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Acute myeloid leukemia (AML) is a hematological malignancy with an undefined heritable risk. Here we perform a meta-analysis of three genome-wide association studies, with replication in a fourth study, incorporating a total of 4018 AML cases and 10488 controls. We identify a genome-wide significant risk locus for AML at 11q13.2 (rs4930561; P = 2.15 × 10−8; KMT5B). We also identify a genome-wide significant risk locus for the cytogenetically normal AML sub-group (N = 1287) at 6p21.32 (rs3916765; P = 1.51 × 10−10; HLA). Our results inform on AML etiology and identify putative functional genes operating in histone methylation (KMT5B) and immune function (HLA)., Genome wide association studies in cancer are used to understand the heritable genetic contribution to disease risk. Here, the authors perform a genome wide association study in European patients with acute myeloid leukemia and identify loci associated with risk of developing the disease.

Acute myeloid leukemia (AML) is a hematological malignancy with an undefined heritable risk. Here we performed a meta-analysis of three genome-wide association studies, with replication in a fourth study, incorporating a total of 4018 AML cases and 10488 controls. We identified a genome-wide significant risk locus for AML at 11q13.2 (rs4930561; P = 2.15 × 10-8; KMT5B). We also identified a genome-wide significant risk locus for the cytogenetically normal AML sub-group (N=1287) at 6p21.32 (rs3916765; P = 1.51 × 10-10; HLA). Our results inform on AML etiology by identifying putative functional genes operating in histone methylation (KMT5B) and immune function (HLA).

Multiple studies have demonstrated the utility of chromosomal microarray (CMA) testing to identify clinically significant copy number alterations (CNAs) and copy-neutral loss-of-heterozygosity (CN-LOH) in myeloid malignancies. However, guidelines for integrating CMA as a standard practice for diagnostic evaluation, assessment of prognosis and predicting treatment response are still lacking. CMA has not been recommended for clinical work-up of myeloid malignancies by the WHO 2016 or the NCCN 2017 guidelines but is a suggested test by the European LeukaemiaNet 2013 for the diagnosis of primary myelodysplastic syndrome (MDS). The Cancer Genomics Consortium (CGC) Working Group for Myeloid Neoplasms systematically reviewed peer-reviewed literature to determine the power of CMA in (1) improving diagnostic yield, (2) refining risk stratification, and (3) providing additional genomic information to guide therapy. In this manuscript, we summarize the evidence base for the clinical utility of array testing in the workup of MDS, myelodysplastic/myeloproliferative neoplasms (MDS/MPN) and myeloproliferative neoplasms (MPN). This review provides a list of recurrent CNAs and CN-LOH noted in this disease spectrum and describes the clinical significance of the aberrations and how they complement gene mutation findings by sequencing. Furthermore, for new or suspected diagnosis of MDS or MPN, we present suggestions for integrating genomic testing methods (CMA and mutation testing by next generation sequencing) into the current standard-of-care clinical laboratory testing (karyotype, FISH, morphology, and flow).

Disease relapse is the major cause of treatment failure after allogeneic stem cell transplantation (allo-SCT) in acute myeloid leukemia (AML). To identify AML-associated genes prognostic of AML relapse post–allo-SCT, we resequenced 35 genes in 113 adults at diagnosis, 49 of whom relapsed. Two hundred sixty-two mutations were detected in 102/113 (90%) patients. An increased risk of relapse was observed in patients with mutations in WT1 (P = .018), DNMT3A (P = .045), FLT3 ITD (P = .071), and TP53 (P = .06), whereas mutations in IDH1 were associated with a reduced risk of disease relapse (P = .018). In 29 patients, we additionally compared mutational profiles in bone marrow at diagnosis and relapse to study changes in clonal structure at relapse. In 13/29 patients, mutational profiles altered at relapse. In 9 patients, mutations present at relapse were not detected at diagnosis. In 15 patients, additional available pre–allo-SCT samples demonstrated that mutations identified posttransplant but not at diagnosis were detectable immediately prior to transplant in 2 of 15 patients. Taken together, these observations, if confirmed in larger studies, have the potential to inform the design of novel strategies to reduce posttransplant relapse highlighting the potential importance of post–allo-SCT interventions with a broad antitumor specificity in contrast to targeted therapies based on mutational profile at diagnosis.

The cytogenetically cryptic t(5;11)(q35;p15) leading to the NUP98-NSD1 fusion is a rare but recurrent gene rearrangement recently reported to identify a group of young AML patients with poor prognosis. We used reverse transcription polymerase chain reaction (PCR) to screen retrospectively diagnostic samples from 54 unselected pediatric AML patients and designed a real time quantitative PCR assay to track individual patient response to treatment. Four positive cases (7%) were identified; three arising de novo and one therapy related AML. All had intermediate risk cytogenetic markers and a concurrent FLT3-ITD but lacked NPM1 and CEBPA mutations. The patients had a poor response to therapy and all proceeded to hematopoietic stem cell transplant. These data lend support to the adoption of screening for NUP98-NSD1 in pediatric AML without otherwise favorable genetic markers. The role of quantitative PCR is also highlighted as a potential tool for managing NUP98-NSD1 positive patients post-treatment.

In this multicentre retrospective study we have studied the impact of T cell chimerism on the outcome of 133 patients undergoing an alemtuzumab based reduced intensity conditioning allograft (RIC). The median age of the patients was 50 years (range 42-55 years). 77 patients were transplanted using an HLA identical sibling donor while 56 patients received a fully matched volunteer unrelated donor graft. 64 patients had a lymphoid malignancy and 69 were transplanted for a myeloid malignancy. 38 patients (29%) relapsed with no significant difference in risk of relapse between patients developing full donor and mixed donor chimerism in the T-cell compartment on D+90 and D+180 post transplant. Day 90 full donor T cell chimerism correlated with an increased incidence of acute GVHD according to NIH criteria (p=0.0004) and the subsequent development of chronic GVHD. Consistent with previous observations, our results confirmed a correlation between the establishment of T cell full donor chimerism and acute GVHD in T deplete RIC allografts. However our study failed to identify any correlation between T cell chimerism and relapse risk and challenge the use of pre-emptive donor lymphocyte infusions (DLI) in patients with mixed T cell chimerism transplanted using an alemtuzumab based RIC regimen.

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

The cytogenetically cryptic t(5;11)(q35;p15) leading to the NUP98-NSD1 fusion is a rare but recurrent gene rearrangement recently reported to identify a group of young AML patients with poor prognosis. We used reverse transcription polymerase chain reaction (PCR) to screen retrospectively diagnostic samples from 54 unselected pediatric AML patients and designed a real time quantitative PCR assay to track individual patient response to treatment. Four positive cases (7%) were identified; three arising de novo and one therapy related AML. All had intermediate risk cytogenetic markers and a concurrent FLT3-ITD but lacked NPM1 and CEBPA mutations. The patients had a poor response to therapy and all proceeded to hematopoietic stem cell transplant. These data lend support to the adoption of screening for NUP98-NSD1 in pediatric AML without otherwise favorable genetic markers. The role of quantitative PCR is also highlighted as a potential tool for managing NUP98-NSD1 positive patients post-treatment.

Metaphase cytogenetic (MC) analysis in patients with myelodysplastic syndrome (MDS) remains the only essential recommended genetic test in Europe(Malcovati et al, Blood 2013). The chromosome abnormalities identified provide evidence of clonality and information regarding disease characterisation, risk stratification, treatment strategies and disease monitoring. With the exception of the 3q/MECOM gene rearrangement, observed in 1% MDS, the common, recurrent chromosome abnormalities and all specified abnormalities in the revised International Prognostic Scoring System (IPSS-R) for MDS are copy number changes (deletion, duplication, amplification) (Greenberg et al, Blood 2012). However, the identification of gene mutations is increasingly becoming important to confirm a diagnosis of MDS in problematic cases, refine risk (particularly in those with lower risk IPSS-R scores) and improve patient management (Bejar et al, JCO 2011;Haferlach et al, Leukemia 2014). Currently there is insufficient prognostic evidence to use gene mutation analysis as the sole genetic test, but SNP array technology may be seen as a stepping stone towards providing some gene mutation information as it detects copy number variation (CNV) at single gene, and sometimes intragenic, resolution and copy neutral loss of heterozygosity (CN-LOH), regions which are known to harbor bi-allelic gene mutations. CN-LOH is common in hematological neoplasia, including MDS. We undertook a prospective validation study using the Affymetrix Cytoscan® HD SNP array, with more than 2.6 million copy number markers, to compare SNP array with MC in patient samples at presentation with confirmed or highly suspected MDS. Subsequent monitoring by SNP array was performed in those patients where samples were received routinely for MC. Expectations were that SNP array analysis would be at least comparable to MC, by detecting all copy number changes detected by chromosome analysis; increase the diagnostic yield of MDS, due to the detection of both sub microscopic CNV and chromosome cryptic CN-LOH; and increase the number of prognostically relevant genetic abnormalities identified. Samples from 358 patients at disease presentation were received for MC at the West Midlands Regional Genetics Laboratory, UK and were used in this study. Of the 358 patients, 207 had a diagnosis of MDS confirmed and 29 had a diagnosis of MDS/MPN confirmed. The remaining 122 cases were classified as follows: 26 other hematological neoplasia (including MPN, AML, CLL and MM), 51 reactive, 32 non-neoplastic disorders and 13 non-diagnostic. In the 236 confirmed MDS or MDS/MPN cases, marrow was available in 219 and peripheral blood (PB) only in 17. PB samples were abnormal by SNP array in 11/17 (65%). Of the 219 marrows, 146/219 (67%) had comparable SNP array and MC results. A normal karyotype or failed MC result was observed in 130 samples, of these 35 (27%) had an abnormal SNP array. Cases were abnormal by both SNP array and MC in 84 (38%) samples, of these additional abnormalities were detected by SNP array in 30 (36%), including gene mutations with prognostic significance, such as KMT2A-itd, TP53 deletion and RUNX1 deletion. A normal SNP array was observed in 5 (2%) samples with abnormal MC due to balanced rearrangements (SNP array will not detect balanced rearrangements) and low level (1-3%) abnormal clones. In marrow samples, the diagnostic yield by MC alone was 91/219 (42%) and by SNP array analysis alone 119/219 (54%). Therefore, SNP array increased the diagnostic yield of MDS and MDS/MPN by 12% and identified further abnormalities in an additional 30/219 (14%) samples. In conclusion, this validation study demonstrates that SNP array may be offered as an alternative test to MC in patients with a confirmed or highly suspected diagnosis of MDS. Patients with features suggesting 3q/MECOM involvement should have FISH for MECOM rearrangements. Disclosures Griffiths: Affymetrix: Research Funding.

There is no single technology capable of detecting the various genetic and genomic aberrations observed in patients with neoplasia. Patients with myelodysplastic syndrome (MDS) may present with chromosomal copy number changes (duplication, deletion, and amplification), balanced chromosome rearrangements, copy neutral loss of heterozygosity (CN-LOH) and/or gene mutations. Currently only microscopic chromosomal changes, as dictated by the international prognostic scoring system (IPSS-R), are used to determine the genetic risk in MDS. However, different genetic aberrations, particularly gene mutations are anticipated to be incorporated into the IPSS-R in the near future. The Affymetrix CytoScan® HD Array is a high definition array with over 2.6 million markers (both copy number and SNP) allowing resolution capabilities way beyond that of metaphase chromosome analysis. The incorporation of 750,000 SNPs also allows for detection of CN-LOH, regions known to harbour bi-allelic gene mutations. A real-time comparative study using the Affymetrix CytoScan® HD Array against traditional metaphase chromosome analysis is being performed on patients with confirmed or highly suspected MDS referred for genetic analysis at the West Midlands Regional Genetics Laboratory, UK. The study is expected to utilise 600 arrays over two years at presentation and on serial surveillance samples. The preliminary results available after the first 100 patients are presented with examples demonstrating the capabilities and clinical utility of SNP array genetic analysis. The study so far, in patients with MDS at presentation, has demonstrated: An increased number of genetic aberrations (CN changes and CN-LOH) detected by SNP array (38/105 (36%) by metaphase analysis and 62/105 (60%) by SNP array analysis).The ability to reliably detect deletions at the single gene level including CUX1, TET2 and RUNX1, and rarely copy number changes within genes including duplication within KMT2A, consistent with partial tandem duplication.The detection of CN-LOH regions, which may contain bi-allelic gene mutations, in 20/105 (19%) of cases, including chromosomal regions 1p (MPL, NRAS), 4q (TET2), 7q (CUX1), 11q (CBL), 17p (TP53). Confirmatory studies are on-going.A lower failure rate for SNP array compared to metaphase analysis (1% v 4%). Of the four failed metaphase cases, three showed recurrent genetic aberrations observed in MDS including del(5q), del(20q) with CN-LOH 17p, and trisomy 8. The failed SNP array had a normal karyotype.Confirmed balanced rearrangements (without gain or loss of genetic material) are not detected.The ability to detect abnormal clones with copy number changes (deletion or gain at one or more loci) at sensitivities as low as between 5 and 20% dependent on aberration size.Twenty-two cases with a normal karyotype had abnormal genetic aberrations detected by SNP array. Two cases with an abnormal karyotype had a normal SNP array profile due to a balanced rearrangement and a low level abnormal clone (4%) beyond the sensitivity of the test.The ability to utilise peripheral blood instead of marrow as no dividing cells are required, thereby allowing genetic analysis in patients too frail or otherwise unsuitable for marrow aspirationThe ability to detect deletion or CN-LOH of the HLA gene region, which is critical in patients being considered for stem cell transplant as HLA typing may be inaccurate or ambiguous.The ability when performed as a complementary tool to metaphase analysis to detect sub-clones or concurrent clonal neoplasia with different copy number changes. Patient benefits are expected to be the potential to improve patient outcomes through improved confidence in diagnosis, prognosis and monitoring. Disclosures Jeffries: Affymetrix: Research Funding.