Copy Number Neutral Loss Of Heterozygosity Research Articles

Tumor Predisposing Post-Zygotic Chromosomal Alterations in Bladder Cancer-Insights from Histologically Normal Urothelium.

Bladder urothelial carcinoma (BLCA) is the 10th most common cancer with a low survival rate and strong male bias. We studied the field cancerization in BLCA using multi-sample- and multi-tissue-per-patient protocol for sensitive detection of autosomal post-zygotic chromosomal alterations and loss of chromosome Y (LOY). We analysed 277 samples of histologically normal urothelium, 145 tumors and 63 blood samples from 52 males and 15 females, using the in-house adapted Mosaic Chromosomal Alterations (MoChA) pipeline. This approach allows identification of the early aberrations in urothelium from BLCA patients. Overall, 45% of patients exhibited at least one alteration in at least one normal urothelium sample. Recurrence analysis resulted in 16 hotspots composed of either gains and copy number neutral loss of heterozygosity (CN-LOH) or deletions and CN-LOH, encompassing well-known and new BLCA cancer driver genes. Conservative assessment of LOY showed 29%, 27% and 18% of LOY-cells in tumors, blood and normal urothelium, respectively. We provide a proof of principle that our approach can characterize the earliest alterations preconditioning normal urothelium to BLCA development. Frequent LOY in blood and urothelium-derived tissues suggest its involvement in BLCA.

Immune pathophysiology of bone marrow failure

Bone marrow (BM) failure is a condition characterized by peripheral pancytopenia due to decreased BM function. It includes conditions such as acquired aplastic anemia (AA), myelodysplastic syndrome (MDS), and paroxysmal nocturnal hemoglobinuria (PNH). AA is characterized by pancytopenia and BM hypoplasia, and is primarily caused by an autoimmune mechanism involving cytotoxic T cells that damage hematopoietic stem cells (HSCs). Recent genomic research has revealed that patients with AA often exhibit clonal hematopoiesis by HSCs with genetic alterations, such as PIGA, DNMT3A, ASXL1, BCOR/BCORL1, copy-number neutral LOH of chromosome 6p (6pLOH), and HLA class I allele mutations. The genomic landscape of AA is distinct from MDS and age-related clonal hematopoiesis. Most notably, the presence of PNH-type cells and HLA class I allele-lacking cells indicates the presence of HSCs that have escaped from autoimmunity. We recently identified a common nonsense mutation at codon19 (c.19C>T, p.R7X) in exon1 (Exon1mut) of different HLA-A and HLA-B alleles, and HLA-DR loss of hematopoietic stem progenitor cells in AA patients carrying HLA-DR15. These results provide important clues for understanding the immune pathophysiology of BM failure.

Combining metaphase cytogenetics with single nucleotide polymorphism arrays can improve the diagnostic yield and identify prognosis more precisely in myelodysplastic syndromes

Background Myelodysplastic syndromes (MDS) encompass a group of heterogeneous haematopoietic stem cell malignancies characterised by ineffective haematopoiesis, cytological aberrations, and a propensity for progression to acute myeloid leukaemia. Diagnosis and disease prognostic stratification are much based on genomic abnormalities. The traditional metaphase cytogenetics analysis (MC) can detect about 40–60% aberrations. Single-nucleotide polymorphism arrays (SNP-A) karyotyping can detect copy number variations with a higher resolution and has a unique advantage in detection of copy number neutral loss of heterozygosity (CN-LOH). Combining these two methods may improve the diagnostic efficiency and accuracy for MDS. Methods We retrospectively analysed the data of 110 MDS patients diagnosed from January 2012 to December 2019 to compare the detection yield of chromosomal abnormalities by MC with by SNP-A, and the relationship between chromosomal abnormalities and prognosis. Results Our results showed that SNP-A improved the detection yield of chromosomal aberrations compared with MC (74.5 vs. 55.5%, p < .001). In addition, the positive yield could be further improved by combining MC with SNP-A to 77.3%, compared with MC alone. Univariate analysis showed that age >65 years, bone marrow blasts ≥5%, with acquired CN-LOH, new aberrations detected by SNP-A, TGA value > the median (81.435 Mb), higher risk by IPSS-R-MC, higher risk by IPSS-R-SNP-A all had poorer prognosis. More critically, multivariable analysis showed that age >65 years and higher risk by IPSS-R-SNP-A were independent predictors of inferior OS in MDS patients. Conclusion The combination of MC and SNP-A based karyotyping can further improve the diagnostic yield and provide more precise prognostic stratification in MDS patients. However, SNP-A may not completely replace MC because of its inability to detect balanced translocation and to detect different clones. From a practical point of view, we recommend the concurrent use of SNP-A and MC in the initial karyotypic evaluation for MDS patients on diagnosis and prognosis stratification. KEY MESSAGES SNP-A based karyotyping can further improve the MDS diagnostic yield and provide more precise prognostic stratification in MDS patients. Acquired CN-LOH is a characteristic chromosomal aberration of MDS, which should be integrated to the diagnostic project of MDS. The concurrent use of SNP-A and MC in the initial karyotypic evaluation for MDS patients can be recommended.

Allele-specific genomic data elucidate the role of somatic gain and copy-number neutral loss of heterozygosity in cancer.

SummaryPan-cancer studies sketched the genomic landscape of the tumor types spectrum. We delineated the purity- and ploidy-adjusted allele-specific profiles of 4,950 patients across 27 tumor types from the Cancer Genome Atlas (TCGA). Leveraging allele-specific data, we reclassified as loss of heterozygosity (LOH) 9% and 7% of apparent copy-number wild-type and gain calls, respectively, and overall observed more than 18 million allelic imbalance somatic events at the gene level. Reclassification of copy-number events revealed associations between driver mutations and LOH, pointing out the timings between the occurrence of point mutations and copy-number events. Integrating allele-specific genomics and matched transcriptomics, we observed that allele-specific gene status is relevant in the regulation of TP53 and its targets. Further, we disclosed the role of copy-neutral LOH in the impairment of tumor suppressor genes and in disease progression. Our results highlight the role of LOH in cancer and contribute to the understanding of tumor progression.

Oligosarcomas, IDH-mutant are distinct and aggressive

Oligodendrogliomas are defined at the molecular level by the presence of an IDH mutation and codeletion of chromosomal arms 1p and 19q. In the past, case reports and small studies described gliomas with sarcomatous features arising from oligodendrogliomas, so called oligosarcomas. Here, we report a series of 24 IDH-mutant oligosarcomas from 23 patients forming a distinct methylation class. The tumors were recurrences from prior oligodendrogliomas or developed de novo. Precursor tumors of 12 oligosarcomas were histologically and molecularly indistinguishable from conventional oligodendrogliomas. Oligosarcoma tumor cells were embedded in a dense network of reticulin fibers, frequently showing p53 accumulation, positivity for SMA and CALD1, loss of OLIG2 and gain of H3K27 trimethylation (H3K27me3) as compared to primary lesions. In 5 oligosarcomas no 1p/19q codeletion was detectable, although it was present in the primary lesions. Copy number neutral LOH was determined as underlying mechanism. Oligosarcomas harbored an increased chromosomal copy number variation load with frequent CDKN2A/B deletions. Proteomic profiling demonstrated oligosarcomas to be highly distinct from conventional CNS WHO grade 3 oligodendrogliomas with consistent evidence for a smooth muscle differentiation. Expression of several tumor suppressors was reduced with NF1 being lost frequently. In contrast, oncogenic YAP1 was aberrantly overexpressed in oligosarcomas. Panel sequencing revealed mutations in NF1 and TP53 along with IDH1/2 and TERT promoter mutations. Survival of patients was significantly poorer for oligosarcomas as first recurrence than for grade 3 oligodendrogliomas as first recurrence. These results establish oligosarcomas as a distinct group of IDH-mutant gliomas differing from conventional oligodendrogliomas on the histologic, epigenetic, proteomic, molecular and clinical level. The diagnosis can be based on the combined presence of (a) sarcomatous histology, (b) IDH-mutation and (c) TERT promoter mutation and/or 1p/19q codeletion, or, in unresolved cases, on its characteristic DNA methylation profile.

Detection of PKD1 and PKD2 Somatic Variants in Autosomal Dominant Polycystic Kidney Cyst Epithelial Cells by Whole-Genome Sequencing.

Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by the development of multiple cysts in the kidneys. It is often caused by pathogenic mutations in PKD1 and PKD2 genes that encode polycystin proteins. Although the molecular mechanisms for cystogenesis are not established, concurrent inactivating germline and somatic mutations in PKD1 and PKD2 have been previously observed in renal tubular epithelium (RTE). To further investigate the cellular recessive mechanism of cystogenesis in RTE, we conducted whole-genome DNA sequencing analysis to identify germline variants and somatic alterations in RTE of 90 unique kidney cysts obtained during nephrectomy from 24 unrelated participants. Kidney cysts were overall genomically stable, with low burdens of somatic short mutations or large-scale structural alterations. Pathogenic somatic "second hit" alterations disrupting PKD1 or PKD2 were identified in 93% of the cysts. Of these, 77% of cysts acquired short mutations in PKD1 or PKD2 ; specifically, 60% resulted in protein truncations (nonsense, frameshift, or splice site) and 17% caused non-truncating mutations (missense, in-frame insertions, or deletions). Another 18% of cysts acquired somatic chromosomal loss of heterozygosity (LOH) events encompassing PKD1 or PKD2 ranging from 2.6 to 81.3 Mb. 14% of these cysts harbored copy number neutral LOH events, while the other 3% had hemizygous chromosomal deletions. LOH events frequently occurred at chromosomal fragile sites, or in regions comprising chromosome microdeletion diseases/syndromes. Almost all somatic "second hit" alterations occurred at the same germline mutated PKD1/2 gene. These findings further support a cellular recessive mechanism for cystogenesis in ADPKD primarily caused by inactivating germline and somatic variants of PKD1 or PKD2 genes in kidney cyst epithelium.

Paroxysmal nocturnal hemoglobinuria caused by CN-LOH of constitutional PIGB mutation and 70-kbp microdeletion on 15q

AbstractParoxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell (HSC) disorder characterized by defective synthesis of the glycosylphosphatidylinositol (GPI) anchors as a result of somatic mutations in the X-linked PIGA gene. The disease is acquired. No constitutional PNH has been described. Here, we report familial PNH associated with unusual inflammatory symptoms. Genetic analysis revealed a germline heterozygous PIGB mutation on chromosome 15 without mutations in PIGA or any of the other genes involved in GPI biosynthesis. In vitro data confirmed that transfection of the mutant PIGB could not restore the surface expression of GPI-anchored proteins (APs) in PIGB-deficient Chinese hamster ovary cells. Homozygosity was caused by copy number–neutral loss of heterozygosity (CN-LOH) of the germline PIGB mutation, leading to deficient expression of GPI-APs in the affected blood cells of the index patient and her mother. The somatic event leading to homozygosity of the germline mutant PIGB gene involved a 70-kbp microdeletion of chromosome 15q containing the TM2D3 and TARSL2 genes, which was implicated in chromosome 15q mosaicism. Interestingly, we detected the deletion in both the patient and her mother. A sister of the mother, who carried the same germline PIGB mutation but without this microdeletion involving TM2D3 and TARSL2, did not have a PNH clone or CN-LOH. In conclusion, we describe PNH caused by CN-LOH of a germline heterozygous PIGB mutation in a patient and her mother and hypothesize that the 70-kbp microdeletion may have contributed to the PNH clone in both.

NF1 Tumor Suppressor Gene Inactivation in Juvenile Myelomonocytic Leukemia: New Genetic Evidence and Consequences for Diagnostic Work-up

Neurofibromatosis type 1 (NF-1) predisposes to juvenile myelomonocytic leukemia (JMML) via loss of function of the NF1 tumor suppressor gene and consecutive deregulation of Ras signal transduction. Affected individuals usually carry one defective NF1 allele in the germline; somatic inactivation of the second NF1 allele in hematopoietic cells is associated with transformation to leukemia. We previously demonstrated that a major mechanism for biallelic loss of NF1 function in patients with JMML/NF-1 is mitotic recombination leading to uniparental disomy (UPD) of the 17q chromosome arm (Flotho, 2007; Steinemann, 2010). Using contemporary resequencing and microarray technology, we have now revisited the genetics of NF1 inactivation in JMML. Specifically, we addressed two questions: 1) Are genetic findings in leukemic cells of JMML/NF-1 patients consistent with the clinical diagnosis and the two-hit concept? 2) Does the quintuple-negative (QN) group of JMML (patients without clinical evidence of NF-1 and negative for mutations in PTPN11, KRAS, NRAS, or CBL) contain unrecognized cases likely driven by NF1? We investigated 156 children with JMML registered in studies EWOG-MDS 98 or 2006 and tested for mutations in PTPN11, KRAS, NRAS, and CBL. Twenty-five children (16%) were clinically diagnosed as NF-1 based on &amp;gt;=6 café-au-lait spots (CALS) or family history plus CALS. Family history was positive in 9 JMML/NF-1 patients; &amp;gt;=6, 4, and 1 CALS were described in 23, 1, and 1 patients, respectively. The median age at diagnosis of JMML in the NF-1 group was 35.9 months (range, 4.2 to 71.4). Sixteen children (10%) were grouped as JMML-QN. Granulocyte DNA from bone marrow or peripheral blood collected at time of diagnosis was used for next-generation sequencing of the entire NF1 coding sequence (custom Ampliseq enrichment and Miseq, Illumina). Pathogenicity of NF1 variants was assessed according to ACMG criteria. Affymetrix Cytoscan HD array analysis was applied to detect segmental deletions or copy number-neutral loss of heterozygosity (LOH). Among 25 JMML/NF-1 cases, 8 exhibited an NF1 loss-of-function mutation at near-100% variant allelic frequency (VAF) in combination with UPD involving almost the entire 17q arm, suggesting single mitotic recombination as the leukemic driver. One case had an NF1 mutation at near-100% VAF and segmental 17q UPD, suggesting the unusual occurrence of double mitotic recombination. Nine cases carried two independent pathogenic NF1 mutations at near-50% VAF each; here, germline and somatic events could not be distinguished due to unavailability of non-hematopoietic or parental DNA. Four cases exhibited an NF1 microdeletion in combination with a pathogenic NF1 mutation at near-100% VAF; non-hematopoietic tissue available in 2 of these 4 cases failed to display the mutation, indicating the microdeletion as the constitutional event. A deleterious mutation at 71% VAF but no LOH was revealed in one sample. Only monoallelic evidence of NF1 deficiency was found in 2 cases. In the JMML-QN group, 9/16 cases had previously unrecognized NF1 alterations. Compound-heterozygous pathogenic NF1 mutations were found in 2 and homozygous pathogenic NF1 mutations combined with focal LOH in 3 patients, strongly suggesting NF1 as the JMML driver. In the absence of clinical NF-1 features, the findings in these 5 children may be explained by postzygotic NF1 mosaicism, onset of JMML before clinical manifestation of NF-1, or double somatic NF1 hits in the hematopoietic lineage. Four cases carried monoallelic pathogenic or likely pathogenic NF1 mutations with VAF at ~50% or less, providing inconclusive evidence of driver function as it is also possible that these are secondary hits in a myeloproliferative neoplasm (MPN) driven by an unidentified event. There was no genetic evidence of NF1 involvement in the remaining 7 JMML-QN cases. Several important conclusions can be drawn from our study: The clinical diagnosis is reliable in children with JMML/NF-1; we propose that it can be made on the basis of CALS and JMML alone as the patients are generally too young to display the conventional spectrum of NF-1 features. Children without features of NF-1 should only be assigned to JMML-QN after genetic work-up of NF1 because this will unmask involvement of NF1 in a significant number of cases. In suspected JMML-QN without identifiable NF1 lesion, other forms of MPN should be considered. Disclosures Locatelli: Jazz Pharmaceeutical: Speakers Bureau; Medac: Speakers Bureau; Miltenyi: Speakers Bureau; Bellicum Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Niemeyer:Celgene: Consultancy; Novartis: Consultancy.

Epigenetic Loss of the HLA-DR15 Expression on Hematopoietic Stem Progenitor Cells in Patients with Acquired Aplastic Anemia Characterized By Cyclosporine Dependency: A Novel Mechanism Underlying the Immune Escape of Hematopoietic Stem Progenitor Cells

[Background] HLA-DR15 (DR15) has been implicated in the susceptibility to immune-mediated bone marrow (BM) failure, such as acquired aplastic anemia (AA), wherein the hematopoietic function depends on cyclosporine (CsA), paroxysmal nocturnal hemoglobinuria (PNH) with BM failure, and low-risk myelodysplastic syndrome responsive to immunosuppressive therapy. However, how DR15 contributes to the development of such immune-mediated BM failure remains unclear. Although the copy-number neutral loss of heterozygosity in chromosome 6p (6pLOH) of hematopoietic stem progenitor cells (HSPCs) in AA patients sometimes involves the HLA-DRB1 region, the frequency of the resultant DR15 loss is low, suggesting little involvement of this DR allele in the escape of HSPCs from cytotoxic T-cell attack. Several studies have recently reported that acute myeloid leukemia cells that relapse after allogeneic hematopoietic stem cell transplantation often lack HLA class II expression through an epigenetic mechanism and thereby escape the graft-versus-leukemia effect. The epigenetic loss of HLA class II expression may also occur in HSPCs that survive the immune attack in AA patients in remission. [Objectives/Methods] To test this hypothesis, we determined the HLA-DR expression on HSPCs defined by lineage-CD45dimCD34+CD38+ cells as well as their subpopulations, including common myeloid progenitors (CMPs), megakaryocyte-erythroid progenitors (MEPs), and granulocyte-monocyte progenitors (GMPs), in the peripheral blood of 52 AA patients (34 with DR15 and 18 without DR15) and 20 healthy individuals using flow cytometry (FCM) with anti-pan-HLA-DR antibodies. All patients were in remission after ATG-based therapy (ATG+CsA±thrombopoietin receptor agonists [TPO-RA] or anabolic steroids [AS], n=19), CsA-based therapy (CsA±TPO-RA or AS, n=27), and others (n=6), and 18 required low-dose CsA to maintain remission. Eighteen (35%) had HLA-class I allele-lacking (HLA-class I[-]) leukocytes due to 6pLOH and/or allelic mutations while 33 (63%) had 0.003-83.8% (median 0.194%) GPI-anchored protein-deficient (GPI[-]) granulocytes. HLA-DR(-) HSPCs detected in some patients were sorted together with their HLA-DR(+) counterparts and subjected to incubation in the presence of interferon gamma (IFN-γ) to see whether or not the DR expression was restored; in addition, they were subjected to RNA sequencing to compare the gene expression profiles between DR(-) and DR(+) HSPCs. [Results] Five (9.6%) of the 52 AA patients had 28.6% to 42.3% (median 34.0%) DR(-) cell populations in HSPCs, which were not detected in either monocytes or B lymphocytes of the same patients or in HSPCs of any healthy individuals (Figure 1a). All 5 patients possessed either HLA-DRB1*15:01 (n=3), DRB1*15:02 (n=1), or DRB1*15:01/15:02 (n=1), with the other DRB1 alleles differing among individuals, and their hematopoietic function depended on CsA, except for 1 patient (Case 5) whose HSPCs consisted of 69% GPI(+) and 31% GPI(-) cells. Of particular interest, Case 5's DR(-) cells were detected in GPI(+) HSPCs but not in GPI(-) HSPCs (Figure 1b). None of the 5 patients possessed HLA-class I(-) leukocytes, which were detected in 18 (38%) of 47 patients not possessing DR(-) HSPCs. In contrast to the patients possessing DR(-) HSPCs, CsA dependency was only observed in 13 (28%) of the 47 AA patients without DR(-) HSPCs. Incubation of sorted DR(-) HSPCs in the presence of IFN-γ for 72 h resulted in full restoration of the DR expression in all HSPC subpopulations (Figure 2). A comparison of the transcriptome profile between DR(-) and DR(+) HSPCs revealed that the signature of differentially expressed genes was enriched in immune response-related genes. [Conclusions] HSPCs that lacked DR due to an epigenetic mechanism were frequently detected in AA patients with DR15 characterized by CsA dependency. Although the loss of expression occurred in both DR alleles, the fact that only DRB1*15:01 or 15:02 was an allele shared by the five patients indicates that the DR loss phenomenon targeted DR15. The DR15(-) HSPCs may escape from antigen-specific CD4+ T-cell attack, which cannot be completely abolished by CsA. As demonstrated by findings of Case 5 showing the presence of a DR(-) cell population only in GPI(+) HSPCs, the lack of GPI may be a mechanism underlying substitution for the DR15 loss. Disclosures Takamatsu: Ono pharmaceutical: Honoraria, Research Funding; SRL: Consultancy, Research Funding; Janssen Pharmaceutical: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Adaptive Biotechnologies: Honoraria. Ishiyama:Alexion: Research Funding; Novartis: Honoraria. Yamazaki:Kyowa Kirin: Honoraria, Research Funding; Novartis: Honoraria. Nakao:Alexion: Research Funding; Novartis: Honoraria; Kyowa Kirin: Honoraria; Symbio: Consultancy.

Towards Identifying the Target of Autoimmunity in Aplastic Anemia

Acquired aplastic anemia (AA) is an autoimmune bone marrow aplasia caused by T cell-mediated destruction of hematopoietic stem and progenitor cells. The antigenic target in AA remains unknown. Recently, we and others identified frequent somatic loss of function of several human leukocyte antigen (HLA) class I alleles in hematopoietic cells that survive the AA immune attack, suggesting these alleles ("risk alleles") present autoantigen in the affected patients. We hypothesize that risk alleles share structural features and peptide-binding characteristics that may inform our understanding of the immune mechanism of AA. To identify additional AA HLA class I risk alleles, we have partnered with two multi-institutional consortia, the North American Pediatric Aplastic Anemia Consortium (NAPAAC) and the Center for International Blood and Marrow Transplant Research (CIBMTR), to evaluate 507 AA patients for somatic HLA loss. Using a combination of targeted massively parallel sequencing of HLA class I genes and single nucleotide polymorphism array genotyping, we identified HLA loss in 19% of the NAPAAC cohort (30 of 156 unselected AA patients) and 13% of the CIBMTR cohort (46 of 351 patients selected to maximize discovery of less common risk alleles). Copy number-neutral loss of heterozygosity of chromosome arm 6p (6p CN-LOH) was the most common cause of HLA loss, occurring in 50 patients (10% of total cohort, 12% NAAPAC and 9% CIBMTR). Somatic mutations were present in 45 patients (9% of total cohort, 12% of NAPAAC and 7% CIBMTR). Patients with HLA loss had a median of 1 mutant clone per patient (range 1-7). Of the somatic mutations, 80% were predicted to disrupt expression of the affected allele (through loss of start, nonsense, or frameshift mutations). The other 20% were missense mutations affecting residues in the peptide binding groove (6 mutations) and the α3 domain (9 mutations) of the HLA class I protein. A total of 19 distinct AA HLA risk alleles were identified, of which 13 were newly identified in this study. Several risk alleles (HLA-B*14:02, HLA-B*40:02, HLA-A*02:01) were enriched in AA patients compared to ethnicity matched controls. Among the 507 patients in the study, at least one risk allele was present in 436/493 (88%) of AA patients and in 34/39 (87%) of the 6p CN-LOH events. Mutations clustered in several groups of alleles (supertypes) known to bind overlapping peptide repertoires, with alleles in B27 and B44 supertypes being most commonly affected. There were no mutations in A01, A01A03, A01A24, A24, B58, B62, or in any of the HLA-C alleles analyzed. Using several metrics to estimate the strength of the autoimmune selection on a given allele, including the frequency of somatic loss and similarity to other HLA risk alleles, we developed an AA HLA risk allele pathogenicity index ranging from very high (B*14:02 and B*40:02) and high pathogenicity (A*33:03, B*08:01, B*13:02, B*14:01, B*27:03, B*27:05, B*38:02, B*41:02 and B*49:01) to those unlikely to be pathogenic. Interestingly, even within high risk supertypes such as B44, there was a wide variation in predicted pathogenicity, ranging from high (for B*40:02 and B*41:02) to low pathogenicity (for B*44:02 and B*44:03), suggesting differences in autopeptide binding. Our study provides a comprehensive analysis of AA HLA risk alleles in a large diverse cohort of AA patients. Our results suggest that HLA risk alleles have shared autoantigen binding specificities that define their pathogenicity in AA, which can be used to identify candidate AA autoantigens. HLA risk allele pathogenicity may have future clinical utility as an adjunctive diagnostic test, as well as for prognostic assessment and haploidentical donor selection. Disclosures Lee: AstraZeneca: Research Funding; Kadmon: Research Funding; Takeda: Research Funding; Novartis: Research Funding; Amgen: Research Funding; Syndax: Research Funding; Pfizer: Consultancy, Research Funding; Incyte: Consultancy, Research Funding. Monos:Omixon: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties.

Assessment of structural chromosomal instability phenotypes as biomarkers of carboplatin response in triple negative breast cancer: the TNT trial

BackgroundIn the TNT trial of triple negative breast cancer (NCT00532727), germline BRCA1/2 mutations were present in 28% of carboplatin responders. We assessed quantitative measures of structural chromosomal instability (CIN) to identify a wider patient subgroup within TNT with preferential benefit from carboplatin over docetaxel.Patients and methodsCopy number aberrations (CNAs) were established from 135 formalin-fixed paraffin-embedded primary carcinomas using Illumina OmniExpress SNP-arrays. Seven published [allelic imbalanced CNA (AiCNA); allelic balanced CNA (AbCNA); copy number neutral loss of heterozygosity (CnLOH); number of telomeric allelic imbalances (NtAI); BRCA1-like status; percentage of genome altered (PGA); homologous recombination deficiency (HRD) scores] and two novel [Shannon diversity index (SI); high-level amplifications (HLAMP)] CIN-measurements were derived. HLAMP was defined based on the presence of at least one of the top 5% amplified cytobands located on 1q, 8q and 10p. Continuous CIN-measurements were divided into tertiles. All nine CIN-measurements were used to analyse objective response rate (ORR) and progression-free survival (PFS).ResultsPatients with tumours without HLAMP had a numerically higher ORR and significantly longer PFS in the carboplatin (C) than in the docetaxel (D) arm [56% (C) versus 29% (D), PHLAMP,quiet = 0.085; PFS 6.1 months (C) versus 4.1 months (D), Pinteraction/HLAMP = 0.047]. In the carboplatin arm, patients with tumours showing intermediate telomeric NtAI and AiCNA had higher ORR [54% (C) versus 20% (D), PNtAI,intermediate = 0.03; 62% (C) versus 33% (D), PAiCNA,intermediate = 0.076]. Patients with high AiCNA and PGA had shorter PFS in the carboplatin arm [3.4 months (high) versus 5.7 months (low/intermediate); and 3.8 months (high) versus 5.6 months (low/intermediate), respectively; Pinteraction/AiCNA = 0.027, Padj.interaction/AiCNA = 0.125 and Pinteraction/PGA = 0.053, Padj.interaction/PGA = 0.176], whilst no difference was observed in the docetaxel arm.ConclusionsPatients with tumours lacking HLAMP and demonstrating intermediate CIN-measurements formed a subgroup benefitting from carboplatin relative to docetaxel treatment within the TNT trial. This suggests a complex and paradoxical relationship between the extent of genomic instability in primary tumours and treatment response in the metastatic setting.

MLPA and DNA index improve the molecular diagnosis of childhood B-cell acute lymphoblastic leukemia

Aneuploidy occurs within a significant proportion of childhood B-cell acute lymphoblastic leukemia (B-ALL). Some copy number variations (CNV), associated with novel subtypes of childhood B-ALL, have prognostic significance. A total of 233 childhood B-ALL patients were enrolled into this study. Focal copy number alterations of ERG, IKZF1, PAX5, ETV6, RB1, BTG1, EBF1, CDKN2A/2B, and the Xp22.33/Yp11.31 region were assessed by Multiplex Ligation-dependent Probe Amplification (MLPA). The MLPA telomere kit was used to identify aneuploidy through detection of whole chromosome loss or gain. We carried out these procedures alongside measurement of DNA index in order to identify, aneuploidy status in our cohort. MLPA telomere data and DNA index correlated well with aneuploidy status at higher sensitivity than cytogenetic analysis. Three masked hypodiploid patients, undetected by cytogenetics, and their associated copy number neutral loss of heterozygosity (CN-LOH) were identified by STR and SNP arrays. Rearrangements of TCF3, located to 19p, were frequently associated with 19p deletions. Other genetic alterations including iAMP21, IKZF1 deletions, ERG deletions, PAX5AMP, which have clinical significance or are associated with novel subtypes of ALL, were identified. In conclusion, appropriate application of MLPA aids the identifications of CNV and aneuploidy in childhood B-ALL.

Resistance of KIR Ligand-Missing Leukocytes to NK Cells In Vivo in Patients with Acquired Aplastic Anemia.

The loss of killer cell Ig-like receptor ligands (KIR-Ls) due to the copy number-neutral loss of heterozygosity of chromosome 6p (6pLOH) in leukocytes of patients with acquired aplastic anemia (AA) may alter the susceptibility of the affected leukocytes to NK cell killing in vivo. We studied 408 AA patients, including 261 who were heterozygous for KIR-Ls, namely C1/C2 or Bw6/Bw4, for the presence of KIR-L-missing [KIR-L(-)] leukocytes. KIR-L(-) leukocytes were found in 14 (5.4%, C1 [n = 4], C2 [n = 3], and Bw4 [n = 7]) of the 261 patients, in whom corresponding KIR(+) licensed NK cells were detected. The incidence of 6pLOH in the 261 patients (18.0%) was comparable to that in 147 patients (13.6%) who were homozygous for KIR-L genes. The percentages of HLA-lacking granulocytes (0.8-50.3%, median 15.2%) in the total granulocytes of the patients with KIR-L(-) cells were significantly lower than those (1.2-99.4%, median 55.4%) in patients without KIR-L(-) cells. KIR2DS1 and KIR3DS1 were only possessed by three of the 14 patients, two of whom had C2/C2 leukocytes after losing C1 alleles. The expression of the KIR3DS1 ligand HLA-F was selectively lost on KIR-L(-) primitive hematopoietic stem cells derived from 6pLOH(+) induced pluripotent stem cells in one of the KIR3DS1(+) patients. These findings suggest that human NK cells are able to suppress the expansion of KIR-L(-) leukocytes but are unable to eliminate them partly due to the lack of activating KIRs on NK cells and the low HLA-F expression level on hematopoietic stem cells in AA patients.

Cryptic aberrations may allow more accurate prognostic classification of patients with myelodysplastic syndromes and clonal evolution.

The karyotype of bone-marrow cells at the time of diagnosis is one of the most important prognostic factors in patients with myelodysplastic syndromes (MDS). In some cases, the acquisition of additional genetic aberrations (clonal evolution [CE]) associated with clinical progression may occur during the disease. We analyzed a cohort of 469 MDS patients using a combination of molecular cytogenomic methods to identify cryptic aberrations and to assess their potential role in CE. We confirmed CE in 36 (8%) patients. The analysis of bone-marrow samples with a combination of cytogenomic methods at diagnosis and after CE identified 214 chromosomal aberrations. The early genetic changes in the diagnostic samples were frequently MDS specific (17 MDS-specific/57 early changes). Most progression-related aberrations identified after CE were not MDS specific (131 non-MDS-specific/155 progression-related changes). Copy number neutral loss of heterozygosity (CN-LOH) was detected in 19% of patients. MDS-specific CN-LOH (4q, 17p) was identified in three patients, and probably pathogenic homozygous mutations were found in TET2 (4q24) and TP53 (17p13.1) genes. We observed a statistically significant difference in overall survival (OS) between the groups of patients divided according to their diagnostic cytogenomic findings, with worse OS in the group with complex karyotypes (P = .021). A combination of cytogenomic methods allowed us to detect many cryptic genomic changes and identify genes and genomic regions that may represent therapeutic targets in patients with progressive MDS.

TP53 Status As Well As Cytogenetic Complexity Significantly Impact on Prognosis in Myelodysplastic Syndromes with Complex (≥3 anomalies) Aberrant Karyotypes

TP53 Status As Well As Cytogenetic Complexity Significantly Impact on Prognosis in Myelodysplastic Syndromes with Complex (≥3 anomalies) Aberrant Karyotypes

CLONAL HEMATOPOIESIS IN A CENTENARIAN COHORT

Mosaicism, the presence of two or more genotypically or karyotypically distinct populations of cells in a single individual, plays an important role in human disease. Mosaicism can result in mutations and/or chromosomal alterations such as loss, gain, or copy-number neutral loss of heterozygosity. Clonal mosaicism and its relationship to aging and cancer, has been previously studied, and earlier work suggests that clonal mosaicism tends to increase with age. The aim of our research is to use genotype data of centenarians to explore the relationship between extreme longevity and mosaic chromosomal alterations (mCAs). To this end, we analyzed genome-wide genotypes from blood-derived DNA of 338 individuals from the New England Centenarian Study. The participants in this dataset ranged from 45 to 112 years of age. For the detection of mCA events, we used MoChA (https://github.com/freeseek/mocha), a bcftools extension, that predicts mCAs based on B-allele frequency (BAF) and log2 intensity(R) ratio (LRR), and uses long-range phase information to increase sensitivity. Chromosomal alteration events, including whole chromosome events, were detected in 180 out of the 338 individuals. A total of 165 duplications, 97 deletions, and 9 copy-number neutral loss of heterozygosity were detected. Additionally, there were 42 events whose copy number state could not be determined with high confidence. 236 events out of the 313 were detected in individuals aged 100 and older. Our analysis of chromosomal alteration frequency by age indicates that, within centenarians, the proportion of individuals with mCAs significantly decreases with increased age (p < 0.05, correlation -0.73).

New Recurrent Structural Aberrations in the Genome of Chronic Lymphocytic Leukemia Based on Exome-Sequencing Data.

Chronic lymphocytic leukemia (CLL) is the most frequent lymphoproliferative syndrome in Western countries, and it is characterized by recurrent large genomic rearrangements. During the last decades, array techniques have expanded our knowledge about CLL’s karyotypic aberrations. The advent of large sequencing databases expanded our knowledge cancer genomics to an unprecedented resolution and enabled the detection of small-scale structural aberrations in the cancer genome. In this study, we have performed exome-sequencing-based copy number aberration (CNA) and loss of heterozygosity (LOH) analysis in order to detect new recurrent structural aberrations. We describe 54 recurrent focal CNAs enriched in cancer-related pathways, and their association with gene expression and clinical evolution. Furthermore, we discovered recurrent large copy number neutral LOH events affecting key driver genes, and we recapitulate most of the large CNAs that characterize the CLL genome. These results provide “proof-of-concept” evidence supporting the existence of new genes involved in the pathogenesis of CLL.

SH2B3 inactivation through CN-LOH 12q is uniquely associated with B-cell precursor ALL with iAMP21 or other chromosome 21 gain

In more than 30% of B-cell precursor acute lymphoblastic leukaemia (B-ALL), chromosome 21 sequence is overrepresented through aneuploidy or structural rearrangements, exemplified by intrachromosomal amplification of chromosome 21 (iAMP21). Although frequent, the mechanisms by which these abnormalities promote B-ALL remain obscure. Intriguingly, we found copy number neutral loss of heterozygosity (CN-LOH) of 12q was recurrent in iAMP21-ALL, but never observed in B-ALL without some form of chromosome 21 gain. As a consequence of CN-LOH 12q, mutations or deletions of the adaptor protein, SH2B3, were converted to homozygosity. 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. Review of published data confirmed a tight association between overrepresentation of chromosome 21 and both CN-LOH 12q and SH2B3 abnormalities in B-ALL. Despite relatively small patient numbers, preliminary analysis linked 12q abnormalities to poor outcome in iAMP21-ALL (p = 0.03). Homology modelling of a leukaemia-associated SH2 domain mutation and in vitro analysis of patient-derived xenograft cells implicated the JAK/STAT pathway as one likely target for SH2B3 tumour suppressor activity in iAMP21-ALL.

Non-leukemic pediatric mixed phenotype acute leukemia/lymphoma: Genomic characterization and clinical outcome in a prospective trial for pediatric lymphoblastic lymphoma.

Rare cases of hematological precursor neoplasms fulfill the diagnostic criteria of mixed phenotype acute leukemia (MPAL), characterized by expression patterns of at least two hematopoietic lineages, for which a highly aggressive behavior was reported. We present a series of 11 pediatric non-leukemic MPAL identified among 146 precursor lymphoblastic lymphomas included in the prospective trial Euro-LBL 02. Paraffin-embedded biopsies of 10 cases were suitable for molecular analyses using OncoScan assay (n = 7), fluorescence in situ hybridization (FISH; n = 7) or both (n = 5). Except for one case with biallelic KMT2A (MLL) breaks, all cases analyzed by FISH lacked the most common translocations defining molecular subsets of lymphoblastic leukemia/lymphomas. Two non-leukemic B-myeloid MPALs showed the typical genomic profile of hyperdiploid precursor B-cell lymphoblastic leukemia with gains of chromosomes 4, 6, 10, 14, 18, and 21. One B-T MPAL showed typical aberrations of T-cell lymphoblastic lymphoma, such as copy number neutral loss of heterozygosity (CNN-LOH) at 9p targeting a 9p21.3 deletion of CDKN2A and 11q12.2-qter affecting the ATM gene. ATM was also mutated in a T-myeloid MPAL case with additional loss at 7q21.2-q36.3 and mutation of NRAS, two alterations common in myeloid disorders. No recurrent regions of CNN-LOH were observed. The outcome under treatment was good with all patients being alive in first complete remission after treatment according to a protocol for precursor lymphoblastic lymphoma (follow-up 3-10 years, median: 4.9 years). In summary, the present series of non-leukemic MPALs widely lacked recurrently reported translocations in lymphoid/myeloid neoplasias and showed heterogeneous spectrum of chromosomal imbalances.

Acquired aplastic anemia

Acquired aplastic anemia (AA) is a hematopoietic disorder caused by an immunologic attack on hematopoietic stem cells (HSCs). The presence of cells with a paroxysmal nocturnal hemoglobinuria (PNH) phenotype or with copy-number neutral loss of heterozygosity of chromosome 6p (6pLOH) suggests an immune-mediated pathophysiology underlying AA. Recently, genomic studies have revealed clonal hematopoiesis by HSCs with altered genes. PIGA, DNMT3A, ASXL1, BCOR, 6pLOH, and HLA class I allele mutations are common in patients with AA. The genomic landscape of AA is distinct from that of the myelodysplastic syndrome or age-related clonal hematopoiesis. This suggests that escape from an autoimmune attack is strongly associated with clonal hematopoiesis in AA. Eltrombopag (EPAG), a thrombopoietin receptor agonist, has recently emerged as a novel therapeutic agent for AA. Further studies are needed to clarify whether EPAG induces clonal expansion of these clones.