Clinical Value of Next Generation Sequencing in the Detection of Recurring Structural Rearrangements and Copy Number Abnormalities in Acute Myeloid Leukemia

Purpose: Acute myeloid leukemia (AML) is the most common acute leukemia in adults, affecting approximately 20,000 patients annually in the United States. AML genetic subtypes, as defined by the World Health Organization (WHO), are identified through fluorescence in situ hybridization (FISH), conventional chromosome analysis, and sequencing techniques. Mate pair sequencing (MPseq) is a next generation sequencing (NGS) technology optimized to detect genome wide structural variants and copy number alterations at high resolution. Our study goal was to investigate the prognostic value of MPseq in comparison to FISH, chromosome, and sequencing studies in the evaluation of AML patients. Methods: We performed a prospective study using blood and bone marrow samples from 105 patients with a diagnosis of AML, using MPseq, along with chromosome, FISH, and NGS or PCR studies to detect small mutations. Cytogenetic and molecular genetic results were correlated with MPseq findings. We also analyzed the MPseq data for chromoplexy, chromothripsis, and progressive complexity. Junction and copy number burden, the incidence of structural variation in the genome and the percent of the genome with aberrant copy number, were evaluated. Overall survival statistics were stratified by AML subtypes and observed anomalies. Results: Although structural variants in AML were characterized at a high resolution using MPseq when compared to conventional cytogenetic methods, risk stratification using current European Leukemia Net (ELN) guidelines was not improved by MPseq. The cohorts involving 5q and/or 7q deletions exhibited high levels of genomic complexity when compared to normal karyotype AML (NK-AML). The incidence of copy number gains, losses and junctions was greatest in 5q and 7q co-deletions (5q/7q) (16.5, 25.0, 69.3) and 5q deletions (5q) (9.8, 16.7, 31.6) subtypes compared to 7q deletions (3.4, 7.0, 6.7) and NK-AML (2.6, 4.3, 3.8) (p<0.001) subtypes. Chromoplexy, chromothripsis, and progressive structural complexity were detected in most samples with 5q deletions and 5q/7q co-deletions, but absent in samples with 7q or NK-AML. Biallelic inactivation of TP53 by sequencing mutation and/or deletion was common in the 5q/7q co-deletion (14/18 cases) and 5q deletion cohorts (7/10 cases), rare in the 7q deletion cohort (1/11 cases), and absent in the NK-AML (n=44) cohort. The median OS was significantly worse for patients with 5q/7q deletions (122.5 days) and 5q deletions (248 days) compared to NK-AML (413.5 days; p<0.001 and p=0.017, respectively) and between 5q/7q deletions and 7q deletions (370.5 days; p<0.001). No significant difference was observed between 5q/7q and 5q deletion subtypes, between NK and 7q deletion subtypes and between 5q and 7q deletion subtypes. The median OS was also significantly shorter for patients with TP53 alterations compared to patients with normal TP53 status. Patients with chromoplexy, chromothripsis and/or progressive structural complexity identified by MPseq had a significantly shorter median OS compared to patients without these features. (p<0.0001) Discussion: Risk stratifications based on current guidelines using cytogenetic and sequencing results were not adjusted due to MPseq results, which is not surprising when primary abnormalities are observed by conventional cytogenetic methods. NK-AML cases did not appear to benefit from a high resolution genomic evaluation. However, MPseq added value when structural variation required additional characterization, detecting novel rearrangements, such as a KAT6A/SORBS3 fusion. Lastly, we recognized common mischaracterizations made by conventional chromosome studies - including missed TP53 deletions in 7 cases, 5q/7q deletions misinterpreted as monosomies, cryptic NUP98 rearrangements, and unappreciated genomic complexity correlating with poor OS. These mischaracterizations challenge the use of conventional chromosome studies as a gold standard without accompanying FISH or MPseq studies. MPseq, similar to other structural methodologies such as optical mapping and long read sequencing, should be considered important complements to standard cytologic techniques given the important additional genomic information obtained. The additional structural variant characterization will be critical in paving the way for genomic discovery with the overall goal of improving prognostication for patient care. Figure Disclosures Vasmatzis: WholeGenome LLC.: Other: Owner; Mayo Clinic: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Prevalence of 7q deletion in patients with Acute myeloid leukemia

Introduction: Deletion of critical regions on long arm of chromosome 7 is important in pathogenesis of acute myeloid leukemia (AML). These regions include 7q22 and 7q31 which carry certain tumor suppressor genes which if deleted can result in uncontrolled division of myeloid cells. Aims: To estimate prevalence of 7q deletion in patients with acute myeloid leukemia (AML). Materials and methods: Retrospective study was done on 25 bone marrow samples diagnosed with Acute myeloid leukemia referred to Division of Human Genetics St.John’s Medical college, Bangalore. Samples were subjected to standard protocol for karyotyping and FISH. Percentage of patients who were positive for 7q deletion was calculated. Results: Out of 25 samples, 5 samples were positive for 7 q deletion accounting to 20% of total patients diagnosed with AML. Conclusion: Presence of 7q deletion can be a poor prognostic marker since tumour suppressor genes are present in these regions. Hence cytogenetic markers are very important in deciding treatment and prognosis in these patients.

Unbalanced Y;7 Translocation between Two Low-Similarity Sequences Leading to SRY-Positive 45,X Testicular Disorders of Sex Development

Unbalanced translocations of Y-chromosomal fragments harboring the sex-determining region Y gene (SRY) to the X chromosome or an autosome result in 46,XX and 45,X testicular disorders of sex development (DSD), respectively. Of these, Y;autosome translocation is an extremely rare condition. Here, we identified a 20-year-old man with a 45,X,t(Y;7)(q11.21;q35) karyotype, who exhibited unilateral cryptorchidism, small testis, intellectual disability, and various congenital anomalies. The fusion junction of the translocation was blunt, and the breakpoint-flanking regions shared only 50% similarity. These results indicate that Y;autosome translocations can occur between 2 low-similarity sequences, probably via nonhomologous end joining. Furthermore, translocations of a Ypterq11.21 fragment to 7q35 likely result in normal or only mildly impaired male-type sexual development, along with various clinical features of 7q deletion syndrome, although their effects on adult testicular function remain to be studied.

Myeloid Malignancies with Isolated 7q Deletion Can be Further Characterized By Their Accompanying Molecular Mutations

Background: 7q deletions (del(7q)) are recurrent cytogenetic abnormalities. They occur either as the sole abnormality or accompanied by additional chromosome aberrations in AML, MDS, MDS/MPN and MPN. Cases with del(7q) as the sole abnormality are rare and poorly characterized. Aim: In patients with myeloid malignancies and del(7q) as the sole abnormality we determined 1. Type and size of the del(7q) 2. Spectrum of accompanying molecular mutations and their impact on the phenotype. Patients and Methods: 81 cases with myeloid malignancies and del(7q) as the sole abnormality were included in this study. Of these 38 had AML (27 de novo, 7 secondary, 4 therapy-related), 17 MDS (14 de novo, 3 therapy-related), 10 MDS/MPN (9 CMML, 1 MDS/MPN unclassifiable) and 16 MPN. The median age was 72 years (range: 29-89 years). All cases were investigated by array CGH (Agilent, Waldbronn, Germany) and for mutations in ASXL1, CALR, CBL, DNMT3A, ETV6, EZH2, JAK2, KRAS, MPL, NPM1, NRAS, RUNX1, SF3B1, SRSF2, TET2, and TP53. Results: Array CGH revealed an interstitial del(7q) in 67 cases, while 14 cases showed terminal del(7q). Further characterization of these deletions using 24 color FISH revealed unbalanced translocations in 10 of the 14 cases with terminal deletion. Partner chromosomes were X, 8, 9, 12, 13, 17 (n=2), 19 (n=2), and 22. The breakpoints on chromosome 7 were diverse ranging from 7q11 to 7q32. In two cases the breakpoint was within the CDK6 gene. In two cases with terminal del(7q) the complete loss of 7q was due to an idic(7)(q11.21). In the remaining two cases the terminal deletion could not be further resolved. In the 67 cases with interstitial del(7q) the size of the del(7q) varied between 1.8 and 158.9 Mb (median: 52.6 Mb). No commonly deleted region could be identified for all cases. However, in 57 cases the deleted region encompassed genomic position 101,912.442 (7q22.1) to 119,608.824 (7q31.31) including 111 genes. The size of the 7q deletion was smaller in cases with interstitial deletion as compared to terminal deletion (57.7 MB vs 70.9 MB, p=0.04) and in MPN as compared to all other entities (48.7 MB vs 62.8 MB, p<0.001). The mutation analyses revealed mutations in TET2 37% (25/67), ASXL1 35% (27/78), RUNX1 26% (18/69), DNMT3A 21% (14/68), SRSF2 18% (13/73), JAK2 V617F 14% (11/79), CBL 9% (7/75), NRAS 9% (7/77), MLL -PTD 5% (4/80), KRAS 5% (3/66), EZH2 4% (3/72), TP53 4% (3/74), SF3B1 4% (3/75), ETV6 3% (2/73), NPM1 3% (2/77), CALR 1% (1/77), MPL 1% (1/76). ASXL1 and TET2 were frequently co-mutated as 56% of ASXL1 mutated cases also harbored a TET2 mutation (p=0.02). 39 cases were analysed for all 16 molecular mutations. The majority of patients (n=27, 69%) had more than one mutation (range: 2-4), 9 patients (23%) had one mutation and in 3 patients (8%) no mutation was detected. The number of mutations per patient was lower in patients <70 years as compared to patients ≥70 years (0, 1,2,3,4 mutations detected in: 23%, 15%, 15%, 46%, and 0% vs 0%, 27%, 27%, 31%, and 15%, p=0.05). CBL mutations were most frequent in CMML (44%) but rare in all other subtypes (5%, p=0.003), while RUNX1 mutations were most frequent in AML (43% vs 9%; p=0.002) and JAK2 V617F mutations most frequent in MPN (50% vs 5%, p<0.001). DNMT3A mutations and MLL -PTD were significantly more frequent in de novo AML than in all other entities (43% vs 11%, p=0.007; 15% vs 0%, p=0.009), while no significant differences in frequency were observed between the different entities for any of the other mutations or the number of mutations per case. In CMML CBL mutations were associated with del(7q) (44%) as CBL mutations were present in only 17% of non del(7q) CMML (n=101, p=0.07). The frequency of RUNX1 mutations was significantly higher in AML with del(7q) as the sole abnormality (43%) as compared to all other AML (n=2273, 21%; p=0.001). Median overall survival (OS) for the total cohort was 25 months and did not differ significantly between AML, MDS, MDS/MPN and MPN (26, 27, not reached, 15 months, respectively). Conclusions: 1. Sizes and localisations of the del(7q) largely overlapped between AML, MDS, MDS/MPN and MPN. 2. 92% of all patients with 7q deletion harbored at least 1 molecular mutation. 3. TET2 and ASXL1 were the most frequently mutated genes and were present at comparable frequencies in all subtypes. 4. AML with del(7q) is closely associated with RUNX1 mutations while CMML with del(7q) frequently harbored CBL mutations suggesting a cooperative leukemogenic potential in these entities. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Fasan:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Targeted Detection of Copy Number Variants Using a Myeloid Malignancy Next Generation Sequencing Mutation Panel Allows Comprehensive Genetic Analysis Using a Single Testing Method

Introduction Myeloid malignancies are clonal disorders of hematopoietic stem and progenitor cells that include myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN) myelodysplastic/myeloproliferative (MDS/MPN) overlap neoplasms, and acute myeloid leukemia (AML). Next generation sequencing (NGS) studies have identified a number of recurrently mutated genes that have diagnostic and/or prognostic significance in these disorders. Chromosomal copy number variations (CNVs) including deletions at 5q, 7q, 12p and 17p as well as trisomy 8, are another major type of recurrent genetic alteration with clinical significance in myeloid malignancies. Detection of CNVs has traditionally required specialized testing methods such as cytogenetics/FISH and/or array-based platforms. Thus, comprehensive genetic profiling of myeloid malignancies requires multiple testing strategies at high cost. In an effort to provide more efficient genetic profiling of these disorders, we designed and tested an algorithm to evaluate for CNVs using sequence coverage data derived from a NGS-based 53-gene myeloid mutation panel with the goal of obtaining information on both mutations and CNVs from a single test. Methods The sample cohort included 73 MDS patients, 36 patients with MDS/MPN neoplasms, 70 MPN patients, and 91 AML patients (n=270 total cases). Genomic DNA was extracted from bone marrow or peripheral blood, and enriched for regions of interest by solution capture (SureSelect, Agilent), then sequenced on the Illumina MiSeq, HiSeq 2000 or NextSeq NGS platforms. Gene variants were identified using the software programs FreeBayes, for single nucleotide variants and small insertions/deletions, and Pindel for larger insertions/deletions. To detect CNVs in the targeted regions, the read coverage data was normalized to a Log2 ratio which was generated by comparing the normalized sample coverage to that obtained from a pool of normal controls. CNVs were detected using a circular binary segmentation algorithm. In a subset of cases (n=43) CNVs detected using NGS data were validated by comparing to the results obtained by SNP microarray (CytoScan HD Array, Affymetrix) testing, the current gold standard, and analyzed by CHAS 2.0 (Affymetrix) and Nexus 7.5 (Biodiscovery). KMT2A (MLL) partial tandem duplications detected by NGS analysis were confirmed by quantitative PCR. Comparisons of proportions were performed by Fisher's exact test. Results In the entire cohort of 270 cases, we detected pathogenic mutations in 208 cases (77%). ASXL1 (n=64), SRSF2 (n=40), TET2 (n=39) and DNMT3A (n=37) were among the most frequently mutated genes as has previously been shown. For targeted CNV analysis, seven cases were excluded due to inadequate normalization of the read coverages. In the validation set of 43 cases, all of the targeted CNVs detected by NGS were confirmed by SNP microarray analysis (Figure 1A). Overall, we detected targeted CNVs in 68 cases (25.8%; AML n=32, MDS n=16, MDS/MPN n=9, MPN n=11). The most frequent CNVs were 7q deletion of a region including the genes LUC7L1 and EZH2 (n=21), TP53 deletion (n=9), ETV6 deletion (n=8), gain of RAD21 (possible trisomy 8) (n=8), and 5q deletion of a region including the genes NSD1 and NPM1 (n=4). In addition, we were able to detect exon-level duplications, the so-called KMT2A partial tandem duplication (also known as MLL -PTD), in 9 cases (Figure 1B). In the 63 cases that were negative by mutation analysis (MDS n=26, AML n=17, MDS/MPN n=5, MPN n=15), targeted CNVs including 7q deletion were observed in 4 cases (6%) (MDS n=3, AML n=1). In addition, targeted CNV analysis detected TP53 deletion in 3 TP53 -non-mutated cases and in 6 TP53 -mutated cases, and TET2 deletion in 2 TET2 -non-mutated cases and in 2 TET2 mutated cases. To investigate the association among gene mutations and targeted CNVs, we found that ETV6 deletion was strongly associated with TP53 alterations (both mutation and gene deletion; p<0.001) and 7q deletion was associated with mutations in TP53, KRAS and IDH1 (p= 0.000073, 0.009, 0.026, respectively). Conclusion Our results demonstrated the feasibility of using the same NGS data to detect both somatic mutations and targeted CNVs with enhanced efficiency and potentially lower costs compared to classical methods. Figure 1. Examples of targeted CNVs detected by NGS and comparison to SNP microarray analysis. Figure 1. Examples of targeted CNVs detected by NGS and comparison to SNP microarray analysis. Disclosures South: Affymetrix: Consultancy, Honoraria; ARUP Laboratories: Employment; Lineagen Corporation: Consultancy; Illumina: Consultancy, Honoraria.