Somatic Copy Number Abnormalities Research Articles

Single-Nucleotide Polymorphism Array for Histologically Ambiguous Melanocytic Tumors

Genome-wide copy number profiling by single-nucleotide polymorphism (SNP) array is increasingly employed in the clinical diagnostic workup of melanocytic tumors. We present our SNP array results on 675 melanocytic tumors, including 615 histologically ambiguous tumors evaluated by our institution's dermatopathology consultation service and a separate validation cohort of 26 known benign nevi and 34 known malignant melanomas. The total number of somatic copy number abnormalities, sub-chromosomal copy number abnormalities, regions of homozygosity, and abnormalities at disease-associated regions was significantly associated with a diagnosis of malignancy across disease categories. In our study, the number of copy number abnormalities was the factor that best discriminated between benign versus malignant diagnoses, confirming recent published research. Histologically ambiguous tumors had a range and spectrum of abnormalities, including recurrent 11p gains, copy state transitions over kinase genes, and 3p deletions overlapping BAP1 in neoplasms with Spitzoid morphology. Our data suggest that histologically ambiguous melanocytic neoplasms and early primary melanomas have a range of abnormalities that is intermediate between unambiguous benign or malignant melanocytic neoplasms. Careful technical review and an integrated diagnostic approach are essential for the accurate interpretation of SNP array results on histologically ambiguous melanocytic tumors.

Abstract IA002: Histopathological classification of endometrial cancers and surrogate markers of genomic subtypes

Abstract Endometrial carcinoma has been subclassified based on tumor cell-type morphology i.e. the resemblance of the tumor cells to their normal counterparts based on microscopic examination. The main histotypes of endometrial carcinoma, endometrioid and serous carcinoma, correspond roughly to Type I and Type II endometrial carcinoma, as described by Bokhman in 1983. Although these histotypes are associated with different patient outcomes and are associated with different mutations, with endometrioid carcinomas having a more favorable prognosis than serous carcinomas, there are a significant number of endometrial carcinomas that are difficult to classify based on histopathological examination. This results in there being only moderate inter-observer reproducibility in the histopathological classification of endometrial carcinoma and this, in turn, compromises the ability to use histotype as a basis for treatment decisions. The Cancer Genome Atlas identified 4 molecular subtypes of endometrial carcinoma based on genomic architecture: ultramutated, hypermutated, low numbers of somatic copy number abnormalities, and high numbers of somatic copy number abnormalities. These correlate with patient outcome and, increasingly, have been show to be correlated with response to treatment. In this presentation the current subclassification of endometrial carcinoma, the development of surrogate markers of genomic molecular subtype and the correlation between molecular subtype and histotype will be discussed,Endometrial carcinoma has been subclassified based on tumor cell-type morphology i.e. the resemblance of the tumor cells to their normal counterparts based on microscopic examination. The main histotypes of endometrial carcinoma, endometrioid and serous carcinoma, correspond roughly to Type I and Type II endometrial carcinoma, as described by Bokhman in 1983. Although these histotypes are associated with different patient outcomes and are associated with different mutations, with endometrioid carcinomas having a more favorable prognosis than serous carcinomas, there are a significant number of endometrial carcinomas that are difficult to classify based on histopathological examination. This results in there being only moderate inter-observer reproducibility in the histopathological classification of endometrial carcinoma and this, in turn, compromises the ability to use histotype as a basis for treatment decisions. The Cancer Genome Atlas identified 4 molecular subtypes of endometrial carcinoma based on genomic architecture: ultramutated, hypermutated, low numbers of somatic copy number abnormalities, and high numbers of somatic copy number abnormalities. These correlate with patient outcome and, increasingly, have been show to be correlated with response to treatment. In this presentation the current subclassification of endometrial carcinoma, the development of surrogate markers of genomic molecular subtype and the correlation between molecular subtype and histotype will be discussed. Citation Format: C. Blake Gilks. Histopathological classification of endometrial cancers and surrogate markers of genomic subtypes [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr IA002.

Diagnostic Variation in p53 Usage for Endometrial Carcinoma Diagnosis: Implications for Molecular Subtyping.

Immunostaining for p53 is widely but variably used when diagnosing endometrial carcinoma (EC). Mutant-pattern p53 staining can support a diagnosis of serous carcinoma, and also serve as a surrogate test for identifying the "serous-like" subset of aggressive EC identified by The Cancer Genome Atlas characterized by high numbers of somatic copy number abnormalities. We, retrospectively, assessed WHO histotype, usage of p53 immunostaining, and p53 status in a consecutive series of biopsies showing EC from a single hospital. Of 79 ECs, 59 (75%) were low-grade EC (LGEC), 13 (16%) high-grade EC (HGEC), and 7 (9%) were serous. p53 immunostaining was performed at the time of diagnosis in 27/79 (34%) biopsies; 6/7 of serous histotype, 11/13 HGEC, and 10/59 LGEC. Mutant-pattern p53 staining was present in 6/6 serous, 2/11 HGEC, and 2/10 LGEC. The remaining 53 tumors subsequently had p53 immunostaining done; all 49 LGEC showed wild-type staining and the serous carcinoma and 1/2 HGEC showed mutant pattern staining. While there are no guidelines on using p53 in endometrial biopsies, this study shows consistent usage in high-grade histotypes and variable usage in LGEC. As 100% (7/7) of serous EC and 3% (2/59) of the LGECs showed mutant-pattern p53 staining, histotype may serve as a surrogate for p53 assessment, such that only HGEC or ambiguous carcinomas should be routinely subjected to p53 immunostaining.

IKZF1 Gene in Childhood B-cell Precursor Acute Lymphoblastic Leukemia: Interplay between Genetic Susceptibility and Somatic Abnormalities.

SNPs in IKZF1 are associated with inherited susceptibility to B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Besides, somatic copy number abnormalities (CNA) in genes related to lymphopoiesis (e.g., IKZF1, CDKN2A/B, BTG1) impact patient's outcome. Therefore, this study aimed to investigate an association between germline susceptibility and CNAs in BCP-ALL. The IKZF1 SNPs (rs11978267 and rs4132601) were genotyped in 276 cases and 467 controls. Bone marrow samples were used to determine the presence of somatic abnormalities. The IKZF1 transcript levels were quantified and associated with the SNPs and CNAs. Categorical variables were compared by χ2 test. ORs were estimated with unconditional logistic regression with 95% confidence interval (CI). The variant allele of IKZF1 rs4132601 conferred increased risk of BCP-ALL (OR, 2.09; 95% CI, 1.16-3.74). Individuals with either rs11978267 or rs4132601 had an increased risk for harboring IKZF1 deletion (OR, 2.80; 95% CI, 1.25-6.23 and OR, 2.88; 95% CI, 1.24-6.69, respectively). Increased risks were observed for individuals harboring both IKZF1 and BTG1 deletions (OR, 4.90; 95% CI, 1.65-14.55, rs11978267 and OR, 5.80; 95% CI, 1.94-17.41, rs4132601). Germline genetic variation increases the risk for childhood ALL in general, but also acts as a susceptibility factor bound for risk of specific somatic alterations. These findings provide new insight into the development of childhood ALL regarding causal variants and the biological basis of the risk association, offering the opportunity for future tailored research. Cancer Prev Res; 10(12); 738-44. ©2017 AACR.

Chromosomal instability analysis and regional tumor heterogeneity in colon cancer

Chromosomal instability (CIN) is classically defined as an increase in the rate at which numerical or structural chromosomal aberrations are acquired in a cancer cell. The number of somatic copy number abnormalities (CNAs) revealed by high resolution genomic array can be considered as a surrogate marker for CIN, but several points, related to sample processing and data analysis, need to be standardized. In this work we analyzed 51 CRC samples and matched normal mucosae by whole genome SNP arrays and compared different bioinformatics tools in order to identify broad (>25% of a chromosomal arm) and focal somatic copy number abnormalities (BCNAs and FCNAs respectively). In 15 tumors, two samples, separated by at least 1 cm, were taken from the same tumor mass (double-sampling pairs) in order to evaluate differences in detection of chromosomal abnormalities between distant regions of the same tumor and their influence on CIN quantitative and qualitative analysis. Our data show a high degree of correlation of the quantitative CIN index (somatic BCNA number) between distant tumor regions. On the contrary, a lower correlation is observed in terms of chromosomal distribution of BCNAs, as summarized by a simplified cytogenetic table. Quantitative or qualitative analysis of FCNAs, including homozygous deletions and high level amplifications, did not add further information on the CIN status. The use of the index "somatic BCNA number" can be proposed for a robust classification of tumors as CIN positive or negative even in the presence of a significant tumor regional heterogeneity.

Somatic copy number abnormalities (SCNAs) and mutations in PI3K/AKT pathway as prognostic factors for overall survival (OS) in platinum-treated locally advanced or metastatic urothelial tumors.

4511 Background: An integrative analysis was conducted to identify genomic alterations and mutational status on the gene pathway level that could predict OS in pts with urothelial cancers (UC) trea...

Somatic copy number abnormalities (SCNAs) and mutations in PI3K/AKT pathway and prognostic significance for overall survival (OS) in platinum-treated locally advanced or metastatic urothelial cancer (UC).

358 Background: An integrative analysis was conducted to identify genomic alterations and mutational status on the gene pathway level that could predict OS in patients (pt) with UC treated with platinum chemotherapy. Methods: DNA was extracted from 94 FFPE samples from invasive UC and screened for mutations and CNAs. Of those, complete clinical data was available from 85 cases. Mutations in the cancer genome were analyzed by a mass-spectrometry based genotyping platform (Oncomap version 3) and genomic imbalances were detected by comparative genomic hybridization (CGH) analysis. Regions with threshold of log2 ratio ≥0.4, or ≤ -0.6 were defined as either having copy number gain or loss and significantly recurrent CNAs across the set of samples was determined using a GISTIC analysis. To better define the co-occurrence pattern of mutations and CNAs, we grouped genes into 3 core signal transduction pathways: (1) TP53 pathway; (2) Abnormalities of in the RTK-RAS-RAF pathway including either ERBB2, FGFR3, MET, FGFR1, KRAS BRAF, RAF1 and NF1; (3) abnormalities on PI3K/AKT pathway including PTEN, PIK3CA, AKT1, TSC1, and MTOR. OS was measured from treatment to time of death or censored. Cox regression was used to assess pathways abnormalities and mutations with outcome. Results: Thirty five samples (41%) harbored mutations on at least one gene, mainly TP53 (16%), PIK3CA (9%), FGFR3 (2%), HRAS/KRAS (5%), and CTNNB1 (1%). Sixty-six per cent of patients had some sort of copy number variation. PI3K/AKT was the most affected pathway and was associated with shorter overall survival (hazard ratio (HR) =2.3 with 95% CI: 1.3 to 4.2). There was no significant association between mutational status and OS. Combined analysis of SNCA and mutations in the same pathway will be presented. Conclusions: Patients with advanced urothelial cancer receiving platinum chemotherapy have a significantly poor survival which correlated with copy number abnormalities in the PI3K/AKT pathway. This can help to identify patients that can benefit from targeted pathway specific agents.

Uncovering the biology of multiple myeloma among African Americans: a comprehensive genomics approach

AbstractEpidemiological data have suggested that African American (AA) persons are twice as likely to be diagnosed with multiple myeloma (MM) compared with European American (EA) persons. Here, we have analyzed a set of cytogenetic and genomic data derived from AA and EA MM patients. We have compared the frequency of IgH translocations in a series of data from 115 AA patients from 3 studies and 353 EA patients from the Eastern Cooperative Oncology Group (ECOG) studies E4A03 and E9487. We have also interrogated tumors from 45 AA and 196 EA MM patients for somatic copy number abnormalities associated with poor outcome. In addition, 35 AA and 178 EA patients were investigated for a transcriptional profile associated with high-risk disease. Overall, based on this cohort, genetic profiles were similar except for a significantly lower frequency of IgH translocations (40% vs 52%; P = .032) in AA patients. Frequency differences of somatic copy number aberrations were not significant after correction for multiple testing. There was also no significant difference in the frequency of high-risk disease based on gene expression profiling. Our study represents the first comprehensive comparisons of the frequency and distribution of molecular alterations in MM tumors between AA and EA patients. ECOG E4A03 is registered with ClinicalTrials.gov, number NCT00098475. ECOG E9487 is a companion validation set to the ECOG study E9486 and is registered with the National Institutes of Health, National Cancer Institute, Clinical Trials (PDQ), number EST-9486.

Recurrent copy number abnormalities and copy neutral-loss of heterozygosity in colorectal cancer identified by high-density SNP array

Two forms of genetic instability have been observed in colorectal cancer (CRC): chromosomal instability, that is present in 85% cases and is characterized by structural and numerical chromosomal abnormalities (aneuploidy); and microsatellite instability (MSI), characterized by a deficiency of the mismatch repair system and associated to a normal or quasi-normal karyotype. Recent advances in molecular cytogenetics techniques are likely to introduce a revolution in the field. In the present study we analysed a series of 57 CRC samples and their related normal mucosae by high resolution genomic arrays (Affymetrix SNP 6.0 arrays) and set up a series of bioinformatics tools that allow the generation of a rapid report on broad (>25% of a chromosomal arm) and small somatic copy number abnormalities (CNAs), somatic homozygous deletions, focal high level amplifications, and regions of loss of heterozygosity (LOH). MSI tumors represented 14% of our CRC series and showed a median values of somatic broad CNAs of 3 (range 1-6), while microsatellite stable (MSS) tumors showed a median value of 12 (range 0-24). Therefore all MSI tumors were below a threshold of 6 CNAs, while 16.2 % of MSS were below this threshold, representing a group of microsatellite and chromosomal stable tumors. No correlation was observed between the number of tumor associated CNAs and the number of somatic copy neutral-LOH regions, suggesting that different mechanisms underlie such chromosomal abnormalities. Analysis of recurrent CNAs identified somatic homozygous deletions that cannot be ascribed to regions of inherent fragility.

Clonal Evolution of Multiple TET2mutations In the Transformation of Chronic Myelomonocytic Leukemia to Acute Myeloid Leukemia and Its Subsequent Relapse

AbstractAbstract 2699Transformation from chronic myelomonocytic leukemia (CMML) to acute myeloid leukemia (AML) is associated with a poor prognosis. Clinical features have been used to score those at high risk, e.g. increased marrow blast percentage. Recently, several gene mutations have been identified including TET2, KRAS, NRAS, CBL and RUNX1, of which TET2 is the most frequent (Kohlmann et al 2010, Kosmider et al 2009). Initial descriptions of TET2 mutations in myeloid malignancy suggested that they were early mutations (Delhommeau et al 2009), however subsequent studies have shown in some patients with myeloproliferative neoplasia (MPN) they can be preceded by mutations in JAK2 (Schaub et al 2010). We report a male patient who was diagnosed with CMML aged 43 years. His marrow blast count was 6% and within 2 months he had transformed to AML (FAB M4) with marrow blasts of 50%. He was treated with daunorubicin and AraC (Barts 12 schedule) and obtained a complete morphological remission (CR). However he relapsed 88 days after CR in his skin, central nervous system and marrow. He died with conservative treatment after 6 months. Using this case as a model for transforming CMML, we aimed to identify clonal changes characteristic of the three stages of his disease, chronic leukaemia, transformation to acute leukemia and subsequent relapse. We used cytogenetic analysis, single nucleotide polymorphism (SNP) arrays (Affymetrix SNP 6.0 and 10K; 10K data previously reported by Raghavan et al 2008) to identify the clonal changes. Gene mutation analysis of genomic and cDNA was performed using PCR, cloning and Sanger and amplicon deep-sequencing for the mutational burden of TET2, CBL and KRAS (454 Life Sciences, Branford, CT). At all stages of his disease, he had a normal karyotype, a NPM1 mutation, but no mutations of FLT3, JAK2, KRAS or CBL (Table). Uniparental disomy (UPD) was demonstrated on chromosome 4q at relapse but not present at diagnosis, suggesting an associated TET2 mutation that would be heterozygous at diagnosis and homozygous at relapse. The heterozygous mutation was indentified in the CMML and at transformation, in a region adjacent to the splice site of the end of exon 7. The transcript resulted in a mis-splicing event leading to a stop codon. A second truncated mutant due to a deletion in exon 6 was found expressed at low level. Deep-sequencing of genomic DNA confirmed a deletion in exon 6 consistent with this product at 2.4% (918-fold coverage) in the CMML, and increased to 5.7% (865-fold coverage) burden at transformation. At relapse, the genomic mutation was homozygous. Expression of only the mis-spliced product was observed, with no wild type TET2, and no evidence of the second truncated product by cloning or deep-sequencing (832 reads). Homozygous TET2 mutations are associated with 4qUPD in MPN and myelodysplastic syndromes (Mohamedali et al 2009, Janowska et al 2010), but this is the first description of clonal evolution of a hetero- to homozygous TET2 mutation at disease progression. The case is consistent with the hypothesis that TET2 mutations evolve early in the pathogenesis of myeloid malignancies. However, it also shows there is considerable instability of TET2 mutations, with multiple mutations at various levels, and with mitotic recombination being a late event leading to relapse.CMMLTransformed AMLRelapsed AMLCGN46,XY46,XY46,XYSNP arrayNot doneNo somatic copy number abnormalities detected4qUPD 88201900-qterTET2c4340+5G>T+10_+21del het result: Cys1298Xc4340+5G>T+10_+21del het result: Cys1298Xc4340+5G>T+10_+21del hom result: Cys1298Xc3732_3733delCT 2.4% result: Tyr1245LeufsX22c3732_3733delCT 5.7% result: Tyr1245LeufsX22AbsentNPM1Dup956_959TCTG hetDup956_959TCTG hetDup956_959TCTG het Disclosure:No relevant conflicts of interest to declare.

Gene Copy Number Changes in Myeloid Leukemias.

Abstract A wide range of genetic alterations contribute to the formation of leukemia. These alterations include, but are not limited to, point mutations, gene rearrangements, deletions, amplifications, and a diverse array of epigenetic changes that influence gene expression. Unfortunately, using present methodologies, the identification of all mutations within a leukemia cell is cost prohibitive. Therefore, a more measured approach is needed. In previous work on pediatric acute lymphoblastic leukemia (ALL), we demonstrated the power of genome-wide copy number analysis for the identification of recurrent mutations. Our analyses revealed deletion, amplification, point mutation, and structural rearrangement in genes encoding key regulators of B lymphocyte development and differentiation in 40% of B-progenitor ALL. PAX5 was the most frequent target of somatic mutation, being altered in 30% of cases. Deletions were also detected in E2A, EBF1, LEF1, Ikaros, and Aiolos. These findings suggest that direct disruption of pathways controlling B cell development and differentiation contributes to ALL pathogenesis. We have now extended our analysis to pediatric acute myeloid leukemia (AML). This analysis revealed a mean number of somatic copy number abnormalities (CNA) of 2.38 per cases, with gains equal to losses. Strikingly, the most frequent recurrent CNA were at the breakpoints of known chromosomal translocations, including t(8;21) and inv(16). Based on this observation, we examined genes with CNA in their 5’ or 3’ regions for their possible involvement in cryptic translocations. Using this approach, two cryptic translocations were identified: t(5;11) [NUP98-NSD1] in four cases, and t(6;11)[MLL-MLLT4] in two cases. A number of other genes were identified as the target of recurrent abnormalities, including amplification of CCDC26, ABCC4, and deletion of FAM20C, BCOR. The most frequent abnormality was the amplification of CCDC26 at 8q24.21 (N=15; focal in four cases, broad in 11 cases), which encodes a putative mediator of retinoic acid receptor signaling. The other recurrent lesions occurred in two or fewer cases. Thus many fewer recurrent lesions were identified in AML than in ALL. In addition to the CNA, we also sequenced genes previously shown to be mutated in AML including NRAS, KRAS, PTPN11, BRAF, SOS1, FLT3, CEPBA, NPM1, AML1, CKIT, ERG, TP53, PTEN, and GATA1. Mutations were identified in all genes except BRAF, or SOS1, with the frequency of mutations varying across the different AML genetic subtypes. FAB M7 cases had the highest frequency of CNA but had sequence mutations limited to GATA1. Mutations of FLT3, CEPBA, and NPM1 were most frequent in cases with no or miscellaneous cytogenetic abnormalities, whereas NRAS mutations were most frequent in t(8;21) and inv(16) cases. Importantly, approximately 20% of the cases with recurring translocations had no other sequence or numerical abnormalities. Collectively, these data demonstrate a marked difference in the spectrum of genetic lesions between AML and ALL, with AML having fewer CNA per cases, a lower frequency of recurrent lesions, and a higher frequency of cases lacking any CNA. These data suggest a fundamental difference in the type of collaborating mutations between AML and ALL.

High-Resolution Genome-Wide Profiling of DNA Copy Number Abnormalities and Gene Resequencing in Pediatric Acute Myeloid Leukemia (AML).

To define the complement of genetic lesions in pediatric AML, we performed high resolution genome-wide analysis on bone marrow blasts from 111 cases using Affymetrix 100K and 500K SNP microarrays and genomic resequencing. Cases included t(8;21) (N=20), inv(16) (N=16), t(15;17) (N=7), MLL rearranged (N=16), FAB-M7 (N=9), miscellaneous cytogenetic abnormalities (N=24) and normal karyotype (N=19). Germline DNA was available for 67 of the cases. The mean number of somatic copy number abnormalities (CNA) was 2.38 (range 0–45) with 1.32 gains (range 0–41) and 1.06 losses (range 0–12). Focal CNAs were detected at the breakpoints of known chromosomal translocations, most commonly in inv(16) cases (7/16) and t(8;21) cases (4/20). Based on this observation, we examined focal CNAs that affected the 5' or 3' regions of genes for their possible involvement in cryptic translocations. CNAs involving NUP98 and MLL led to the identification of two cases with t(5;11)[NUP98-NSD1], and two with t(6;11)[MLL-MLLT4] not evident on cytogenetic analysis. RT-PCR for these fusions identified two additional NUP98-NSD1 cases. Identification of focal CNAs are thus useful in identifying clinically significant translocations in AML. Other recurring somatic CNAs were uncommon. Twelve regions containing a single gene were identified, including amplification of EBF, PRDM5, CCDC26*, GDF10, ABCC4*, and deletion of FAM20C*, TUSC1, GPC5, A2BP1, INSR, BCOR* (* involved in N>2 cases). The most frequent abnormality was amplification of CCDC26 at 8q24.21 (N=15; focal in 4 cases, broad in 11) which encodes a putative mediator of retinoic acid receptor signaling. The focal amplifications are predicted to abolish expression of normal transcripts. Seventeen additional recurring CNAs involving 2 to 25 genes were identified, many of which contain known or putative tumor suppressor genes or oncogenes, including deletions of regions at 7q36, 9p21 (CDKN2A/B), 11p14-p12 (WT1, WIT1), and amplification at 19p13 (JUNB, LYL1). These regions showed significant correlation between copy number and local gene expression in an integrated analysis of CNA and Affymetrix U133A gene expression data. Copy-neutral LOH was uncommon and recurrent only with LOH of chromosome 13 in 3 cases. Sequence analysis has been completed for several genes involved in recurring CNAs (CCDC26, TUSC1, FBXW7) and for genes known to be mutated in AML (N/KRAS, PTPN11, BRAF, SOS1, FLT3, CEPBA, NPM1, AML1, CKIT, GATA1). No mutations were identified in CCDC26, TUSC1, FBXW7, BRAF, or SOS1. Marked differences in the combination of CNA and sequence mutation were identified across the different genetic subtypes of AML. FAB M7 cases had the highest frequency of CNA (mean 9.3 lesions/case) but had sequence mutations limited to GATA1. Mutations of FLT3, CEPBA and NPM1 were most frequent in cases with no or miscellaneous cytogenetic abnormalities, where as RAS mutations were most frequent in t(8;21) and inv(16) cases. Importantly, approximately 30% of the cases with recurring translocations had no other sequence or numerical abnormalities. These data demonstrated that, in contrast to pediatric ALL, AML is characterized by relatively few recurring CNAs, and that spectrum of CNA and sequence mutation is significantly associated with disease subtype.