Copy-neutral Loss Of Heterozygosity Research Articles

Copy number alterations and copy-neutral loss of heterozygosity in Ukrainian patients with primary myelofibrosis

To examine frequencies and spectrum of genomic alterations in Ukrainian patients diagnosed with primary myelofibrosis(PMF). We enrolled 30 Ukrainian patients diagnosed with PMF who were previously tested for usual mutations in mye-loproliferative neoplasms driver genes (JAK2, MPL and CALR). Genomic DNA samples were obtained from peripheral blood leukocytes of these patients. Copy number alterations and copy-neutral loss of heterozygosity (cnLOH) were assessed using a high-density CytoScanHD microarray platform. Statistical significance was evaluated by the Fisher exact test. We identified frequent genomic alterations, but no significant difference in the rates of copy-number loss, copy-number gain, cnLOH, or multiple genomic alterations were found in the groups of PMF patients that were positive for one of the usual mutations in driver genes or negative for such mutations (33.3% and 55.6%, p = 0.4181, 19.0% and 11.1%, p = 1.0000, 61.9% and 44.4%, p = 0.4434, 33.3% and 55.6%, p=0.4181, respectively). The most frequent alterations were cnLOH at 1p36-1p22, 9p24.3-9p13.3 and 11q12.3-11q25; copy number loss at 7q21-7q36.3 and 13q12.3-13q14.3. Copy number alterations and cnLOH commonly affected the EZH2, LAMB4, CBL, CUX1, ATM, RB1 and TP53 genes, in addition to JAK2, MPL and CALR. We demonstrated the spectrum of genomic alterations in the groups of the Ukrainian PMF patients with or without the usual mutations in the specific driver genes. We identified several potential genes, which may be involved in the myeloproliferative neoplasms development and their phenotype modification (EZH2, LAMB4, CBL, CUX1, ATM, RB1 and TP53).

JAK2-mutated acute myeloid leukemia: comparison of next-generation sequencing (NGS) and single nucleotide polymorphism array (SNPa) findings between two cases

JAK2 mutations are rare in de novo acute myeloid leukemia (AML), and JAK2-mutated acute myeloid leukemia (AML) patients usually have a previous history of myeloproliferative neoplasms (MPNs). Current advances in laboratory techniques, such as single nucleotide polymorphism array (SNPa) and next-generation sequencing (NGS), have facilitated new insight into the molecular basis of hematologic diseases. Herein, we present two cases of JAK2-mutated AML in which both SNPa and NGS methods added valuable information. Both cases had leukemogenic collaboration, namely, copy-neutral loss of heterozygosity (CN-LOH), detected on chromosome 9. One of the cases exhibited both JAK2 and IDH2 mutations, most likely having originated as an MPN with leukemic transformation, while the other case was classified as a de novo AML with JAK2, CEBPA, and FLT3 mutations.

Shwachman-Diamond syndrome with clonal interstitial deletion of the long arm of chromosome 20 in bone marrow: haematological features, prognosis and genomic instability.

In Shwachman-Diamond syndrome (SDS), deletion of the long arm of chromosome 20, del(20)(q), often acquired in bone marrow (BM), may imply a lower risk of developing myelodysplastic syndrome/acute myeloid leukaemia (MDS/AML), due to the loss of the EIF6 gene. The genes L3MBTL1 and SGK2, also on chromosome 20, are in a cluster of imprinted genes, and their loss implies dysregulation of BM function. We report here the results of array comparative genomic hybridization (a-CGH) performed on BM DNA of six patients which confirmed the consistent loss of EIF6 gene. Interestingly, array single nucleotide polymorphisms (SNPs) showed copy neutral loss of heterozygosity for EIF6 region in cases without del(20)(q). No preferential parental origin of the deleted chromosome 20 was detected by microsatellite analysis in six SDS patients. Our patients showed a very mild haematological condition, and none evolved into BM aplasia or MDS/AML. We extend the benign prognostic significance of del(20)(q) and loss of EIF6 to the haematological features of these patients, consistently characterized by mild hypoplastic BM, no or mild neutropenia, anaemia and thrombocytopenia. Some odd results obtained in microsatellite and SNP-array analysis demonstrate a peculiar genomic instability, in an attempt to improve BM function through the acquisition of the del(20)(q).

PRR14L mutations are associated with chromosome 22 acquired uniparental disomy, age-related clonal hematopoiesis and myeloid neoplasia.

Acquired uniparental disomy (aUPD, also known as copy-neutral loss of heterozygosity) is a common feature of cancer cells and characterized by extended tracts of somatically-acquired homozygosity without any concurrent loss or gain of genetic material. The presumed genetic targets of many regions of aUPD remain unknown. Here we describe the association of chromosome 22 aUPD with mutations that delete the C-terminus of PRR14L in patients with chronic myelomonocytic leukemia (CMML), related myeloid neoplasms and age-related clonal hematopoiesis (ARCH). Myeloid panel analysis identified a median of 3 additional mutated genes (range 1-6) in cases with a myeloid neoplasm (n=8), but no additional mutations in cases with ARCH (n=2) suggesting that mutated PRR14L alone may be sufficient to drive clonality. PRR14L has very limited homology to other proteins and its function is unknown. ShRNA knockdown of PRR14L in human CD34+ cells followed by in vitro growth and differentiation assays showed an increase in monocytes and decrease in neutrophils consistent, with a CMML-like phenotype. RNA-Seq and cellular localization studies suggest a role for PRR14L in cell division. PRR14L is thus a novel, biallelically mutated gene and potential founding abnormality in myeloid neoplasms.

A unique subset of low-risk Wilms tumors is characterized by loss of function of TRIM28 (KAP1), a gene critical in early renal development: A Children's Oncology Group study.

This study explores the genomic alterations that contribute to the formation of a unique subset of low-risk, epithelial differentiated, favorable histology Wilms tumors (WT), tumors that have been characterized by their expression of post-induction renal developmental genes (Subset 1 WT). We demonstrate copy neutral loss of heterozygosity involving 19q13.32-q13.43, unaccompanied by evidence for imprinting by DNA methylation. We further identified loss-of-function somatic mutations in TRIM28 (also known as KAP1), located at 19q13, in 8/9 Subset 1 tumors analyzed. An additional germline TRIM28 mutation was identified in one patient. Retrospective evaluation of previously analyzed WT outside of Subset 1 identified an additional tumor with anaplasia and both TRIM28 and TP53 mutations. A major function of TRIM28 is the repression of endogenous retroviruses early in development. We depleted TRIM28 in HEK293 cells, which resulted in increased expression of endogenous retroviruses, a finding also demonstrated in TRIM28-mutant WT. TRIM28 has been shown by others to be active during early renal development, and to interact with WTX, another gene recurrently mutated in WT. Our findings suggest that inactivation of TRIM28 early in renal development contributes to the formation of this unique subset of FHWTs, although the precise manner in which TRIM28 impacts both normal renal development and oncogenesis remains elusive.

Genomic loss of HLA alleles may affect the clinical outcome in low-risk myelodysplastic syndrome patients

The Revised International Prognostic Score and some somatic mutations in myelodysplastic syndrome (MDS) are independently associated with transformation to acute myeloid leukemia (AML). Immunity has also been implicated in the pathogenesis of MDS, although the underlying mechanism remains unclear. We performed a SNP array on chromosome 6 in CD34+ purified blasts from 19 patients diagnosed with advanced MDS and 8 patients with other myeloid malignancies to evaluate the presence of loss of heterozygosity (LOH) in HLA and its impact on disease progression. Three patients had acquired copy-neutral LOH (CN-LOH) on 6p arms, which may disrupt antigen presentation and act as a mechanism for immune system evasion. Interestingly, these patients had previously been classified at low risk of AML progression, and the poor outcome cannot be explained by the acquisition of adverse mutations. LOH HLA was not detected in the remaining 24 patients, who all had adverse risk factors. In summary, the clinical outcome of patients with advanced MDS might be influenced by HLA allelic loss, wich allows subclonal expansions to evade cytotoxic-T and NK cell attack. CN-LOH HLA may therefore be a factor favoring MDS progression to AML independently of the somatic tumor mutation load.

Myeloid/Histiocytic Neoplasms Associated with Germ Cell Tumors Harbor Shared Genetic Abnormalities Indicating a Clonal Relationship, Unique Molecular Pathogenesis and Cellular Origin

IntroductionNeoplasms of different lineages that arise in the same patient and show a clonal relationship are exceedingly rare, but have been described in instances of histiocytic and myeloid/lymphoid neoplasms (Durham BH, et al. Blood 2017;130(2):176-80; Ansari J, et al. Eur J Haematol 2016;97(1):9-16). The clonal relationships can be demonstrated by shared genetic abnormalities such as translocations, somatic mutations, and other chromosomal level alterations. Nevertheless, a clonal link between germ cell tumors (GCT), a group of solid tumors derived from primitive stem cells, and hematologic malignancies has been less well-characterized. Relevant literature has mostly reported associations of GCT with acute leukemias, without a comprehensive assessment of genetic alterations (Mukherjee S et al. Ann Hematol 2017;96: 1435-39). To further characterize this phenomenon, we identified a small cohort of patients diagnosed with both GCT and any myeloid/histiocytic neoplasm. We evaluated their molecular and cytogenetic alterations, identifying shared and unique abnormalities, providing evidence of a clonal relationship between these two groups of neoplasms, which traditionally represent different cellular origins.MethodsA search of the pathology database at a major referral center (Memorial Sloan-Kettering Cancer Center) was performed to identify patients diagnosed with GCT between 2012-2018, and had at least 1 prior or subsequent bone marrow biopsy. The medical records were reviewed for details of clinical presentations and evidence of myeloid neoplasm, with corresponding morphologic, cytogenetic, and molecular findings. The findings were correlated with the genetic alterations detected in the GCT during diagnostic work-up. Cytogenetic analyses include karyotyping, fluorescence in situ hybridization (FISH) studies for common abnormalities among myeloid neoplasms, and single nucleotide polymorphism (SNP) array for copy number gain/loss and copy neutral-loss of heterozygosity (CN-LOH). Molecular analyses include an amplicon capture-based next generation sequencing (NGS) assay for 49 genes relevant for hematologic malignancies, and MSK-IMPACTTM, a hybrid capture-based NGS assay for mutation and copy number alteration in 400+ genes. Somatic nature of the identified variants was confirmed on MSK-IMPACTTM by germline variant filtering with the aid of appropriate normal control samples (blood or nail).Results8 patients with GCT diagnoses showed marrow findings consistent with or suspicious for involvement by myeloid/histiocytic neoplasms, with clinical presentations not typical for therapy-related myeloid neoplasms. The patients were predominantly male (n=6) and young (age range: 13-55, median=26.5). The GCT included mixed GCT (n=6), mature teratoma (n=1) and immature teratoma (n=1). The primary sites of involvement were mediastinum (n=4), testes (n=2), and ovaries (n=2). In all cases, the myeloid/histiocytic neoplasms and GCT were diagnosed in close proximity in time (range: <1 to 24 months). So far, cytogenetic and molecular data in 4 patients with mediastinal-based tumors showed shared alterations between the GCT and myeloid/histiocytic neoplasms, including isochromosome 12p in 3 patients, an alteration characteristic of GCT. Other shared alterations observed included identical mutations in TP53, PIK3CD, KRAS, BCOR; trisomy 1, 8, 21; gain of 21q; loss of 13q; and CN-LOH of 2q, 5q, 17p. Interestingly, in all 4 patients, the GCT and myeloid/histiocytic neoplasm each showed additional unique genetic alterations.ConclusionThese findings suggest that a subset of patients with GCT can develop clonally-related myeloid/histiocytic neoplasms, with a relatively short latency period. The genetic changes seen in our cases are relatively less common in conventional myeloid neoplasms, suggesting unique molecular pathogenesis. Yolk sac component in the GCT, which has been described to harbor hematopoietic precursor cells, may contribute to pathogenesis (Orazi A, et al Cancer 1993;71(12):3873-81). Although the detailed pathogenic mechanism remains uncertain, we observed that in the cohort, the GCT and myeloid/histiocytic neoplasm each harbored additional unique genetic changes. This suggests the presence of a common neoplastic progenitor giving rise to neoplasms of different lineages, which subsequently accumulated additional alterations. [Display omitted] DisclosuresHo:Invivoscribe, Inc.: Honoraria. Yabe:Y-mAbs Therapeutics: Consultancy. Arcila:Invivoscribe, Inc.: Consultancy, Honoraria.

Characterization of Novel Subtypes in B Progenitor Acute Lymphoblastic Leukemia

▪IntroductionB progenitor acute lymphoblastic leukemia (B-ALL) is a leading cause of childhood cancer death. Many chimeric genes have been identified and led to a refined classification of B-ALL and tailored therapies. Still, up to 30% of B-ALL cannot be classified into established subtypes, and the outcome for many is poor.MethodsTo identify novel subtypes of B-ALL, we performed integrative genomic analysis including transcriptome sequencing (RNA-seq) of 1,988 cases from St. Jude, Children's Oncology Group and adult cooperative group studies and analyzed chromosomal rearrangements, gene-expression profiles (GEP), somatic mutations and chromosome-level copy-number alterations. Cases lacking known or putative subtype-defining alterations underwent whole genome sequencing. Effects on proliferation and transformation of novel lesions were assessed by retroviral expression in cell lines and point-mutation knock-in mice using CRISPR/Cas9 genome editing.ResultsUsing integrated genetic alterations and gene expression profiling, we classified 23 B-ALL subtypes (Table and Figure). Three groups included cases with similar GEP as canonical subtypes (ETV6-RUNX1, KMT2A-rearranged, and ZNF384-rearranged), but lacking the expected drivers (e.g., ETV6-RUNX1-like, n=42). Eighteen cases (0.9%) had rearrangements of BCL2, MYC and/or BCL6 showing a distinct GEP; they were mostly adults (n=16) with very poor outcome. These alterations, rarely seen in ALL, are identical to those observed in “double/triple hit” lymphoma, and are of pre-B immunophenotype. Eight cases with tightly clustered GEP comprised a novel subtype defined by IKZF1 N159Y missense mutation. N159Y is in the DNA-binding domain of IKZF1, and is known to perturb IKZF1 function, with distinct nuclear mislocalization and induction of aberrant intercellular adhesion.We identified two subtypes with distinct GEP characterized by PAX5 alterations. One, herein termed PAX5 altered (PAX5alt), comprised 148 (7.4%) cases, was characterized by diverse PAX5 alterations including rearrangements (n=57), sequence mutations (n=46) and/or focal intragenic amplifications (n=8). These PAX5 alterations were found in 73.6% of PAX5alt cases and different alteration types were mutually exclusive. Other PAX5 alterations, including deletions and large-scale amplifications were also assessed using SNP array, but were not enriched in the PAX5alt group. Clinically, PAX5alt pediatric and adult patients had favorable (96.8±3.2%) and intermediate (42.1±10.2%) 5-year overall survival (OS), respectively. The other GEP distinct subtype comprised 44 cases, all with PAX5 P80R missense mutations. In 30 of these cases, PAX5 P80R was homozygous due to deletion of the wild-type (WT) PAX5 allele or copy-neutral loss of heterozygosity. Of the other 14 cases with heterozygous PAX5 P80R mutations, 13 had a second frameshift (n=7), nonsense (n=2) or deleterious missense (n=4) PAX5 mutation. Four of the remaining 1,944 cases also had the PAX5 P80R mutation, but all were heterozygous with preservation of a WT PAX5 allele, consistent with the notion that homozygous or compound heterozygous PAX5 P80R mutation is the hallmark of this subtype. Adult PAX5 P80R cases (n=14) showed better 5-year OS (61.9±13.4%) than those in PAX5alt subtype (42.1±10.2%). To examine the effects of PAX5 P80R on B-cell maturation, WT PAX5, PAX5 P80R, V26G and P34Q were expressed in Pax5-/- lineage-depleted bone marrow cells. Expression of WT PAX5, PAX5 V26G and P34Q resulted in near complete rescue of B cell differentiation; however, expression of PAX5 P80R blocked the differentiation at the pre-pro-B stage of B-cell maturation. Further, Pax5 P80R heterozygous or homozygous mice developed pre-B-ALL with a median latency of 166 and 87 days, respectively, with heterozygous mice acquiring alterations on the second allele. In contrast, Pax5+/- mice, and those harboring G183S mutation observed in familial leukemia, do not spontaneously develop B-ALL.ConclusionsThese results show the utility of transcriptome sequencing in defining subtypes and founding genetic alterations in B-ALL, provide a revised taxonomy of the disease across the age spectrum, and reinforce the central role of PAX5 as a checkpoint in B lymphoid maturation and leukemogenesis. DisclosuresMcKay:ImmunoGen Inc.: Employment. Tallman:Orsenix: Other: Advisory board; AROG: Research Funding; BioSight: Other: Advisory board; Cellerant: Research Funding; AbbVie: Research Funding; Daiichi-Sankyo: Other: Advisory board; ADC Therapeutics: Research Funding. Stock:Jazz Pharmaceuticals: Consultancy. Konopleva:Stemline Therapeutics: Research Funding. Relling:Shire Pharmaceuticals: Research Funding. Mullighan:Cancer Prevention and Research Institute of Texas: Consultancy; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding; Loxo Oncology: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau.

Molecular and pathologic characterization of AML with double Inv(3)(q21q26.2)

The inv(3)(q21q26.2) altering a single chromosome 3 homolog is an established myeloid malignancy-associated entity. Comparatively, double inv(3) cases involving both homologs are exceedingly rare with 13 reports across AML, CML and MDS. This scarcity was confirmed by finding only 2 new cases out of 34,898 bone marrows collected during a 55 year period at a large medical center (0.0005%). The double inv(3) was detected by karyotype and confirmed by FISH on both homologs in a 41 year old female and a 72 year old male with AML. In the latter case, a 2.26-fold increase in MECOM RNA level was found using an NGS myeloid gene panel. Chromosomal microarray analysis identified segmental copy-neutral loss-of-heterozygosity (CN-LOH) at 3q21 extending to near the q-arm terminus. This is the third report of distal 3q CN-LOH, substantiating that the double inv(3) arises through somatic repair of acquired segmental LOH. Long term clinical and genetic evaluation revealed no discernible morphologic difference between single and double inv(3) cases, conventional chemotherapy resistance and rapid dominance of the double inv(3) clone. The two new cases are consistent with relatively longer survival of double inv(3) patients in the absence of concurrent chromosome 7 loss compared to those with both abnormalities. Importantly, the first known outcome data of bone marrow transplantation in double inv(3) AML is also presented.

Detection and characterization of homozygosity of mutated CALR by copy neutral loss of heterozygosity in myeloproliferative neoplasms among cases with high CALR mutation loads or with progressive disease

Detection and characterization of homozygosity of mutated <i>CALR</i> by copy neutral loss of heterozygosity in myeloproliferative neoplasms among cases with high <i>CALR</i> mutation loads or with progressive disease

Characteristics of myeloproliferative neoplasms in patients exposed to ionizing radiation following the Chernobyl nuclear accident.

Myeloproliferative neoplasms (MPNs) driver mutations are usually found in JAK2, MPL, and CALR genes; however, 10%-15% of cases are triple negative (TN). A previous study showed lower rate of JAK2 V617F in primary myelofibrosis patients exposed to low doses of ionizing radiation (IR) from Chernobyl accident. To examine distinct driver mutations, we enrolled 281 Ukrainian IR-exposed and unexposed MPN patients. Genomic DNA was obtained from peripheral blood leukocytes. JAK2 V617F, MPL W515, types 1- and 2-like CALR mutations were identified by Sanger Sequencing and real time polymerase chain reaction. Chromosomal alterations were assessed by oligo-SNP microarray platform. Additional genetic variants were identified by whole exome and targeted sequencing. Statistical significance was evaluated by Fisher's exact test and Wilcoxon's rank sum test (R, version 3.4.2). IR-exposed MPN patients exhibited a different genetic profile vs unexposed: lower rate of JAK2 V617F (58.4% vs 75.4%, P = .0077), higher rate of type 1-like CALR mutation (12.2% vs 3.1%, P = .0056), higher rate of TN cases (27.8% vs 16.2%, P = .0366), higher rate of potentially pathogenic sequence variants (mean numbers: 4.8 vs 3.1, P = .0242). Furthermore, we identified several potential drivers specific to IR-exposed TN MPN patients: ATM p.S1691R with copy-neutral loss of heterozygosity at 11q; EZH2 p.D659G at 7q and SUZ12 p.V71 M at 17q with copy number loss. Thus, IR-exposed MPN patients represent a group with distinct genomic characteristics worthy of further study.

Next Generation Sequencing of Sporadic Vestibular Schwannoma: Necessity of Biallelic NF2 Inactivation and Implications of Accessory Non-NF2 Variants.

1) Describe the genetic alterations discovered in a series of sporadic vestibular schwannomas (VS). 2) Identify if more clinically aggressive variants possess different genetic alterations compared to more indolent-behaving VS. Fresh frozen tumor and matched peripheral blood leukocytes from 23 individuals with sporadic VS were analyzed using whole-exome sequencing, tumor whole transcriptome expression profiling (mRNA-Seq), and tumor mate-pair analysis. Source cases included tumors with fast preoperative growth, giant tumors in young patients, tumors with macrocystic change, recurrent tumors following radiation or microsurgery, and indolent small tumors with minimal or no growth before surgery. Somatic and germ-line alterations of the NF2 gene and beyond the NF2 locus were identified and analyzed using complementing analyses. Biallelic somatic events involving the NF2 gene were discovered in every analyzed tumor specimen with no concurrent NF2 variants identified in matching peripheral blood specimens. Thirteen tumors showed loss of one chromosome 22 (ch22), 4 tumors showed copy-neutral 22q loss of heterozygosity, and 31 unique small variants in the NF2 gene were discovered. Of the latter, 10 were essential splice site, 11 frame shift, 7 stop gain, 2 missense, and 1 in-frame mutation. No other common or recurring NF2 mutations were identified. However, several other notable large chromosomal aberrations were discovered including 2 tumors with loss of a chromosome 21, 3 with loss of an X or Y chromosome, 1 with copy-neutral loss of heterozygosity in chromosome 15, and 1 with loss of 18p and 16q. All of these other major chromosomal abnormalities only occurred in tumors demonstrating a more aggressive phenotype. To date, few studies have used whole-exome sequencing, mate-pair analysis, and RNA-seq to profile genome-wide alterations in sporadic VS. Using high-throughput deep sequencing, "two-hit" alterations in the NF2 gene were identified in every tumor and were not present in peripheral blood supporting that all events were somatic. Type of NF2 gene alteration and accessory mutations outside the NF2 locus may predict phenotypic expression and clinical course.

Abstract PR03: Genetic mechanisms of immune evasion in colorectal cancer

Abstract Background: Immune checkpoint blockade has shown activity in microsatellite-instability high (MSI-H) colorectal cancers (CRCs). However, despite a very high mutational and neoantigen load among virtually all MSI-H tumors, the response rate is around 40-50%. In addition, for the majority of CRCs, which are microsatellite stable (MSS), immune checkpoint inhibitors have so far proven ineffective. Thus, to better understand the genetic drivers of immune evasion in CRC, we integrated next generation sequencing data from over 1200 tumors with transcriptional and immunohistochemical measures of immune infiltration. Methods: We molecularly characterized 1,211 colorectal cancers, including 592 tumors from The Cancer Genome Atlas with whole exome sequencing (WES) and whole transcriptome (RNAseq) data and 619 cancers from two prospective cohort studies with WES and immunohistochemical (IHC) annotations. To identify driver gene alterations and their selection pressures specific to MSI-high tumors, we developed a statistical method to identify significantly mutated microsatellite tracts and we further developed a method to identify copy neutral loss of heterozygosity (CN-LOH) events. We used an established immune-gene transcriptional signature as well as IHC stains against specific subsets of immune-infiltrating cells to identify genetic events associated with immune evasion. Results: We demonstrated that WNT-signaling and immune-related genes are significantly mutated in colorectal cancer. In MSI-H CRCs, we found biallelic antigen-presentation machinery (APM) mutations in the HLA Class I genes, B2M and TAP2, in addition to recurrent mutations in NLRC5 and RFX5, which downregulated HLA Class I expression. In all CRCs, we showed that WNT-signaling activity and APC-biallelic mutations are inversely associated with both transcriptional and IHC measures of T-cell infiltration. Specifically, nuclear CTNNB1 expression was inversely correlated with overall tumor-infiltrating lymphocytes (p = 0.027), CD8+ subset of T-cells (p = 0.0019) and CD45RO+ subset of T-cells (0.0080). Meanwhile, colorectal tumors with biallelic disruptive mutations in APC had a significantly decreased activated T-cell transcriptional signature (p = 4e-12) relative to samples with no disruptive mutations in APC. We further showed that in MSS tumors, AXIN2 (a key WNT-signaling effector) super-enhancer hypomethylation, independent of the APC mutation status, was further associated with decreased T-cell activity. Conclusions: In this largest CRC genomic analysis to date, we identify genetic events that are associated with immune evasion in this disease. Specifically, we find evidence of immuno-editing in MSI-H tumors through disruptive mutations in APM. In the MSS and MSI-H subtype of CRCs, we use transcriptional and immunohistochemical orthogonal analyses to demonstrate exclusion of an effective immune infiltrate in CRC through an active WNT-signaling pathway. Our results shed light on the underlying molecular mechanisms of immune exclusion in CRC and have direct implications for novel combination immunotherapy trials for patients with this disease. This abstract is also being presented as Poster B23. Citation Format: Marios Giannakis, Catherine Grasso, Daniel Wells, Tsuyoshi Hamada, Xinmeng Jasmine Mu, Michael Quist, Jonathan Nowak, Reiko Nishihara, Charles M. Connolly, Sachet Shukla, William M. Grady, David Wheeler, Catherine J. Wu, Jesse Zaretsky, Levi Garraway, Thomas Hudson, Charles Fuchs, Antoni Ribas, Riki Peters, Shuji Ogino. Genetic mechanisms of immune evasion in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr PR03.

Machine Learning for Better Prognostic Stratification and Driver Gene Identification Using Somatic Copy Number Variations in Anaplastic Oligodendroglioma.

1p/19q-codeleted anaplastic gliomas have variable clinical behavior. We have recently shown that the common 9p21.3 allelic loss is an independent prognostic factor in this tumor type. The aim of this study is to identify less frequent genomic copy number variations (CNVs) with clinical importance that may shed light on molecular oncogenesis of this tumor type. A cohort of 197 patients with anaplastic oligodendroglioma was collected as part of the French POLA network. Clinical, pathological, and molecular information was recorded. CNV analysis was performed using single-nucleotide polymorphism arrays. Computational biology and feature selection based on the random forests method were used to identify CNV events associated with overall survival and other clinical-pathological variables. Recurrent chromosomal events were identified in chromosomes 4, 9, and 11. Forty-six focal amplification events and 22 focal deletion events were identified. Twenty-four focal CNV areas were associated with survival, and five of them were significantly associated with survival after multivariable analysis. Nine out of 24 CNV events were validated using an external cohort of The Cancer Genome Atlas. Five of the validated events contain a cancer-related gene or microRNA: CDKN2A deletion, SS18L1 amplification, RHOA/MIR191 copy-neutral loss of heterozygosity, FGFR3 amplification, and ARNT amplification. The CNV profile contributes to better survival prediction compared with clinical-based risk assessment. Several recurrent CNV events, detected in anaplastic oligodendroglioma, enable better survival prediction. More importantly, they help in identifying potential genes for understanding oncogenesis and for personalized therapy. Genomic analysis of 197 anaplastic oligodendroglioma tumors reveals recurrent somatic copy number variation areas that may help in understanding oncogenesis and target identification for precision medicine. A machine learning multivariable model built using this genomic information enables better survival prediction.

Abstract B52: Copy-neutral loss of heterozygosity is decreased in African American colorectal cancers

Abstract Background: Recent reports have shown that chromosomal copy number gains and losses associated with the development of colorectal cancer (CRC) occur at similar frequencies in Whites and African Americans (AAs), but no studies have compared copy-neutral loss of heterozygosity (cnLOH) between these two populations. cnLOH can arise by at least two different mechanisms. One involves recombination between homologous chromosomes, but cnLOH most commonly arises from mis-segregation of chromosomes via a mitotic form of uniparental disomy. Preliminary analysis of copy number data from Chicago AA CRC cases by our lab suggested that cnLOH occurred less frequently in AA CRCs. The present study aimed to use publicly available data from The Cancer Genome Atlas (TCGA) to test whether cnLOH was less frequent in AA CRC cases in the TCGA. Methods: Raw Affymetrix GenomeWide SNP Array 6.0 files were obtained from TCGA Data Portal and processed using R package Rawcopy and PennCNV to determine segments of loss, gain, and cnLOH. Samples from AAs and Whites were processed separately, using normals from their respective populations as reference. cnLOH events involving less than 20 markers were removed, and the proportion of each chromosome arm affected by cnLOH was calculated for each tumor. Chromosome arms with over 50% cnLOH were counted as cnLOH events. Linear regression models were used to determine associations between the number of chromosome arm events and clinical variables. AA and White cases were identified per TCGA clinical data. Additionally, logistic regression models were used to determine associations between affected and unaffected tumors, defining affected as a tumor with &amp;gt; 2 cnLOH events. The value of 2 was chosen because it was the median value of cnLOH events per tumor among all samples. Chromosome arm-specific differences were tested by Fisher exact tests, using a Bonferroni correction to adjust for multiple testing. Results: Data from 56 AA and 210 White CRCs were analyzed. Race was associated with a decreased number of cnLOH events per tumor, with AA CRCs on average having significantly fewer chromosome arms affected than White CRCs (p = 0.01). This difference remained significant after correction for age at diagnosis, sex, stage, and BMI. Additionally, AA CRCs were half as likely to have developed more than two cnLOH events compared to White CRCs. In AA CRCs, the frequency of cnLOH events was lower on 32 of the 39 chromosome arms compared to frequency of cnLOH events in White CRCs. Chromosomes 17p and 5q were the arms most frequently subject to cnLOH, comprising 13% of all cnLOH events in White tumors and 15% of all cnLOH events in AA tumors. 19.6% and 17.9% of AA CRC cases had cnLOH events on 17p and 5p, respectively, whereas 24.8% and 24.3% of White CRC cases had events on 17p and 5p. These differences were not significant. cnLOH of chromosome 6p was nominally significantly different (3% of AA cases vs 17% of White cases, p = 0.009); however, that difference did not remain significant after adjustment for multiple testing. No other differences were significant. No differences were found in chromosome arm gains or losses. Conclusions: The results in our present study show that cnLOH affects AA CRCs less than White CRCs. cnLOH is not a rare event in CRC. Only 64 of 266 CRCs examined from TCGA (~24%) had zero chromosome arms affected. This mechanism of genomic instability is more frequent for certain chromosome arms, specifically chromosomes 5q and 17p, where the known tumor suppressors APC and TP53 are located. In AA CRCs, the majority of chromosome arms showed lower levels of cnLOH, suggesting that the difference in the frequency of cnLOH events is the result of a reduced susceptibility to cnLOH and not of the selective action of different cancer driver genes. Taken together, these results suggest that there is a racial difference in mechanisms that drive genomic instability as it affects and drives CRC. Citation Format: Gaius J. Augustus, Nathan A. Ellis. Copy-neutral loss of heterozygosity is decreased in African American colorectal cancers [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B52.

Abstract 3093: Genomic characteristics of myeloproliferative neoplasms in patients exposed to ionizing radiation following the Chernobyl nuclear accident

Abstract Philadelphia-chromosome negative chronic myeloproliferative neoplasms (MPNs) are a unique group of hematological malignancies including Polycytemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) characterized by impaired function and structure of bone marrow. MPN driver mutations are usually found in Janus Kinase 2 (JAK2), Thrombopoietin Receptor (MPL) and Calreticulin (CALR) genes, however, 10-15% of MPN cases are triple negative (TN) for these mutations. A previous study showed a lower rate of JAK2 V617F mutations in PMF patients exposed to sublethal doses of ionizing radiation (IR) from the Chernobyl accident in Ukraine. We hypothesized a lower rate of the usual driver mutations in IR-exposed MPN patients. To examine whether there are distinct driver mutations, 281 Ukrainian IR-exposed and unexposed MPN patients, were enrolled in the study. Their records were reviewed for classification by the WHO 2016 MPN criteria. Genomic DNA was obtained from the peripheral blood leukocytes of 281 MPN patients to identify JAK2 V617F, MPL W515, and type 1- and 2-like CALR mutations by allele-specific PCR, Sanger Sequencing and RT-PCR, respectively. Copy number alterations and copy-neutral loss of heterozygosity (cnLOH) were assessed in 30 PMF patients by high-density Affymetrix CytoScan HD oligo-SNP microarray platform. Whole exome sequencing was used to identify additional genetic variants in these 30 PMF patients. Statistical significance for categorical variables and continuous variables were evaluated by Fisher's exact test and Wilcoxon's rank sum test using Statistical Analysis R, version 3.4.2. Clinical features of exposed and unexposed MPN patients were similar. More PMF IR-exposed patients were transfusion dependent (32.4%) than PMF unexposed patients (14.1%) (p = 0.04). JAK2 V617F was detected in 58% of IR-exposed and in 74% of unexposed MPN patients (p = 0.007). JAK2 V617F was also less frequent in IR-exposed PV patients, but not statistically significant. Type 1-like CALR mutation was detected in 12% of exposed and 3% of unexposed patients (p = 0.033). Overall, exposed patients were TN in 28% of IR-patients versus 16% of unexposed patients (p = 0.027). Among other genetic variants, ATM S1691R mutation with cnLOH at 11q22.3 was identified in one TN IR-exposed PMF patient. Previously the mutation was reported, but not in MPN patients. Missense mutations in EZH2 at 7q36.1 and SUZ12 at 17q11.2 with copy number loss were also identified in TN IR-exposed PMF patients. Our results confirm a lower rate of JAK2 V617F and higher rate of TN cases among IR-exposed MPN patients versus unexposed. We also demonstrated a higher frequency of type 1-like CALR mutation in IR-exposed MPN patients and new potential MPN driver mutations. Thus, IR-exposed MPN patients represent a disease group with distinct genomic characteristics worthy of further study. Citation Format: Larysa Poluben, Maneka Puligandla, Donna Neuberg, Christine R. Bryke, Nancy Hsu, Sergiy Klymenko, Olga Mishcheniuk, Oleksandr Shumeiko, Steven Balk, Xin Yuan, Olga Voznesensky, German Pihan, Miriam Adam, Ernest Fraenkel, Paula G. Fraenkel. Genomic characteristics of myeloproliferative neoplasms in patients exposed to ionizing radiation following the Chernobyl nuclear accident [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3093.

Abstract 4358: Landscape of allelic imbalance in pan-cancer adjacent normal tissue: Insights into field cancerization and cancer pathogenesis

Abstract It has been observed that tumors and adjacent normal appearing tissues share molecular signatures. This phenomenon has been observed in tissues of the skin, colon, and lung among others, particularly for patterns of gene expression. A recent pan cancer study of gene expression in normal tissue adjacent to the tumor (NAT) has shown that NATs have unique expression profiles that are distinct from non-tumor-bearing tissues. In this work, we sought to further characterize the molecular profile of NAT samples across cancer types. We have previously applied our haplotype based statistical technique to survey allelic imbalance (AI) in putatively normal lung tissues adjacent to tumor. Using this method, we can detect changes indicative of chromosomal alteration (gain, loss and copy-neutral LOH) even when present at low mutant cell fractions (&amp;lt; 5%). We focused our analysis on a subset of TCGA patients with both a solid tumor and NAT sample (n = 1530). In addition, to serve as a control, we surveyed alterations in normal samples derived from blood (n = 7,796). We detected 218 (megabase scale) chromosomal alterations in 44 of the NAT samples. These somatic alterations were present at a higher rate in NAT (2.9%) compared to the control normal blood set (1.9%). NAT samples with detectable AI had an average of 5 AI events compared to an average of 2 events in blood samples. We did not detect AI in the blood samples from the 44 patients with AI in the NAT. The most common recurrent alterations in NAT were 8p23.1, 17q21.31 and 1q42.12-1q42.13; each found in 14% of NAT AI positive samples. Chromosomal alterations in NAT were present at different rates between cancer types. We detected AI in more than 10% of NATs from breast invasive carcinoma, sarcoma, and bladder urothelial carcinoma. Of the 218 AI events in NAT, 67% were detected in the matching tumor sample. Two cancers, kidney renal papillary cell carcinoma and thyroid carcinoma, had a positive correlation between presence of AI in NAT and AI burden in the matched tumor sample (P = 0.005 and 0.03 respectively). The most common mutations in the tumors of patients with chromosomal aberrations in NAT were TP53 and MUC16 found in 39% and 20%, of tumors respectively. Lung squamous carcinoma and ovarian serous cystadenocarcinoma patients with detectable AI in NAT had a worse survival rate (P = 0.0241 and 0.0439 respectively). Our survey of AI in NAT tissues provides further support for NAT tissues as a model for understanding cancer pathogenesis. Citation Format: Yasminka A. Jakubek, Francis A. San Lucas, Smruthy Sivakumar, Richard G. Fowler, Humam Kadara, Paul Scheet. Landscape of allelic imbalance in pan-cancer adjacent normal tissue: Insights into field cancerization and cancer pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4358.

PO-309 Identification of genomic aberrations associated with lymph node metastasis in diffuse-type gastric cancer

IntroductionDiffuse type gastric cancer (DGC) is a GC subtype with heterogeneous clinical outcomes. Lymph node metastasis of DGC heralds a dismal progression, which hampers curative treatment of patients. However, the genomic heterogeneity of DGC is still unknown.Material and methodsTo identify genomic variations associated with lymph node metastasis of DGC, we performed whole exome sequencing on the 23 cases of DGC and paired non-tumoral tissues, and compared the mutation profiles according to the presence (N3, n=13) or absence (N0, n=10) of regional lymph node metastasis.Results and discussionsOverall, we identified 185 recurrently mutated genes in DGC, which included novel mutation at CMTM2 as well as previously known mutations at CDH1, RHOA, and TP53. Noticeably, CMTM2 expression could predict prognostic outcomes of DGC but not intestinal type GC (IGC), indicating pivotal roles of CMTM2 in DGC progression. In addition, we identified recurrent loss of heterozygosity (LOH) of DNA copy numbers at the 3p12-pcen locus in DGC. In comparison of N0 and N3 tumours, N3 tumours exhibited more frequent DNA copy number aberrations including copy-neutral LOH and mutations of CpTpT trinucleotides than those of N0 tumours (p=0.2 × 10-3).ConclusionIn conclusion, DGCs have distinct profiles of somatic mutations and DNA copy numbers according to the status of lymph node metastasis, which might be helpful in delineating pathobiology of the DGC.

SH2B3 aberrations enriched in iAMP21 B lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) represents the most common childhood malignancy. Although survival for pediatric B-ALL has approached 90%, variability in outcome among and within cytogenetically defined subgroups persists. While G-banding and fluorescence in situ hybridization (FISH) have been used to characterize leukemic clones, there is added value of chromosomal microarray and next generation sequencing in screening genome-wide for copy number aberrations, copy neutral loss of heterozygosity and nucleotide variations. Evaluation of novel genetic aberrations can provide information about the biologic mechanisms of cytogenetically defined subgroups associated with poor prognosis, explain heterogeneity in patient outcome and identify novel targets for therapeutic intervention. The high risk B-ALL intrachromosomal amplification of chromosome 21, (iAMP21), subtype is characterized by amplification of a region of chromosome 21 that typically encompasses the RUNX1 gene and is associated with poor prognosis. Analysis of chromosomal microarray and FISH data revealed that deletions of SH2B3, encoding a negative regulator of multiple tyrosine kinase and cytokine signaling pathways, are enriched among leukemias harboring iAMP21. Enrichment of SH2B3 aberrations in the iAMP21 subtype may indicate that loss of SH2B3 contributes to disease progression and raises the possibility that these leukemias may be sensitive to tyrosine kinase inhibitors.

Genetic Mechanisms of Immune Evasion in Colorectal Cancer.

To understand the genetic drivers of immune recognition and evasion in colorectal cancer, we analyzed 1,211 colorectal cancer primary tumor samples, including 179 classified as microsatellite instability-high (MSI-high). This set includes The Cancer Genome Atlas colorectal cancer cohort of 592 samples, completed and analyzed here. MSI-high, a hypermutated, immunogenic subtype of colorectal cancer, had a high rate of significantly mutated genes in important immune-modulating pathways and in the antigen presentation machinery, including biallelic losses of B2M and HLA genes due to copy-number alterations and copy-neutral loss of heterozygosity. WNT/β-catenin signaling genes were significantly mutated in all colorectal cancer subtypes, and activated WNT/β-catenin signaling was correlated with the absence of T-cell infiltration. This large-scale genomic analysis of colorectal cancer demonstrates that MSI-high cases frequently undergo an immunoediting process that provides them with genetic events allowing immune escape despite high mutational load and frequent lymphocytic infiltration and, furthermore, that colorectal cancer tumors have genetic and methylation events associated with activated WNT signaling and T-cell exclusion.Significance: This multi-omic analysis of 1,211 colorectal cancer primary tumors reveals that it should be possible to better monitor resistance in the 15% of cases that respond to immune blockade therapy and also to use WNT signaling inhibitors to reverse immune exclusion in the 85% of cases that currently do not. Cancer Discov; 8(6); 730-49. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 663.