Copy-neutral Loss Of Heterozygosity Research Articles

Prognostic Impact of Pre-Transplant Chromosomal Aberrations Detected By SNP-Array in Patients Undergoing Unrelated Donor Hematopoietic Cell Transplant for Acute Myeloid Leukemia

<h3>Background</h3> High-resolution genome-wide SNP-arrays detect large chromosomal aberrations including copy-neutral loss of heterozygosity (CNLOH), which is not captured in conventional cytogenetics. <h3>Methods</h3> We used SNP-array genotyping data generated by the DISCOVeRY-BMT study to detect chromosomal aberrations in pre-HCT blood samples from 1,974 acute myeloid leukemia (AML) patients. Clinical data and blood samples were available through the Center for International Blood and Marrow Transplant Research. We used Cox proportional hazard model for statistical analyses. <h3>Results</h3> AML patients in this study received unrelated donor HCT between 2000-2011 at a median age of 48.2 (range=0.6-78.0) years. About 53% of the patients were males, 7.4% had therapy-related disease, and 73% were in complete remission. Chromosomal aberrations were detected in 14.4% of the patients (Figure 1). Most common aberrations in patients with advanced disease at HCT (n=536) were copy losses in chr7 (5.8%), chr5 (4.5%), CNLOH in the following: chr13 (4.7%), chr11 (3.9%), or chr17 (3.5%), and copy gain in chr8 (3.5%). Common aberrations in patients in complete remission (n=1438) were copy-loss in chr7 (1.0%), chr5 (0.7%), and CNLOH in chr9 (0.9%) or in chr17 (0.8%) (Figure 2). Among patients with normal cytogenetics at diagnosis (n=572), 27.5% received HCT in advanced disease (of them 8.3% had chr13-CNLOH). In multivariable models including commonly detected aberrations and adjusted for important factors (footnote Figure 3), chr13-CNLOH was associated with an approximately 3-fold increased risk of leukemia relapse and post-HCT mortality in patients with advanced disease (relapse: HR=2.67, 95% CI=1.67-4.26; mortality: HR=2.79, 95% CI=1.81-4.28, p<0.0001). In contrast, chr17-CNLOH was associated with a statistically significant risk of post-HCT mortality in patients with remission (HR=2.85, 95% CI=1.51-5.37, p=0.001); the association with relapse was not statistically significant (HR=1.84, p=0.21). Having aberrations in ≥3 chromosomes was associated with a statistically significant excess risk of mortality in patients with advanced disease (HR=1.59, 95% CI=1.07-2.36, p=0.02) (Figure 3). In patients with normal cytogenetics at diagnosis, chr13-CNLOH pre-HCT refined risk stratification of patients with advanced disease; compared to patients in remission, the HR for OS in patients with advanced disease in the presence or absence of chr13-CNLOH= 5.7 vs. 1.7, respectively, p<0.0001 for both; HR=3.6, p=0.0004 when comparing presence or absence of chr13-CNLOH in patients with advanced disease. <h3>Conclusion</h3> Pre-HCT CNLOH in chr13 or chr17 are associated with inferior post-HCT survival in AML; possibly as a consequence of high risk of post-HCT leukemia relapse. CNLOH in chr13 or chr17 may provide new genomic prognostic markers that guide patient risk stratification and possibly therapeutic options.

Dynamic Extreme Aneuploidy (DEA) in the vegetable pathogen Phytophthora capsici and the potential for rapid asexual evolution.

Oomycete plant pathogens are difficult to control and routine genetic research is challenging. A major problem is instability of isolates. Here we characterize >600 field and single zoospore isolates of Phytophthora capsici for inheritance of mating type, sensitivity to mefenoxam, chromosome copy number and heterozygous allele frequencies. The A2 mating type was highly unstable with 26% of 241 A2 isolates remaining A2. The A1 mating type was stable. Isolates intermediately resistant to mefenoxam produced fully resistant single-spore progeny. Sensitive isolates remained fully sensitive. Genome re-sequencing of single zoospore isolates revealed extreme aneuploidy; a phenomenon dubbed Dynamic Extreme Aneuploidy (DEA). DEA is characterized by the asexual inheritance of diverse intra-genomic combinations of chromosomal ploidy ranging from 2N to 3N and heterozygous allele frequencies that do not strictly correspond to ploidy. Isolates sectoring on agar media showed dramatically altered heterozygous allele frequencies. DEA can explain the rapid increase of advantageous alleles (e.g. drug resistance), mating type switches and copy neutral loss of heterozygosity (LOH). Although the mechanisms driving DEA are unknown, it can play an important role in adaptation and evolution and seriously hinders all aspects of P. capsici research.

Difference of genomic copy numbers alterations between hairy cell leukemia-variant and classical hairy cell leukemia: a pilot retrospective study in Chinese.

Background: Hairy cell leukemia (HCL) is a rare chronic B-cell lymphoproliferative disorder. It has two pathological subtypes: classical HCL (HCL-C) and HCL-variant (HCL-V). HCL-C and HCL-V are distinct in morphology and immunophenotype. Their differentiation is important for patient management and clinical outcome, with HCL-V responding poorly to conventional HCL treatments. Recently, whole genomic sequencing has been used to identify the difference between HCL-C and HCL-V and mutation of BRAFV600E has been proved to be a molecular hallmark of HCL-C. However, BRAF inhibitors were not effective in all HCL-C cases and HCL-V seems be lack of the high-frequency mutations. Therefore, it is necessary to compare the genomic changes between HCL-C and HCL-V by high-resolution studies, especially in Chinese population, the genomic alterations of HCL have rarely be reported.Methods: In this study, the clinical features of a total of 18 Chinese HCL patients were described. Single nucleotide polymorphism (SNP) array analysis was performed to evaluate the genomic copy number alterations (CNA) and copy neutral loss of heterozygosity (CN-LOH) on six HCL-Vs with CD25-/BRAFV600E- and four HCL-Cs with CD25+/BRAFV600E+.Results: A total of 24 CNAs including seven chromosomal gains and 17 chromosomal losses, and 22 CN-LOHs were revealed. Five of the six cases of HCL-V showed 15 CNAs including four cryptic chromosomal gains and 11 chromosomal losses. Overlapping regions involving micro-deletion of chromosome 2q13 and large chromosomal loss of 14q were showed in HCL-V. In HCL-C, a total of nine CNAs were revealed in three of the four cases including three chromosomal gains and six chromosomal losses. No overlapping area was observed among the CNVs. 15 CN-LOHs were showed in five of the six cases of HCL-V and seven CN-LOHs was demonstrated in all of the four HCL-Cs.Conclusions: Comparing to Westerners, a relatively higher proportion of HCL-V in all HCL is observed in this study. CNAs and CN-LOHs were common in both HCL-V and HCL-C but the CNAs were different in them. HCL-C was characterized with the higher ratio of large chromosomal changes but lacked of recurrent CNAs, while HCL-V was presented with the higher incidence of cryptic CNAs and recurrent CNAs involving tumor-associated genes. It is necessary to further investigate the association of the genes, such as NPHP1 and TRAF3 genes, and HCL-V in the future study.

De Novo and Therapy-Related Acute Myeloid Leukemia and Myelodysplastic Syndrome: Similarities and Differences in SNP-Array Detected Chromosomal Aberrations in Pre-Transplant Blood Samples

De Novo and Therapy-Related Acute Myeloid Leukemia and Myelodysplastic Syndrome: Similarities and Differences in SNP-Array Detected Chromosomal Aberrations in Pre-Transplant Blood Samples

Role of Genetic Evolution and Germline Mutations in SAMD9 and SAMD9L Genes

Acquired deletions on chromosome 7 (monosomy 7/del7q) are common in myeloid neoplasms, especially pediatric MDS and AML. Although these tumors have historically been reported to occur within families, suggesting a genetic predisposition, the genetic lesion(s) that initiate these diseases has remained elusive until the last few years. Following a series of publications in which germline mutations in SAMD9 and SAMD9L were reported in a MIRAGE syndrome and Ataxia Pancytopenia syndrome, respectively, our group and others described similar heterozygous missense germline mutations in pediatric MDS, especially non-syndromic familial MDS with monosomy 7. Mutations in SAMD9 and SAMD9L have now also been reported in transient monosomy 7, inherited bone marrow failure and AML. Collectively, it is estimated that germline mutations in these genes are present in nearly 20% of children with MDS, with a strong enrichment in those with monosomy 7. Surprisingly, SAMD9 and SAMD9L are paralogous genes adjacently located on human chromosome 7 at band 7q21, and the monosomy 7 clone that expands in children universally lacks the pathologic germline variant. Expression of the mutant proteins in cells results in profound growth suppression, suggesting that there is strong selective pressure for hematopoietic cells to not express the mutant alleles. In addition to chromosome loss, additional methods that suppress expression of the pathologic allele have been described. These include copy neutral loss of heterozygosity (CN-LOH) with duplication of the wild-type allele or the somatic acquisition of additional mutations in cis with the germline mutation that counteract the growth suppressive effect of the germline mutation. The clinical phenotype is largely dictated by the revertant mutation in the dominant hematopoietic clone within the patient's bone marrow. Those with an expansion of a CN-LOH clone are more commonly asymptomatic, in contrast to those patients with a dominant monosomy 7 clone. Progression to higher grade MDS or AML is associated with the acquisition of additional somatic mutations including mutations in SETBP1, KRAS and RUNX1. The recognition of these germline mutations has had an immediate impact on the clinical management of children with MDS, including their family members, and ongoing clinical work in the pediatric MDS community is aimed at establishing guidelines for the pathologic diagnosis, clinical monitoring and treatment for these patients. In addition to these ongoing clinical pursuits, there is significant research interest in these genes, the function of their proteins in hematopoietic cells and how the germline mutations alter the function of the wild-type protein. The SAMD9 and SAMD9L proteins are largely uncharacterized and have been shown to be important in endocytosis, growth factor signaling and to have antiviral properties. Intriguingly, SAMD9 and SAMD9L are both induced by inflammatory signals, including interferons, suggesting a link between inflammatory stress and the disease phenotype. Ongoing studies are aimed at developing models, including in vitro and in vivo models, to understand the mechanisms by which these germline mutations can ultimately lead to the development of pediatric MDS and related disorders. Disclosures No relevant conflicts of interest to declare.

Significance of Abnormalities of PPM1D in Myeloproliferative Neoplasms

The TP53 pathway has been shown to be dysregulated in myeloproliferative neoplasms (MPN) through several different mechanisms, including TP53 mutations, TP53 deletions and Murine Double Minute 2 (MDM2) overexpression. Downregulation of the TP53 pathway likely plays a role in MPN progression as evidenced by the association of TP53 loss of heterozygosity with transformation to acute leukemia and the presence of inactivating mutations of TP53 found in a proportion of MPN-blast phase (MPN-BP) cases (Kubesova et al, Leukemia. 2018;32(2):450-61). Furthermore, MDM2 inhibition induces TP53 pathway upregulation and consequent targeting of hematopoietic stem and progenitor cells in polycythemia vera (PV) and myelofibrosis (MF). The MDM2 inhibitor, idasanutlin, has shown activity in a Phase I trial of high-risk PV patients (Mascarenhas et al. Blood. 2019 Jun 5). An additional regulator of TP53 is protein phosphatase, Mg2+/Mn2+ dependent 1D (PPM1D) which by dephosphorylating TP53 and stabilizing MDM2 regulates the DNA damage response pathway and apoptosis. Somatic activating mutations of PPM1D are present in both solid and hematologic malignancies and are specifically associated with therapy-related myeloid disorders (Hsu et al. Cell Stem Cell. 2018 Nov 1;23(5):700-13). Grinfeld et al. (N Engl J Med. 2018 Oct 11;379(15):1416-30) recently reported that in their genomic analysis of 2035 MPN patients, PPM1D was the eighth most common mutated gene in MPNs at 1.9% frequency. To determine whether there was an association of PPM1D mutations with MPNs we analyzed the genomic data of patients who participated in several clinical trials executed by the Myeloproliferative Neoplasm Research Consortium (MPN-RC). Of 89 MPN-BP patients, 5 patients harbored a PPM1D mutation with a median variant allele frequency (VAF) of 17%. In comparison, 4 out of 135 high risk PV and ET patients harbored a PPM1D mutation with a median VAF of 24%. All mutations were truncating which is consistent with previous reports of PPMID mutations in malignancies. The PPM1D gene is located on the long arm of chromosome 17 (17q23.2) and we hypothesized that cytogenetic aberrations involving this gene locus might also contribute to abnormalities of PPM1D function. Through analysis of our cytogenetic database, 16/1,124 (1.4%) MPN patients were found to have cytogenomic abnormalities involving the region containing the PPM1D gene. Eight of these patients had karyotypic abnormalities, including 3 pts with isochromosome 17q resulting in a gain of 17q and a loss of 17p, including the TP53 gene. The other 4 patients had structural variations of 17q which might lead to aberrant expression of PPM1D. One patient by FISH analysis had gain of 17q. Ten patients had cytogenomic aberrations of 17q detected through analysis of array comparative genomic hybridization and single nucleotide polymorphism (aCGH+SNP), 2 of which had concurrent karyotypic abnormalities of 17q. All patients had a gain or copy neutral loss of heterozygosity (cnLOH) of the 17q22-24.2 region. CnLOH of this region could lead to aberrant overexpression of the PPM1D gene. One of the 8 patients with cnLOH of 17q harbored a PPM1D mutation. Of the 16 patients with 17q cytogenomic abnormalities, 7 (44%) had MPN-BP and 7 (44%) patients had ET or ET that progressed to myelofibrosis (MF) or MPN-BP indicating an association of these abnormalities with advanced disease. By quantitative real time-PCR we determined the PPM1D transcript levels in mononuclear cells from 31 MPN patients without known PPM1D mutations (7 polycythemia vera (PV), 6 ET, 14 MF, 4 MPN-BP) and compared the transcript levels to mononuclear cells from healthy control patients. Forty-two percent (13/31) patients had overexpression of PPM1D (&gt;1.5 fold increase range). Fold increase ranged from 1.5-8 and overexpression was present in the following diagnoses: 4/7 PV, 1/6 ET, 8/14 MF and 0/4 MPN-BP. We have provided evidence that a number of abnormalities of PPM1D including activating mutations, cytogenetic aberrations and transcript overexpression are present in a significant proportion of MPN patients. These abnormalities in PPM1D can be additional events that lead to the downregulation of TP53 and contribute to MPN disease progression. We propose that inhibitors of PPM1D may be used in combination with other drugs that upregulate TP53 to treat MPN patients. Disclosures Rampal: Constellation, Incyte, and Stemline Therapeutics: Research Funding; Agios, Apexx, Blueprint Medicines, Celgene, Constellation, and Jazz: Consultancy. Mascarenhas:CTI Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Roche: Consultancy, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Pharmaessentia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Promedior: Research Funding; Merus: Research Funding. Hoffman:Merus: Research Funding.

DOCK2 Expression Affects Leukemogenesis and Disease Progression in a Murine Model of FLT3/ITD Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm characterized by clonal expansion of myeloid blasts. The FMS-like tyrosine kinase-3 (FLT3) receptor gene is the most commonly mutated gene in AML, and an internal tandem duplication (ITD) mutation portends a poor prognosis. FLT3/ITD activates Rac1 GTPase, and both proteins play key roles in DNA damage response (DDR). Disease progression in FLT3/ITD AML is often heralded by copy-neutral loss of heterozygosity (CN-LOH) at the FLT3 locus, which is typically caused by duplication of the mutant allele through homologous recombination with loss of the wild-type (WT) allele. We previously identified dedicator of cytokinesis 2 (DOCK2) as a FLT3/ITD-interacting protein. The DOCK family of proteins acts as guanine nucleotide exchange factors for the Rho family of GTPases, including Rac1. Expression of DOCK2 is limited to hematopoietic cells, and it is expressed in leukemic blasts in FLT3/ITD AML. Knockdown of DOCK2 in FLT3/ITD cell lines results in reduced Rac1 activity, decreased expression and activity of FLT3 and DDR factors, and increased sensitivity to cytarabine. In this study, we used mouse models to further investigate the relationship between DOCK2 and FLT3/ITD. Mice with a Dock2 mutation (Dock2/Hsd) that results in a marked reduction in Dock2 expression were bred with mice that carry a Flt3/ITD mutation (Flt3+/ITD). Quantitative PCR of mouse bone marrow (BM) revealed that homozygous expression of Dock2/Hsd markedly decreased the expression of Flt3 and key DDR factors in both WT and Flt3+/ITD mice. Flow cytometric analysis of the BM demonstrated that Dock2/Hsd resulted in partial reversal of the myeloproliferative neoplasm that develops in Flt3+/ITD mice, with a significant reduction in multipotent progenitors (MPP) in Flt3+/ITD;Dock2Hsd/Hsd (0.026%) vs. Flt3+/ITD mice (0.075%; P=0.048) at 3 months. To assess the role of DOCK2 in leukemogenesis and disease progression, the Dock2/Hsd mutation was introduced into a murine model of FLT3/ITD acute leukemia that carries both Flt3/ITD and the Nup98-HoxD13 (NHD13) fusion gene. It was previously shown that Flt3+/ITD;NHD13 mice develop acute leukemia with 100% penetrance and short latency. We found that the median survival of Flt3+/ITD;NHD13 mice (117 days) was not significantly affected by heterozygous expression of Dock2/Hsd (123 days); however, the median survival of Flt3+/ITD; NHD13; Dock2Hsd/Hsd mice was extended to 198 days (P=0.001). In contrast, Dock2/Hsd provided no survival benefit to mice that carried either Flt3+/ITD or NHD13 alone. The prolonged survival of Flt3+/ITD;NHD13;Dock2Hsd/Hsd mice was accompanied by delayed onset of LOH at the Flt3/ITD locus. At 3 months, approximately half of Flt3+/ITD;NHD13;Dock2+/+ (47%; n=19) and Flt3+/ITD;NHD13;Dock2+/Hsd (57%; n=14) mice exhibited LOH in the peripheral blood, whereas only 11% of Flt3+/ITD; NHD13;Dock2Hsd/Hsd mice showed LOH (n=18; P=0.008 vs. Dock2+/Hsd and P=0.029 vs. Dock2+/+). Most of the moribund Flt3+/ITD;NHD13 mice (n=13) developed AML with minimal maturation (70%), and the remainder developed T-lymphoblastic leukemia with an early T-precursor phenotype (ETP-ALL). In contrast, moribund Flt3+/ITD;NHD13;Dock2Hsd/Hsd mice (n=14) exhibited a more diverse disease spectrum: 30% mixed phenotype acute leukemia, 21% AML with minimal maturation, 21% ETP-ALL, 21% AML with maturation, and 7% acute erythroid leukemia (AEL). All moribund Flt3+/ITD;NHD13 mice showed complete LOH in BM; however, only 68% of moribund Flt3+/ITD;NHD13;Dock2Hsd/Hsd mice exhibited complete LOH, with the rest showing partial or no LOH. Notably, all mice that exhibited partial or no LOH developed AEL or AML with maturation, diseases that were present in NHD13 mice but were not observed in Flt3+/ITD;NHD13 mice. This suggests that suppression of Dock2 mitigates the effects of Flt3/ITD, allowing the NHD13 mutation to drive the development of disease. These findings demonstrate that decreased DOCK2/Rac1 activity delays LOH at the FLT3 locus and disease progression in a murine model of FLT3/ITD leukemia. Thus, pharmacologic inhibition of the Rac1 signaling pathways via DOCK2 may provide a novel and promising therapeutic target for FLT3/ITD AML. Disclosures Small: Pharos I, B &amp; T: Consultancy, Research Funding; InSilico Medicine: Membership on an entity's Board of Directors or advisory committees. Duffield:Boston Biomedical/Sumitomo Dainippon Pharma Co., Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; MedImmune: Consultancy.

Biallelic PTCH1 Inactivation Is a Dominant Genomic Change in Sporadic Keratocystic Odontogenic Tumors.

Keratocystic odontogenic tumors (KCOTs) are locally aggressive odontogenic neoplasms with recurrence rates of up to 60%. Approximately 5% of KCOTs are associated with nevoid basal cell carcinoma (Gorlin) syndrome and 90% of these show genomic inactivation of the PTCH1 gene encoding Patched 1. Sporadic KCOTs reportedly have PTCH1 mutations in 30% of cases, but previous genomic analyses have been limited by low tumor DNA yield. The aim of this study was to identify recurrent genomic aberrations in sporadic KCOTs using a next-generation sequencing panel with complete exonic coverage of sonic hedgehog (SHH) pathway members PTCH1, SMO, SUFU, GLI1, and GLI2. Included were 44 sporadic KCOTs from 23 female and 21 male patients with a median age of 50 years (range, 10 to 82 y) and located in the mandible (N=33) or maxilla (N=11). Sequencing identified PTCH1 inactivating mutations in 41/44 (93%) cases, with biallelic inactivation in 35 (80%) cases; 9q copy neutral loss of heterozygosity targeting the PTCH1 locus was identified in 15 (34%) cases. No genomic aberrations were identified in other sequenced SHH pathway members. In summary, we demonstrate PTCH1 inactivating mutations in 93% of sporadic KCOTs, indicating that SHH pathway alterations are a near-universal event in these benign but locally aggressive neoplasms. The high frequency of complete PTCH1 loss of function may provide a rational target for SHH pathway inhibitors to be explored in future studies.

1167P - Classification of esthesioneuroblastoma (ENB) based on chromosome (chr) arm gain and loss (CNA) in the setting of a hypomutated genomic landscape

BackgroundENB is a rare skull base tumor arising from the olfactory neuroepithelium with variable prognosis and no consensus guidelines for treatment. Previous genomic studies (Classe 2018, Capper 2018) have reported scant genomic alterations (GA) in advanced and metastatic ENB (mENB), including mutation of IDH2. Here, we explore the cytogenetic landscape of in 94 cases of mENB as assessed by NGS-based comprehensive genomic profiling (CGP). MethodsTumour specimens from 94 patients with mENB were assayed using hybrid capture-based CGP to delineate all classes of GA as point mutations, insertions deletions (indels), rearrangements and focal (<20MB) amplifications or loss. Each chr was assessed for arm wide copy gain, loss of heterozygosity (LOH1, only one allele remaining) and copy neutral loss of heterozygosity (LOHx, 2+ identical alleles). % score by arm was utilized to generate two principal components and analysis was performed using distance based hierarchical clustering. ResultsmENB cases had 1 or fewer GA detected in 80% (75/94) of cases with a corresponding median tumor mutational burden of 1.5 mut/mb. TP53 was the most frequently altered gene in the cohort, while IDH2 mutations were found in only 2 cases (2.1%). Focal and homozygous deletions were identified in 11.7% (11/94) and 14.9% (14/94) of cases respectively. We then assessed chr arm gain and LOH1/X and identified a median of 11 events per case. Based on chr arm level changes, we identified 3 distinct subtypes of mENB: type IA (n=53), defined by wide spread LOHx, type IB (n=22), defined by wide spread LOH1, and type II (n=19) generally lacking arm CNA. Type IB lacked chr5 or chr20 gain, which were both seen in 36% of type IA (p=0.001). Patients with IA/B types were older than type II (mean 54.9 v 46.5 years, p=0.015) and both IDH2 mutated cases segregated into type II. ConclusionsGiven the pauci-mutational genomic landscape of mENB, clustering by chr arm level changes identifies distinct classes of mENB, which were associated with biologic factors including age and IDH2 mutation. Further studies to correlate clinico-pathologic characteristics with the cytogenetic characteristics of these newly defined subtypes of mENB are needed. Legal entity responsible for the studyFoundation Medicine Inc. FundingFoundation Medicine Inc. DisclosureR. Madison: Full / Part-time employment: Foundation Medicine Inc; Shareholder / Stockholder / Stock options: Roche. D.C. Pavlick: Full / Part-time employment: Foundation Medicine Inc; Shareholder / Stockholder / Stock options: Roche. S. Khan: Honoraria (self): Ariad; Honoraria (self): Genentech; Honoraria (self): Foundation Medicine; Honoraria (self): EMD Serono; Honoraria (self): Genzyme; Honoraria (self): Guardant; Honoraria (self): Takeda; Honoraria (self): Oak Ridge Universities; Honoraria (self): Onyx Pharmaceuticals. J. Lee: Full / Part-time employment: Foundation Medicine Inc; Shareholder / Stockholder / Stock options: Roche. J.S. Ross: Leadership role, Full / Part-time employment: Foundation Medicine Inc; Shareholder / Stockholder / Stock options: Roche; Officer / Board of Directors: Celcius Therapeutics. V.A. Miller: Leadership role, Full / Part-time employment: Foundation Medicine Inc; Shareholder / Stockholder / Stock options: Roche; Advisory / Consultancy, Scientific Advisory Board: Revolution Medicines. B.M. Alexander: Full / Part-time employment, Officer / Board of Directors: Foundation Medicine Inc; Shareholder / Stockholder / Stock options: Roche; Advisory / Consultancy, Personal Fees: AbbVie; Advisory / Consultancy, Personal Fees: Schlesinger Associates; Advisory / Consultancy, Personal Fees: Bristol-Myers Squibb; Advisory / Consultancy, Personal Fees: Precision Health Economics; Research grant / Funding (self), grants outside submitted work: Puma Biotechnology; Research grant / Funding (self), grants outside submitted work: Celgene; Research grant / Funding (self), grants outside submitted work: Eli Lily. J. Chung: Full / Part-time employment: Foundation Medicine Inc; Shareholder / Stockholder / Stock options: Roche. All other authors have declared no conflicts of interest. A.B. Schrock: Full / Part-time employment: Foundation Medicine Inc; Shareholder / Stockholder / Stock options: Roche. S.M. Ali: Full / Part-time employment: Foundation Medicine Inc; Shareholder / Stockholder / Stock options: Roche.

Technical laboratory standards for interpretation and reporting of acquired copy-number abnormalities and copy-neutral loss of heterozygosity in neoplastic disorders: a joint consensus recommendation from the American College of Medical Genetics and Genomics (ACMG) and the Cancer Genomics Consortium (CGC)

The detection of acquired copy-number abnormalities (CNAs) and copy-neutral loss of heterozygosity (CN-LOH) in neoplastic disorders by chromosomal microarray analysis (CMA) has significantly increased over the past few years with respect to both the number of laboratories utilizing this technology and the broader number of tumor types being assayed. This highlights the importance of standardizing the interpretation and reporting of acquired variants among laboratories. To address this need, a clinical laboratory-focused workgroup was established to draft recommendations for the interpretation and reporting of acquired CNAs and CN-LOH in neoplastic disorders. This project is a collaboration between the American College of Medical Genetics and Genomics (ACMG) and the Cancer Genomics Consortium (CGC). The recommendations put forth by the workgroup are based on literature review, empirical data, and expert consensus of the workgroup members. A four-tier evidence-based categorization system for acquired CNAs and CN-LOH was developed, which is based on the level of available evidence regarding their diagnostic, prognostic, and therapeutic relevance: tier 1, variants with strong clinical significance; tier 2, variants with some clinical significance; tier 3, clonal variants with no documented neoplastic disease association; and tier 4, benign or likely benign variants. These recommendations also provide a list of standardized definitions of terms used in the reporting of CMA findings, as well as a framework for the clinical reporting of acquired CNAs and CN-LOH, and recommendations for how to deal with suspected clinically significant germline variants.

IMPLICATIONS OF REVERTANT SOMATIC MOSAICISM IN BONE MARROW FAILURE SYNDROMES

Revertant somatic mosaicism (RM) has been reported in genetic diseases including skin disorders and bone marrow failure (BMF) syndromes. We present examples of RM of haematopoietic cells in the context of inherited BMF syndromes with dramatically different clinical outcomes. Diamond Blackfan Anaemia (DBA) is an inherited BMF disorder caused by mutations/deletions in ribosomal proteins. We have a 9 year old male who required regular transfusions until he became transfusion independent at age 4. We identified a 184kb deletion on chromosome 12 encompassing 11 genes including RPS26 (known DBA gene). Significantly, we observed multiple independent copy neutral loss of heterozygosity (CNLOH) events on chromosome 12, correcting the 184kb deletion in a subset of blood cells, but not in hair. We propose RM as a mechanism for correction of the germline deletion resulting in spontaneous remission in the patient. We also recently identified germline mutations in SAMD9L, in a family with thrombocytopaenia, macrocytosis and ALL. In this family, we see examples for somatic mosaicism leading to a severe blood phenotype as well as CNLOH RM causing milder phenotypes in different family members. RM needs to be considered when selecting tissue source for diagnosis in carriers with mild phenotype/remission, and use of patient-derived cell lines for drug screens with multiple rounds of cell replication. We are currently trying to understand and manipulate revertant clone selection and hope this will open windows for rational correction and selection protocols for effective therapeutic intervention. Experimental manipulations to select clones that improve clinical phenotype have substantive implications on efficiencies of gene manipulations, manufacture and quality control requirements of autologous cellular therapies.

The SRCIN1/p140Cap adaptor protein negatively regulates the aggressiveness of neuroblastoma

Neuroblastoma is the most common extra-cranial pediatric solid tumor, responsible for 13–15% of pediatric cancer death. Its intrinsic heterogeneity makes it difficult to target for successful therapy. The adaptor protein p140Cap/SRCIN1 negatively regulates tumor cell features and limits breast cancer progression. This study wish to assess if p140Cap is a key biological determinant of neuroblastoma outcome. RNAseq profiles of a large cohort of neuroblastoma patients show that SRCIN1 mRNA levels are an independent risk factor inversely correlated to disease aggressiveness. In high-risk patients, CGH+SNP microarray analysis of primary neuroblastoma identifies SRCIN1 as frequently altered by hemizygous deletion, copy-neutral loss of heterozygosity, or disruption. Functional experiments show that p140Cap negatively regulates Src and STAT3 signaling, affects anchorage-independent growth and migration, in vivo tumor growth and spontaneous lung metastasis formation. p140Cap also increases sensitivity of neuroblastoma cells to doxorubicin and etoposide treatment, as well as to a combined treatment with chemotherapy drugs and Src inhibitors. Our functional findings point to a causal role of p140Cap in curbing the aggressiveness of neuroblastoma, due to its ability to impinge on specific molecular pathways, and to sensitize cells to therapeutic treatment. This study provides the first evidence that the SRCIN1/p140Cap adaptor protein is a key player in neuroblastoma as a new independent prognostic marker for patient outcome and treatment. Altogether, these data highlight the potential clinical impact of SRCIN1/p140Cap expression in neuroblastoma tumors, in terms of reducing cytotoxic effects of chemotherapy, one of the main issues for pediatric tumor treatment.

Abstract LB-210: An enhanced tumor evolution model for colorectal cancer evolution

Abstract The classical genetic model of colorectal cancer presents somatic APC mutations as the earliest genomic alterations, followed by KRAS and TP53 mutations. However, the timing and relative order of clonal expansion and other types of somatic genome alterations such as genomic rearrangements are still unclear. Here, we performed detailed analysis of somatic genetic alterations, including point mutations, copy number alterations and genomic rearrangements, in 63 whole-genome sequenced colorectal cancers from the Cancer Genome Atlas Research Network. The relative order of these alterations occurring during tumorigenesis was inferred from variant allele fractions. We found that driver point mutations, gene fusions, and arm level copy losses typically arise early. Copy-neutral loss of heterozygosity is often a two-step event: a deletion followed by a duplication. Different mechanisms act in different genomic regions to optimize the DNA dosage. Chromothripsis, clustered genomic rearrangements previously thought to occur as a single catastrophic event, is frequent and may occur multiple times independently in the same tumor through different mechanisms. In contrast to recent studies reporting neutral growth of tumors, selection is often present on subclones. Our results suggest that different evolutionary models can operate in a single tumor at different stages. Combining these results, we present a refined tumor progression model for human colorectal cancer. Our enhanced genetic model significantly expands our understanding of tumorigenesis process. Citation Format: Lixing Yang. An enhanced tumor evolution model for colorectal cancer evolution [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 LB-210.

Integrated Analysis of TP53 Gene and Pathway Alterations in The Cancer Genome Atlas

The TP53 tumor suppressor gene is frequently mutated in human cancers. An analysis of five data platforms in 10,225 patient samples from 32 cancers reported by The Cancer Genome Atlas (TCGA) enables comprehensive assessment of p53 pathway involvement in these cancers. More than 91% of TP53-mutant cancers exhibit second allele loss by mutation, chromosomal deletion, or copy-neutral loss of heterozygosity. TP53 mutations are associated with enhanced chromosomal instability, including increased amplification of oncogenes and deep deletion of tumor suppressor genes. Tumors with TP53 mutations differ from their non-mutated counterparts in RNA, miRNA, and protein expression patterns, with mutant TP53 tumors displaying enhanced expression of cell cycle progression genes and proteins. A mutant TP53 RNA expression signature shows significant correlation with reduced survival in 11 cancer types. Thus, TP53 mutation has profound effects on tumor cell genomic structure, expression, and clinical outlook.

PF528 INTEGRATING DETECTION OF COPY NEUTRAL CHROMOSOMAL LOSSES IN A CLINICAL SETTING IN LEUKEMIA AND LYMPHOMA BY MEANS OF ALLELIC IMBALANCE AND READ DEPTH RATIO COMPARISON

Background:Chromosomal instability, leading to full or partial aneuploidy, is a common feature of solid tumors and hematological malignancies, with several recurrent aberrations recognized as important diagnostic or prognostic molecular markers. While genome‐wide genotyping and genomic hybridization microarray analyses have been performed in clinical laboratories for several years the usage of next generation sequencing for detection of acquired copy number alterations (CNA) has not yet reached full clinical integration. Algorithmic strategies based on whole exome sequencing (WES) have been proposed, primarily focusing on read depth alterations affected by copy gain or loss.Aims:We aimed to identify copy‐neutral loss of heterozygosity (CN‐LoH), which is not detectable by sequencing read depth correlation or the analogous microarray CGH, by means of simple statistical analyses for increased transparency and direct clinical implementation.Methods:23 paired samples from different hematological disorders were selected from 14 individuals with MCL, CLL, CML, AML, T‐ALL and monoclonal B cell lymphocytosis. Detection of CN‐LoH by means of significant allele frequency (AF) shift using Fisher's exact test and χ2 was performed in combination with paired gene‐wise read depth ratios correlations in order to resolve both copy altering and neutral chromosomal aberrations. Alignment (GRCh37) and variant calling was performed using BWA (Li and Durbin 2009) and GATK (McKenna et al. 2010) (v. 3.6/3.8). Variants and reads located outside targeted regions were excluded, and a base quality threshold of 25 and minimum allele read depth of 20 was applied. Gene‐wise read depths were retrieved with BEDTools (Quinlan and Hall 2010) from RefSeq coordinates (UCSC, Karolchik et al. 2004). Statistics and plotting were performed in Mathematica and software developed for demonstration purposes (http://doi.org/10.7910/DVN/KFMGNY, Harvard Dataverse).Results:Sequencing yield spanned 48.5–188.6 million (median 84.3x106) paired‐end reads. The 23 paired variant call sets comprised a median of 19,101 coding variants, matching the expected number (Frebourg 2014; Ng et al. 2009), with a median read depth of 96 and lower and upper quartiles at 71 and 136, respectively. The combination of significant AF shifts identified 69 altered chromosomes and offered mutual confirmation. Six evident CN‐LoHs (&gt;1% AF shifts, p &lt; 0.01) were found in MCL, CML, AML and T‐ALL with one additional suspected low frequency deletion (estimated 10–15% burden) in a case of CLL. Robust test of equal variances (Brown and Forsythe 1974) between the paired diagnosis and remission sample was needed to detect this low burden aberration (p &lt; 10–10 in contrast to the other paired autosomes (pmedian = 0.019)). This also clearly indicates that low burden CN‐LoH pose a challenge – as with other methods. Another patient (MCL) harbored both a balanced tetrasomy (not evident by AF alone, see figure) and a CN‐LoH on chromosome 22 (not evident by DP‐ratios alone).Summary/Conclusion:The samples from 6 of the 14 patients with hematological malignancies/disorders were found to harbor 7 copy neutral deletions in total – all escaping previous clinical cytogenetic analyses. One was detected in a diagnostic sample from a patient otherwise diagnosed as cytogenically normal AML, which emphasize the importance of such methods. We conclude that this low‐complexity method, juxtaposing DP and AF, is directly applicable for the detection of copy neutral chromosomal loss of single genes from WES with intermediate coverage and medium to high burden chromosomal aberrations.image

PS921 INACTIVATION OF SH2B3 THROUGH CN‐LOH 12Q IS UNIQUELY ASSOCIATED WITH B‐CELL PRECURSOR ALL WITH IAMP21 OR OTHER CHROMOSOME 21 GAIN

Background:In more than 30% of childhood B‐cell precursor acute lymphoblastic leukaemia (B‐ALL), chromosome 21 sequence is overrepresented through constitutional or acquired aneuploidy or structural rearrangements, exemplified by intrachromosomal amplification of chromosome 21 (iAMP21). How these abnormalities promote B‐ALL remains obscure but a mechanistic link is suggested by the existence of shared co‐occurring abnormalities.Aims:We aimed to identify and characterise acquired genetic abnormalities that cooperate with amplification or aneuploidy of chromosome 21 to promote B‐ALL.Methods:Regions of copy neutral loss of heterozygosity and deletion were defined by analysis of SNP6.0 arrays, methylation arrays were used to assess loss of imprinting. The mutational status of SH2B3 was determined by amplicon sequencing and a homology model based on the resolved structure of the Sh2 domain of Sh2b1 was used to assess the impact of an acquired in‐frame substitution. STAT activation status was determined by flow cytometric immunophenotyping.Results:Analysis of B‐ALL patients with iAMP21, other structural amplifications of chromosome 21 or constitutional or acquired additional whole copies of chromosome 21 (n = 99), revealed recurrent CN‐LOH of 6p, 9p and 12q. In patients without overrepresentation of chromosome 21, CN‐LOH 6p and 9p were also recurrent but intriguingly, CN‐LOH 12q was absent, among our own (n = 203) or published cases (n = 974). Further analysis demonstrated CN‐LOH 12q was not associated with loss of imprinting but with homozygous mutations or deletions of the adaptor protein, SH2B3. In patients without CN‐LOH 12q, bi‐allelic abnormalities of SH2B3 occurred, but only in iAMP21‐ALL, giving an overall incidence of 18% in this sub‐type. Taking into account our own cohort, analysis of archival array data and review of published cases, we conclude that in B‐ALL, SH2B3 abnormalities are invariably bi‐allelic, frequent in iAMP21‐ALL, recurrent but less common in association with acquired aneuploidy 21 but extremely rare in the absence of overrepresentation of chromosome 21. Importantly, as iAMP21 patients are now uniformly treated as high risk, preliminary analysis of 26 patients treated on UKALL97/99 (n = 5) or UKALL2003 (n = 21), linked 12q abnormalities to poor outcome in iAMP21‐ALL (p = 0.03). Homology modelling of an iAMP21‐ALL associated R392W mutation in the SH2 domain of SH2B3 demonstrated loss of critical interactions required for binding and suppression of activated JAK. Further implicating the JAK/STAT pathway as one target for SH2B3 tumour suppressor activity, stimulation with IL7 of iAMP21‐ALL patient derived xenograft cells, with but not without SH2B3 inactivation, resulted in activation of STAT5 though not other STATs.Summary/Conclusion:Although chromosome 21 copy number profiles are highly heterogeneous in iAMP21‐ALL a limited number of candidate oncogenes are consistently amplified, overexpressed and spared from disruption by structural rearrangements. From our data we infer that in iAMP21‐ALL, high expression of one or more of these candidate genes creates strong pressure for loss of SH2B3 function while lower but still elevated expression of the same gene/s, resulting from gain of one or two copies of whole chromosome 21, produces a similar though weaker selective environment. Since JAK/STAT activation is common in B‐ALL without gain of chromosome 21 and given the near‐exclusive association we observed, we are currently exploring the possibility that SH2B3 also interacts with other pathways specifically activated by increased doses of chromosome 21 genes.

An integrative approach reveals genetic complexity and epigenetic perturbation in acute promyelocytic leukemia: a single institution experience

Acute promyelocytic leukemia (APL) is a distinct type of acute myeloid leukemia that is defined by the presence of the translocations that mostly involve the RARA gene. The most frequent translocation is the t(15;17), which fuses the RARA gene with the PML gene. Previous studies have shown that other cooperative mutations are required for the development of APL after the initiating event of the t(15;17). In this study, we combined cytogenetics with next-generation sequencing and single-nucleotide polymorphism array to study the genetic complexity in 20 APL cases diagnosed in our institution. All but 3 cases had additional genetic aberrations. Our study demonstrated that somatic mutations are frequent events in APL. In addition to the previously reported recurrent cooperative mutations in the FLT3, WT1, and RAS genes, we identified mutations in several epigenetic modifiers, including TET2, EZH2, and DNMT3A, co-occurring with either FLT3 or WT1 mutations. Mutations of the WT1 gene and chromosome 11p copy neutral loss of heterozygosity affecting WT1 are present in a third of the cases in our series. Two-thirds of APL cases in our study demonstrated a global reduction but focal accumulation of H3K27 methylase (H3K27me) expression, indicating a disorganized chromatin methylation pattern with generally more accessible chromatin status. Our study confirmed genetic complexity of APL and revealed that epigenetic aberrations are more common than previously expected. Although epigenetic modulation is not a common treatment strategy in APL, targeting this pathway may have some clinical utility in refractory or relapsed APL cases.

27. ACMG/CGC technical laboratory standards for interpretation and reporting of acquired copy number abnormalities (CNAs) and copy-neutral loss of heterozygosity (CN-LOH) in neoplastic disorders

With the expanded clinical use of Chromosome Microarray Analysis (CMA) to detect diagnostic, prognostic and therapeutic markers in cancer, there is a growing need for standardized, objective and evidence-based interpretation and reporting of CMA findings. Here we present the classification and reporting recommendations for acquired CNAs and CN-LOH regions in neoplastic disorders, created through collaboration between the American College of Medical Genetics and Genomics (ACMG) and the Cancer Genomics Consortium (CGC).

Pretransplant HLA typing revealed loss of heterozygosity in the major histocompatibility complex in a patient with acute myeloid leukemia

IntroductionChromosomal abnormalities are frequent events in hematological malignancies. The degree of HLA compatibility between donor and recipient in hematopoietic stem cell transplantation is critical. Purpose of the studyIn this report, we describe an acute myeloid leukemia case with loss of heterozygosity (LOH) encompassing the entire HLA. Materials and methodsHLA molecular typing was performed on peripheral blood (PB) and buccal swabs (BS). Chromosomal microarray analysis (CMA) was performed using a whole genome platform. ResultsTyping results on PB sample collected during blast crisis demonstrated homozygosity at the -A, -B, -C, -DR, and -DQ loci. A BS sample demonstrated heterozygosity at all loci. A subsequent PB sample drawn after count recovery confirmed heterozygosity. The CMA performed on PB samples collected during and after blast crisis revealed a large terminal region of copy-neutral LOH involving chromosome region 6p25.3p21.31, spanning approximately 35.9 Mb. The results of the CMA assay on sample collected after count recovery did not demonstrate LOH. ConclusionsLOH at the HLA gene locus may significantly influence the donor search resulting in mistakenly choosing homozygous donors. We recommend confirming the HLA typing of recipients with hematological malignancies when homozygosity is detected at any locus by using BS samples, or alternatively from PB when remission is achieved.

Pediatric acute myeloid leukemia with t(7;21)(p22;q22)

The t(7;21)(p22;q22) resulting in RUNX1-USP42 fusion, is a rare but recurrent cytogenetic abnormality associated with acute myeloid leukemia (AML) and myelodysplastic syndromes. The prognostic significance of this translocation has not been well established due to the limited number of patients. Herein, we report three pediatric AML patients with t(7;21)(p22;q22). All three patients presented with pancytopenia or leukopenia at diagnosis, accompanied by abnormal immunophenotypic expression of CD7 and CD56 on leukemic blasts. One patient had t(7;21)(p22;q22) as the sole abnormality, whereas the other two patients had additional numerical and structural aberrations including loss of 5q material. Fluorescence in situ hybridization analysis on interphase cells or sequential examination of metaphases showed the RUNX1 rearrangement and confirmed translocation 7;21. Genomic SNP microarray analysis, performed on DNA extracted from the bone marrow from the patient with isolated t(7;21)(p22;q22), showed a 32.2 Mb copy neutral loss of heterozygosity (cnLOH) within the short arm of chromosome 11. After 2-4 cycles of chemotherapy, all three patients underwent allogeneic hematopoietic stem cell transplantation (HSCT). One patient died due to complications related to viral reactivation and graft-versus-host disease. The other two patients achieved complete remission after HSCT. Our data displayed the accompanying cytogenetic abnormalities including del(5q) and cnLOH of 11p, the frequent pathological features shared with other reported cases, and clinical outcome in pediatric AML patients with t(7;21)(p22;q22). The heterogeneity in AML harboring similar cytogenetic alterations may be attributed to additional uncovered genetic lesions.