series and in the Ph negative subgroup. Interestingly, there were significant differences in relapse rates between whole and partial IKZF1 deletions. IKZF1 haploinsufficient patients had a probability of CR duration at 6 years of 70% 36% vs. 14% 17% of partial gene deletion carriers (p1⁄40.013). Advanced age and CDKN2A/B deletions were predictors for overall survival in the whole series but also in the Ph negative subgroup. Conclusions: IKZF1, EBF1 and CDKN2A/B genetic abnormalities could help to better define prognostic subgroups in adult patients with B-ALL. Deletion Duplication IKZF1 46 (34%) 1 (1%) EBF1 15 (11%) 10 (7%)

Prognostic impact and the relevance of PTEN copy number alterations in patients with advanced colorectal cancer (CRC) receiving bevacizumab

Loss of phosphatase and tensin homologue (PTEN) expression may be prognostic in colorectal cancer (CRC) and may have a correlation with vascular endothelial growth factor (VEGF) expression via hypoxia-inducible factor 1 (HIF-1) alpha, and the PI3K/mTOR pathways. We therefore have explored the prognostic association of PTEN loss and the potential that PTEN loss may be predictive of outcome with bevacizumab. Patients enrolled in the AGITG MAX trial, a randomized Phase III trial of capecitabine (C) +/− bevacizumab (B) (+/− mitomycin C [M]) with available tissues were analyzed for PTEN expression (loss vs. no loss) as assessed using a Taqman® copy number assay (CNA). Of the original 471 patients enrolled, tissues from 302 (64.1%) patients were analyzed. PTEN loss was observed in 38.7% of patients. There was no relationship between PTEN loss and KRAS or BRAF mutation. PTEN status was not prognostic for progression-free survival (PFS) or overall survival (OS) in multivariate analyses adjusting for other baseline factors; loss versus no loss PFS hazard ratio (HR) 0.9 (0.7–1.16), OS HR 1.04 (0.79–1.38). PTEN was not prognostic when assessed by KRAS and BRAF status. By using the comparison of C versus CB+CBM, PTEN status was not significantly predictive of the effectiveness of B for PFS or OS. PTEN status was not prognostic for survival in advanced colorectal cancer, irrespective of KRAS or BRAF status. PTEN status did not significantly predict different benefit with bevacizumb therapy.

Abstract 1665: High resolution genome profile in familial colorectal cases without mutations on mismatch repair genes

Abstract Colorectal cancer (CRC) represents the fourth leading cause of cancer mortality. Approximately 30% of all CRC cases have a hereditary component, but only 5% are associated with inherited mutations in known genes. The most common hereditary syndrome of CRC is the Lynch syndrome (LS) and is caused by mutations in mismatch repair genes, mainly MLH1 and MSH2. In ∼50% of families classified with the Amsterdam criteria not show germline mutations in mismatch repair genes, suggesting that other genetic factors are associated with predisposition to CRC. In this study, it was evaluated germline copy number alterations (CNAs) in 57 LS patients without pathogenic mutations in mismatch repair genes (MLH1, MSH2, MSH6, PMS1 and PMS2), as well as in CHEK2 and TP53, by array comparative genomic hybridization (aCGH) using a platform of 4x180K (Agilent). Genomic data were extracted with Feature Extraction software and analyzed using Nexus 6.0 software, statistical algorithm FASST2 Segmentation and sensitivity threshold of 5.0E-6. The aCGH data showed a total of 361 CNAs (in average, 6.3±3.8 CNA/individual) including 144 gains and 217 genomic losses. After the comparison with a reference dataset obtained from 100 healthy Brazilian individuals (Krepischi et al, 2011; personal communication) and exclusion of the CNAs detected in more than 5% of this reference dataset, it was identified ∼100 rare CNAs. Most of them were detected in only one case, however, six CNAs were common at least in five cases: 1p11.2, 1p36.32, 7p22.3, 8q24.3, 9p11.2 and 18q11.1. The ROCK1 gene, mapped on 18q11.1, was found as involved in nine cases (three losses and six gains). This gene was associated with carcinogenesis and progression of human tumors, including colon cancer (Nakashima et al, Int J Oncol. 36:585, 2010). Amplification on 7p22.3 (5 cases), where is mapped the homeobox gene UNCX, a transcription factor involved in somitogenesis and neurogenesis. Mutations in rare constitutional CNAs may affect genes or major pathways associated with cancer, offering an explanation for families with high cancer risk. In CRC, the large scale analysis of germline CNAs in patients and their relatives can allow the identification of new genes involved in tumor predisposition. Financial support: FAPESP and CNPq Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1665. doi:1538-7445.AM2012-1665

X chromosome array-CGH for the identification of novel X-linked mental retardation genes

Array-CGH technology for the detection of submicroscopic copy number changes in the genome has recently been developed for the identification of novel disease-associated genes. It has been estimated that submicroscopic genomic deletions or duplications will be present in 5–7% of patients with idiopathic mental retardation (MR). Since 30% more males than females are diagnosed with MR, we have developed a full coverage X chromosome array-CGH with a theoretical resolution of 82 kb, for the detection of copy number alterations in patients with suspected X-linked mental retardation (XLMR). First, we have validated the genomic location of X-derived clones through male versus female hybridisations. Next, we validated our array for efficient and reproducible detection of known alterations in XLMR patients. In all cases, we were able to detect the deletions and duplications in males as well as females. Due to the high resolution of our X-array, the boundaries of the genomic aberrations could clearly be identified making genotype–phenotype studies more reliable. Here, we describe the production and validation of a full coverage X-array-CGH, which will allow for fast and easy screening of submicroscopic copy number alterations in XLMR patients with the aim to identify novel MR genes or mechanisms involved in a deranged cognitive development.