Copy-neutral Loss Of Heterozygosity Research Articles

Comparison of the copy-neutral loss of heterozygosity identified from whole-exome sequencing data using three different tools.

Loss of heterozygosity (LOH) is a genomic aberration. In some cases, LOH can be generated without changing the copy number, which is called copy-neutral LOH (CN-LOH). CN-LOH frequently occurs in various human diseases, including cancer. However, the biological and clinical implications of CN-LOH for human diseases have not been well studied. In this study, we compared the performance of CN-LOH determination using three commonly used tools. For an objective comparison, we analyzed CN-LOH profiles from single-nucleotide polymorphism array data from 10 colon adenocarcinoma patients, which were used as the reference for comparison with the CN-LOHs obtained through whole-exome sequencing (WES) data of the same patients using three different analysis tools (FACETS, Nexus, and Sequenza). The majority of the CN-LOHs identified from the WES data were consistent with the reference data. However, some of the CN-LOHs identified from the WES data were not consistent between the three tools, and the consistency with the reference CN-LOH profile was also different. The Jaccard index of the CN-LOHs using FACETS (0.84 ± 0.29; mean value, 0.73) was significantly higher than that of Nexus (0.55 ± 0.29; mean value, 0.50; p = 0.02) or Sequenza (0 ± 0.41; mean value, 0.34; p = 0.04). FACETS showed the highest area under the curve value. Taken together, of the three CN-LOH analysis tools, FACETS showed the best performance in identifying CN-LOHs from The Cancer Genome Atlas colon adenocarcinoma WES data. Our results will be helpful in exploring the biological or clinical implications of CN-LOH for human diseases.

Single cell proteogenomic sequencing identifies a relapse-fated AML subclone carrying FLT3-ITD with CN-LOH at chr13q.

Internal tandem duplication of the Feline McDonough Sarcoma (FMS)‐like tyrosine kinase 3 (FLT3‐ITD) is one of the most clinically relevant mutations in acute myeloid leukemia (AML), with a high FLT3‐ITD allelic ratio (AR) (≥0.5) being strongly associated with poor prognosis. FLT3‐ITDs are heterogeneous, varying in size and location, with some patients having multiple FLT3‐ITDs. Bulk cell‐based approaches are limited in their ability to reveal the clonal structure in such cases. Using single‐cell proteogenomic sequencing (ScPGseq), we attempted to identify a relapse‐fated subclone in an AML case with mutations in WT1, NPM1, and FLT3 tyrosine kinase domain and two FLT3‐ITDs (21 bp and 39 bp) (low AR) at presentation, then relapsed only with WT1 and NPM1 mutations and one FLT3‐ITD (high AR). This relapse‐fated subclone at presentation (∼2.1% of sequenced cells) was characterized by the presence of a homozygous 21 bp FLT3‐ITD resulting from copy neutral loss of heterozygosity (CN‐LOH) of chr13q and an aberrant, immature myeloid cell surface signature, contrast to the cell surface phenotype at presentation. In contrast to results from multicolor flow‐cytometry, ScPGseq not only enabled the early detection of rare relapse‐fated subclone showing immature myeloid signature but also highlighted the presence of homozygous 21 bp FLT3‐ITDs in the clone at presentation.

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.

A Clinicopathological and Molecular Analysis of Fumarate Hydratase (FH)-deficient Renal Cell Carcinomas with Heterogeneous Loss of FH Expression

Aims. Fumarate hydratase (FH)-deficient renal cell carcinoma (RCC) is a rare aggressive renal malignancy associated with hereditary leiomyomatosis and RCC syndrome (HLRCC). Tumors exhibiting heterogeneous (ie, patchy) FH loss by immunohistochemistry have rarely been described, may be diagnostically challenging, and have never been the focus of a study. We aimed to investigate the FH mutational status of FH-deficient RCC with heterogeneous versus complete FH loss, to characterize additional genetic drivers, and to evaluate 2SC immunohistochemistry in this setting. Methods and Results. We studied FH-deficient RCC with heterogeneous (n = 3) and complete (n = 4) FH loss. Targeted next-generation sequencing (NGS) was performed on all tumors. No patients had a known history of HLRCC. All tumors had histological features within the morphologic spectrum described for FH-deficient RCC. All 7 tumors were immunoreactive for 2SC. Molecularly, all 7 tumors revealed multiple hits involving the FH locus resulting in complete loss of wild-type alleles. All tumors with heterogeneous FH loss harbored FH missense variants within domain 2 of the protein, and each had concomitant copy neutral loss of heterozygosity (CN-LOH). In complete FH loss tumors, FH alterations included splice variants with concomitant loss of heterozygosity (n = 2) and homozygous gene deletions (n = 2). Other non-recurrent alterations included biallelic alterations of TP53, NF2, SMAD4 and activation of PIK3CA. Conclusions. Our series highlights how heterogeneous FH loss (patchy positive staining) is an important staining pattern to recognize since it is compatible with a diagnosis of FH-deficient RCC and should prompt additional ancillary studies (confirmatory 2SC immunohistochemistry and/or molecular testing) and genetic evaluation.

12. Cancer cytogenomic array analysis reveals origin of ovarian teratoma and reproductive lessons learned from it

Ovarian teratoma may be derived from germ cells at any developmental stage from premeiotic oogonia through meiotic oocytes to mature, post-meiotic ova. We analyzed fresh tissue samples from 27 primary ovarian teratomas and 3 extragonadal deposits (age: 4-16 years) using CytoScan array and karyotype. Array analysis detected five patterns of copy neutral loss of heterozygosity (CN-LOH). Failure of meiosis I (Type I error) with CN-LOH in the p arm and/or q arm of chromosomes without spanning centromeres was observed in 13 cases, Type II error characterized by homozygosity spanning centromeres in 6 cases, Type III error with homozygosity of the entire genome via endoreduplication of a haploid ovum in 3 cases, Type IV error with no acquired CN-LOH in 4 cases, and Type V error with both meiotic I and meiotic II non-disjunction in 1 case.

50. Chromosomal microarray uncovers novel mechanisms of progression in myelodysplastic syndromes with normal karyotype

Myelodysplastic Syndrome (MDS) with normal karyotype (NK-MDS) represents ∼50% of all MDS. Chromosomal microarray (CMA) provides clinically important information in NK-MDS by uncovering cryptic copy-number aberrations (CNA) and copy-neutral loss-of-heterozygosity (CN-LOH). Data regarding the role of CN-LOH in AML progression in NK-MDS is scant.

49. Clinical validation of Infinium CytoSNP-850K SNP-Array for routine copy number profiling of hematological malignancies

Background: Chromosomal microarray (CMA) plays a fundamental role in genomic characterization of hematological malignancies (HM) by uncovering sub-microscopic copy-number aberrations (CNA) and copy-neutral-loss-of-heterozygosity (CN-LOH). The goal of this study was to validate the Infinium CytoSNP-850K SNP-array (Illumina) for clinical evaluation of HM in a CLIA-certified laboratory.

Hematopoietic mosaic chromosomal alterations in the New England Centenarian Study.

Mosaic chromosomal alterations (mCAs) are structural alterations that include deletions, duplications, or copy-neutral loss of heterozygosity. mCAs are reported to be associated with survival, age, cancer, and cardiovascular disease. Previous studies of mCAs in large population-based cohorts (UK Biobank, MGBB, BioBank Japan, and FinnGen) have demonstrated a steady increase of mCAs as people age. The distribution of mCAs in centenarians and their offspring is not well characterized. We applied MOsaic CHromosomal Alteration (MoChA) caller on 2298 genome-wide genotype samples of 1582 centenarians, 443 centenarians’ offspring, and 273 unrelated controls from the New England Centenarian Study (NECS). Integrating Log R ratio and B-allele frequency (BAF) intensities with genotype phase information, MoChA employs a Hidden Markov Model to detect mCA-induced deviations in allelic balance at heterozygous sites consistent with genotype phase in the DNA microarray data. We analyzed mCAs spanning over 100 k base pairs, with an estimated cell fraction less than 50%, within samples with genome-wide BAF phase concordance across phased heterozygous sites less than 0.51, and with LOD score of more than 10 for the model based on BAF and genotype phase. Our analysis showed that somatic mCAs increase with older age up to approximately 102 years, but the prevalence of the subjects with mCAs tend to decrease after that age, thus suggesting that accumulation of mCAs is less prevalent in long-lived individuals. We also used Poisson regression to show that centenarians and their offspring tend to accumulate less mCA (RR = 0.63, p=0.045) compared to the controls.

Reversion Mosaicism in Primary Immunodeficiency Diseases.

Reversion mosaicism has been reported in an increasing number of genetic disorders including primary immunodeficiency diseases. Several mechanisms can mediate somatic reversion of inherited mutations. Back mutations restore wild-type sequences, whereas second-site mutations result in compensatory changes. In addition, intragenic recombination, chromosomal deletions, and copy-neutral loss of heterozygosity have been demonstrated in mosaic individuals. Revertant cells that have regained wild-type function may be associated with milder disease phenotypes in some immunodeficient patients with reversion mosaicism. Revertant cells can also be responsible for immune dysregulation. Studies identifying a large variety of genetic changes in the same individual further support a frequent occurrence of reversion mosaicism in primary immunodeficiency diseases. This phenomenon also provides unique opportunities to evaluate the biological effects of restored gene expression in different cell lineages. In this paper, we review the recent findings of reversion mosaicism in primary immunodeficiency diseases and discuss its clinical implications.

Copy-Neutral Loss-of-Heterozygosity Adds Diagnostic and Prognostic Information to the Molecular Profiling of Hematological Malignancies

Background: Copy-neutral loss-of-heterozygosity (CN-LOH) - not detectable by chromosome banding analysis - is gaining importance as a prognostic factor and can either cause the duplication of an activating mutation in an oncogene, the deletion of a tumor suppressor gene or the gain/loss of specific methylated regions. However, examination for possible CN-LOH in hematological diagnostics is at present not routinely performed and, hence, data regarding the occurrence of CN-LOH across different entities as well as the association of relevant genes is limited.Aim: (1) Frequency assessment of CN-LOH by target enrichment sequencing (TES) in a diagnostic setting, (2) evaluation of whole genome sequencing (WGS) data to estimate the prevalence of CN-LOH in a larger cohort, to pinpoint relevant genes for CN-LOHs with so far unknown associations, and to determine cross-entity variability.Patients and Methods: 1196 patients (507 female, 689 male, median age: 66 years), sent between 04/2021-07/2021 for diagnostic work-up, were analyzed by TES with a median coverage of 1765x for the gene panel and 52x for the CNV spike-in panel (IDT, Coralville, IA). Amplification-free WGS libraries of 3851 different patients were sequenced with a median coverage of 102x. Reads were aligned to the human reference genome (GRCh37, Ensembl annotation, Isaac aligner). Cnvkit (v 0.9.9) was used to call copy number variations (CNVs) and CN-LOH for TES and HadoopCNV (Yang et al. 2017) was used to call CN-LOH for WGS.Results: 1196 patients were analyzed by TES. For 10% of the patients at least one CN-LOH event was detected without any association to age or gender but a slightly higher incidence in myeloid compared to lymphoid neoplasms (10% vs 6%). In 14 patients, CN-LOH affected more than one chromosome arm. CN-LOH occurred most frequently in 4q (n = 15), 7q (n = 16), 9p (n = 25) and 11q (n = 10). As expected, 4q CN-LOH co-occurred with high variant allele frequencies (VAF) of TET2. Based on WGS data, 4q CN-LOH occurred predominately in AML (35%), CMML (22%), and MDS (20%). In rare cases, 4q CN-LOH was associated with FBXW7 variants in T-ALL. 7q CN-LOH occurred nearly exclusively in myeloid neoplasms (95%) and was associated with high VAFs in EZH2 in 69% of TES and 82% of WGS cases. CUX1 variants with high VAFs were detected in 80% (TES) and 45% (WGS) of the remaining cases, respectively. The well-known 9p CN-LOH led to JAK2V617F homozygosity in all myeloid neoplasms and occurred most often in MPNs. In T-ALL, regions of 9p CN-LOH harbored CDKN2A/B deletions. 11q CN-LOH occurred more often in myeloid than lymphoid neoplasms (79% vs 21%) and was associated with CBL variants in 61% and KMT2A-PTD in 19% of the cases. In contrast, ATM was the relevant gene in all lymphoid cases with 11q CN-LOH. CN-LOH in 11p was detected less frequently and only in 25% of cases an association with WT1 variants could be identified.Our WGS data confirmed the known associations between 1p CN-LOH and high allele burden in MPL, CSF3R and NRAS, 2p CN-LOH and DNMT3A variants, 13q CN-LOH and FLT3-ITD, the near exclusive occurrence of 16p CN-LOH in follicular lymphoma (FL, 98%) with high CREBBP-mutant allele burden , 17p CN-LOH and TP53 homozygosity, and the exclusive occurrence of 21q CN-LOH in AML and its association with RUNX1 mutations. Besides, 12q CN-LOH was associated with KMT2D in FL, with SH2B3 in MDS/MPN overlaps and in rare cases with KDM2B. For 17q CN-LOH the relevant gene was not unequivocally identifiable with high mutant allele variants in SRSF2, STAT5B, and NF1. 18q CN-LOH was a very rare event but consistently associated with a high VAF of MBD2, which presumably influences cell proliferation (Cheng et al. 2018). 19q CN-LOH was mostly (63%) associated with a high VAF of CEBPA variants, except for patients with hairy cell leukemia: in these cases nonsense mutations in CIC (VAF > 90%) were detected. CN-LOH in 22q was more common in myeloid malignancies (65% vs 35%) and associated with PRR14L mutations in the majority of myeloid cases (62%). Of note, this association occurred neither in AML samples nor in lymphoid neoplasms. No recurrent mutations were found for 6p and 14q CN-LOHs. For all other chromosomes, CN-LOH events were very rare.Conclusions: By using a CNV spike-in panel, TES adds additional diagnostic and prognostic information by enabling simultaneous detection of selected gene mutations and genome-wide CNVs, as well as CN-LOH, without increase in sequencing costs and turn-around times. [Display omitted] DisclosuresHaferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.

TP53 Mutations, Deletions, and CN-LOH: Comparison of TP53 single Hit and Double Hit Events and Their Impact on Prognosis in Hematological Malignancies

Background: TP53 is altered in ~50% of human cancers. Alterations mainly include mutations and/or deletions, but also copy-neutral loss of heterozygosity (CN-LOH) was reported. Frequently, both TP53 alleles are altered (by mutation + deletion, mutation + CN-LOH or ≥2 mutations), leading to a “double hit” event.Aim: Analysis of TP53 aberrations using WGS in 4646 cases with 29 different hematological malignancies, comparing (1) the frequencies of TP53 alterations, (2) occurrence of single hit vs. double hit, (3) correlation with complex karyotype and (4) impact on survival.Methods: Whole-genome sequencing (WGS) was performed for all 4,646 patients (median coverage 100x). 151bp paired-end reads were generated on NovaSeq 6000 and HiSeqX machines (Illumina, San Diego, CA). As no sample specific normal tissue was available, a so-called Tumor/Unmatched normal (TUN) workflow was used to reduce technical artefacts and germline calls. All reported p-values are two-sided and were considered significant at p<0.05.Results: In the total cohort of 4,646 cases, in 582 (13%) at least one alteration (alt) involving TP53 was detected (comprising mutations (mut), deletions (del) and CN-LOH (LOH); Fig 1A,B). Cases were categorized as follows: cases with (1) 1 TP53 mut only (n=166), (2) del only (n=100), (3) LOH only (n=15), constituting the single hit events. Further, (4) cases with mut+del (might include 1 or more mut, n=211), cases with mut+LOH (≥1 mut, n=41), cases with ≥2 mut only (without del or LOH, n=49), resulting in double hit events (Fig 1B). Regarding the respective entities, high frequencies of TP53 alt were mainly detected in lymphoid malignancies (e.g. HGBL, MPAL, vHZL, MZL, MCL), whereas they were infrequent or absent in many myeloid malignancies (e.g. aCML, MPN, CMML, CML, MLN_eo; Fig 1A). For further analysis, only entities in which >10 cases showed TP53 alt events were used. Comparison of single hit vs. double hit revealed that T-NHL, MM, MPN and MDS predominantly showed a single hit, whereas the double hit was frequent in MPAL, MZL, MDS/MPN-U, CLL and MCL cases (Fig 1A). However, the type of double hit differed between myeloid and lymphoid malignancies, as myeloid neoplasms showed a high frequency of cases with ≥2 mut only, whereas in many lymphoid malignancies the double hit was predominantly generated by mut+del (Fig 1A,C). All TP53-associated events (mut, del, LOH and the respective combinations) were found to be associated with a complex karyotype in the total cohort (LOH: 14% complex karyotype in cases without TP53 alt vs. 59% in cases with TP53 alt, p<0.001). This association was also detected for most of the selected entities (exceptions: MZL, T-NHL). Regarding overall survival (OS), in the total cohort, all events involving TP53 impact on OS (TP53 alt: 22 months vs. 84 months, p<0.001; TP53 mut: 20 vs. 82 months, p<0.001; TP53 del: 20 vs. 79 months, p<0.001; TP53 LOH: 20 vs. 75 months, p<0.001). Moreover, although the single hit already impacts on OS, the double hit leads to an even inferior outcome (no hit vs. single hit vs. double hit: 84 vs. 39 vs. 14 months, p<0.001). In the selected entities, an influence of TP53 alt on OS was detected for all malignancies except HGBL, MZL and T-NHL, for which also the presence of a double hit did not show an effect on OS. For the majority of the other entities, the double hit leads to a shorter OS than the single hit (as observed for the total cohort), with the exceptions of MCL and MPAL: in these entities, the single hit did not impact on OS, but only a double hit is associated with inferior outcome.Conclusions: (1) Frequency of TP53 alterations and of double hit vs. single hit differs markedly between entities. (2) The kind of TP53 complexity differs between both lineages (double hit in myeloid neoplasms: often ≥2 mut only; in lymphoid malignancies: predominantly mut+del). (3) In 7% (41/582) of cases with TP53 alt, CN-LOH transforms a single hit into a double hit. (4) In the total cohort and in the majority of selected entities (except MZL and T-NHL), TP53 alt are associated with complex karyotype. (5) In the total cohort, all events involving TP53 impact on OS; cases with double hit show an inferior outcome compared to single hit. (6) Regarding OS, the selected entities can be divided into three categories: (i) no influence of TP53 alt (HGBL, MZL, T-NHL); (ii) double hit required for impact on OS (MCL, MPAL); (iii) influence of both single hit and double hit with inferior outcome of double hit (all other). [Display omitted] DisclosuresKern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.

Germline-Somatic Interactions in Myelofibrosis Susceptibility

▪Introduction: Myelofibrosis (MF) is a rare myeloproliferative neoplasm (MPN) characterized by bone marrow fibrosis, progressive bone marrow failure, and increased risk of acute myeloid leukemia. While MF arises from somatic driver mutations in JAK2, MPL, and CALR, some MPN patients may have a heritable component. To comprehensively examine the genetic etiology of MF, we performed the first integrative analysis of SNP array genotyping (using Infinium Global Screening Array), targeted long-read sequencing (using PacBio SMRT sequencing) and telomere length (TL, using qPCR assay).Methods: Our study included 937 MF patients who received an allogeneic hematopoietic cell transplant (HCT) between 2000 and 2016 and had an available pre-HCT blood sample at the Center for International Blood and Marrow Transplant Research Repository. Somatic mosaic chromosomal alterations (mCAs, including deletions, duplications, or copy-neutral losses-of-heterozygosity (CNLOH)) were called with the Mosaic Chromosomal Alteration (MoChA) algorithm using raw genotyping intensity data. A genome-wide association study (GWAS) was restricted to include 827 MF patients of European ancestry and utilized 4,135 genetically-matched healthy controls.Results: GWAS identified six independent MF susceptibility loci at genome-wide significance (P< 5×10 -8); four of which replicate prior MPN susceptibility loci [9p24.1(JAK2), 5p15.33(TERT), 3q25.33(IFT80), and 4q24(TET2)] and two novel MF loci [6p21.35(HLA-DQB1-AS1) and 17p13.1(TP53)] (Figure 1). A transcriptome-wide association analysis using whole blood GTEx data highlighted the 9p24.1 locus with increased JAK2 expression associated with elevated risk of MF (P= 2.18×10 -19). A strong colocalization statistic further indicated shared genetic component between eQTL and this JAK2 locus (HyPrColoc Posterior Probability= 0.6) (Figure 2).Based on the strong signal identified at TERT (Figure 1), we investigated the relationship between MF risk and genetically-inferred telomere length using a panel of 19 germline variants previously found to be associated with telomere length. Of the 19 telomere-length associated variants investigated, 7 were found to be associated with MF risk (binomial P= 2.31×10 -5, linear trend P= 5.48×10 -4) (Figure 3). Both Mendelian randomization and genome-wide genetic correlation analyses further indicated that increased risk of MF was associated with longer inherited telomere length.Utilizing available clinical mutation data on a subset of 185 patients, MF cases carrying the germline risk haplotype of the 9p24.1(JAK2) susceptibility locus were observed to more frequently have the JAK2 V617F mutation (71% vs 59%; P= 0.02). Targeted PacBio long-read sequencing around JAK2 provided further evidence of linkage between the germline risk allele and the JAK2 V617F mutation. Detectable autosomal mCAs were also abundant in MF cases with 67.4% having at least one mCA (compared to ~3% in the general population) and 27.6% having an mCA spanning JAK2 (mostly CNLOH) (Figure 4). In addition, using a binomial test for biased allelic imbalance, a cis relationship was identified at 9p24.1 in which the MF risk haplotype was predominantly duplicated by CNLOH (binomial P=1.36×10 -9). Regional sequencing of JAK2 further confirmed duplication of JAK2 V617F by CNLOH. Finally, we observed an inverse association between autosomal mCAs and qPCR measured telomere length (OR= 0.22, 95% CI= 0.07-0.65, P= 6.40×10 -3). These results were consistent by mCA chromosomal region and copy number state.Conclusion: Our results suggest a molecular framework for the genetic etiology of MF in which both genetically-inferred telomere length and germline variation at JAK2 are associated with increased MF risk. The 9p24.1 risk haplotype predisposes to the acquisition of a somatic JAK2 V617F mutation in cis and subsequent duplication of JAK2 V617F by mCAs (usually CNLOH). This process leads to aberrant JAK2 activity and increased clonal proliferation, accelerating telomere length shortening and increasing genomic instability in patients with MF. [Display omitted] DisclosuresGupta: AbbVie: Consultancy, Honoraria; Constellation Pharma: Consultancy, Honoraria; Roche: Consultancy; Pfizer: Consultancy; BMS-Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria, Research Funding. Lee: Janssen: Other; Incyte: Research Funding; AstraZeneca: Research Funding; Kadmon: Research Funding; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Syndax: Research Funding; Takeda: Research Funding; Amgen: Research Funding. Saber: Govt. COI: Other.

Single-Cell Proteogenomic Sequencing Allows Early Detection of Relapse Clone with CN-LOH at FLT3-ITD Locus from Initial Diagnosis in AML

IntroductionFLT3-ITD is one of the most common and clinically relevant mutations in acute myeloid leukemia (AML). A high FLT3-ITD allelic ratio (AR) (>0.5) is strongly associated with poor prognosis. FLT3-ITDs are heterogeneous mutations, varying in sizes and locations with some patients having multiple FLT3-ITDs. Unfortunately, conventional clinical techniques are often not adequate in measuring these characteristics at sufficient resolution. The current study aimed to characterize an AML case with 2 FLT3-ITDs at diagnosis, map the multi-omic evolution of AML clones, and understand genetic patterns underlying AML relapse using single-cell proteogenomic sequencing (ScPGseq).MethodsLeukemic samples from a 46-year old female patient diagnosed with de novo AML obtained at initial diagnosis and relapse were archived and used in this study. Based on the 2017 ELN risk stratification, the patient was classified into a low-risk group due to normal karyotype/low FLT3-ITD AR/NPM1+. The patient achieved complete remission (CR) after induction chemotherapy in combination with midostaurin. The patient relapsed 2 months after 4 cycles of consolidation, with a CR duration of 6 months. After relapse, the disease was refractory despite reinduction and salvage therapies, even with gilteritinib (Fig A). ScPGseq was performed using the Mission Bio's AML panel and antibody oligoconjugates for 16 cell surface proteins. Raw sequencing reads were processed using Mission Bio's Tapestri pipeline and results were exported from Tapestri Insights (v3.0.2). All downstream computational and statistical analyses were performed using R and Python.ResultsConsistent with clinical sequencing (R 2 = 0.997) and PCR, ScPGseq identified 5 mutations in FLT3 (21bp and 39bp ITDs and D835Y), NPM1, and WT1 at diagnosis (2367 cells). At relapse, 1 FLT3-ITD (21bp) and mutations in NPM1 and WT1 were detected while the other FLT3-ITD (39bp) and FLT3 D835Y were absent (2611 cells). Clonal analyses of mutation patterns identified 4 AML clones at diagnosis (C1-4) and 1 at relapse (C3R; Fig B). Two closely related clones, C3 and C3R (WT1+/NPM1+/21bp FLT3-ITD+) differed in zygosity of 21bp FLT3-ITD, where nearly all cells in C3R carries homozygous FLT3-ITD. SNP array confirmed the presence of copy neutral loss of heterozygosity (CN-LOH) in chr13q.Analysis of 16 cell surface proteins along with clonal information identified 3 clusters including one devoted to non-leukemic fractions (i.e., no mutations) (top) and another nearly exclusively consisting of mutant cells from diagnosis, which we termed “monocyte-like signature” (right bottom). The other cluster was composed of nearly all mutant cells from the relapse sample and about 1/3 of mutant cells from diagnosis, which we termed “immature myeloid cell signature” (left) (Fig C).When investigating further, C3 cells with immature myeloid cell signature (98/287 cells, 34.1%) exhibited much higher allelic burden of (mean 73% vs. 56%, adj. p < 1.1e-6) and were significantly enriched with homozygous 21bp FLT3-ITDs compared to C3 cells with monocyte-like signature (189/287 cells, 65.9%) (41/98 cells, 42% vs. 11/189 cells, 5.8%, adj. p = 2.7e-13). No other mutations showed similar patterns. It indicates that C3R existed from the initial diagnosis at low frequency, instead of losing heterozygosity during relapse. Without the single cell proteogenomic analyses, C3R cells, present in <5% cells at the initial diagnosis could have not been detected.The multi-omic information elucidates the complete clonal history of this AML (Fig D). Starting with WT1 and NPM1 mutations (C1), 3 FLT3 mutations (2 ITDs and D835Y) were subsequently acquired in 3 subclones (C2-4). A subset of C3 cells (WT1+/NPM1+/21bp FLT3-ITD+) further gained CN-LOH in chr13q (C3R at diagnosis). When treated, AML cells without homozygous 21bp FLT3-ITD were cleared. Cells with homozygous 21bp FLT3-ITD survived/escaped from the treatment and became the dominant clone at relapse (C3R at relapse).ConclusionThe current study demonstrates that ScPGseq allows1) simultaneous and comprehensive analyses of multiple FLT3-ITDs at the single-cell level2) early detection of relapse clone with subclonal homozygous 21bp FLT3-ITD from the initial diagnosis, which explains one of the mechanisms of relapse in AML cases with low FLT3-ITD AR3) multi-omic clonal analyses, which further refine clonal models relying only on mutation profiles. [Display omitted] DisclosuresMinden: Astellas: Consultancy. Kim: Pfizer: Honoraria, Research Funding; Bristol-Meier Squibb: Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Paladin: Honoraria, Research Funding.

TP53 Allelic Status in Myeloid Malignancies

IntroductionTP53 is a tumor suppressor gene involved in regulating cell division and apoptosis in response to DNA damage. In hematologic malignancies, TP53 alterations are present in both myeloid and lymphoid malignancies. TP53 alterations including both sequence level mutations and deletions occur in 8-10% of de novo acute myeloid leukemia (AML), and are significantly enriched in patients with therapy-related myeloid neoplasms with a frequency of 25-40%. TP53 alterations are associated with complex karyotype, resistance to traditional cytotoxic chemotherapy and dismal outcome, and are well established poor prognostic markers for both AML and myelodysplastic syndrome (MDS). While both biallelic and monoallelic TP53 alterations are seen, biallelic TP53 alteration is more frequent in both AML and MDS. In the instances of monoallelic TP53 alterations, the remaining wild-type allele can be inhibited by the dominant negative effect of the mutant p53.The clinical implication of TP53 allelic status remains controversial. Biallelic TP53 alterations and/or high TP53 mutation variant allele frequency (VAF) are associated with older patients, complex karyotype, few co-occurring mutations and poor outcome in both AML and MDS. Other studies demonstrate that biallelic vs. monoallelic TP53 alterations or high vs. low TP53 VAF have similar prognosis in myeloid malignancies. Current European LeukemiaNet (ELN) recommends testing TP53 deletion using karyotype analysis and TP53 mutations by molecular testing.Methods:We performed a retrospective review of patients with myeloid malignancies, myeloid next generation sequencing (NGS) panel and cytogenetic tests performed at ARUP Laboratories. We identified 18 patients with myeloid malignancies and TP53 mutations. We used a combination of karyotype analysis, FISH, chromosomal microarray (CMA) and NGS to determine the TP53 allelic status.Results:18 patients diagnosed with myeloid malignancies and TP53 mutations were identified. Among them, 6 were diagnosed with AML, 10 had MDS, one with CMML and one had post essential thrombocythemia myelofibrosis. Age range is from 31 to 78 with a median age 65.5 years.23 TP53 mutations were identified among 18 patients. The majority (78%) of TP53 mutations are located in the DNA binding domain. Co-occurring mutations are uncommon in patients with TP53 mutations. 11 out of 18 patients did not have co-occurring mutations in other myeloid malignancy related genes. Three cases had 1, three cases had 2, and one case had 3 co-occurring mutations at the time of diagnosis.Karyotype analysis and FISH were performed on all 18 patients. CMA was performed on 9 patients. 17p abnormalities, defined by the deletion or copy-neutral loss of heterozygosity (CN-LOH) of 17p, were seen in 9 out of 18 cases. The 17p abnormalities in 8 out of these 9 cases were visible by karyotype. Case 11 had a CN-LOH identified by CMA which was cryptic by karyotype. Typical complex karyotype was seen in 16 out of 18 cases.Biallelic TP53 alterations are defined by either the presence of a TP53 mutation with a 17p abnormality (deletion or CN-LOH), or two TP53 mutations with similar VAF, or one TP53 mutation with VAF >50%. Biallelic TP53 alterations were seen more frequently compared with monoallelic TP53 alterations (14 vs. 4 patients) and enriched in older patients (Figure 1). All 6 AML patients had biallelic TP53 alterations.Outcome data is available in 10 patients including 8 patients with biallelic and 2 patients with monoallelic TP53 alterations. Patients with biallelic TP53 alterations have dismal outcome (Figure 2). Patient 7 represents an atypical patient with TP53 alteration in our study cohort. She was diagnosed with MDS at the age of 31. She had monoallelic TP53 mutation with a relatively low VAF and normal karyotype through her disease course. Her MDS never progressed and she remained in remission after transplant for more than four years and still doing well.Conclusion:In this study, we evaluated the TP53 allelic status in 18 patients with myeloid malignancies. Biallelic TP53 alterations are more frequent than monoallelic TP53 alterations, and are associated with older patients, and fewer co-occurring mutations. Biallelic TP53 alterations are associated with typical complex karyotype and dismal outcome. Our study supports the importance to differentiate between biallelic and monoallelic TP53 alterations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Benchmarking of Whole Genome Sequencing (WGS) and Whole Transcriptome Sequencing (WTS) As Diagnostic Tools for the Genetic Characterization of Acute Myeloid Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL) in Adults

Background: In AML and ALL the application of WHO classification and ELN guidelines requires a combination of cytogenetics and targeted sequencing for specific mutations to determine the diagnostic and prognostic subgroup. WGS and WTS have emerged as comprehensive techniques that allow the simultaneous analysis and identification of all genetic alterations in a single approach with possible turnaround times of 1 week.Aim: Evaluate the accuracy of WGS and WTS in providing all relevant genetic information in a clinical setting.Patients and Methods: The cohort comprised 738 AML, 293 BCP-ALL and 124 T-ALL. The diagnosis was established following WHO guidelines. WGS (100x, 2x151bp) and WTS (50 Mio reads, 2x101bp) were performed on a NovaSeq instrument. Variants were called with Strelka2, Manta and GATK using a tumor w/o normal pipeline, fusions with Arriba, STAR-Fusion and Manta.Results: The combination of WGS and WTS detected all chromosomal and molecular abnormalities in the AML and ALL cohorts relevant for disease stratification and prognostication as identified by chromosome banding analysis (CBA) and targeted panel sequencing (TPS). A very high concordance between CBA and WGS was revealed for the detection of balanced structural variants (SV) with the added benefit of WGS to also detect cytogenetically cryptic rearrangements (i.e.: ETV6-MN1, NUP98-KDM5A), which all were confirmed either by FISH or RT-PCR. Fusion calling by WTS identified 96% of the WHO subtype defining rearrangements and detected 20 additional fusion transcripts relevant for disease stratification (e.g. EP300-ZNF384, TCF3-HLF) including 9 fusion transcripts that led to prognostic reassignment or could serve as a potential treatment target. Breakpoints of unbalanced SV can occur in repetitive sequences of the genome, hampering the detection by WGS. However, adding copy number alteration (CNA) calls to the analyses allows also reliable identification of unbalanced SV.WGS outperformed CBA in cases with insufficient in vitro proliferation due to suboptimal pre-analytics (i.e. longer transport time) and identified 36 chromosomal aberrations in 12 cases with CBA not evaluable. WGS's independence of in vitro cell proliferation was most impactful in ALL: 40 T-ALL cases showed a normal karyotype according to CBA. WGS detected SVs in 16 (40%) and CNAs in 20 (50%) of these cases, confirming the normal karyotype for only 9 samples. In the BCP-ALL cohort, CNV analysis identified 29 low hypodiploid and 16 high hyperdiploid karyotypes, 6 of which were missed by CBA. Due to the higher resolution and unrestricted, genome-wide assessment, WGS detected relevant gene deletions (RB1, ERG, PAX5, CDKN2A, IKZF1, ETV6, BTG1) in 59% of ALL cases, providing additional diagnostic and prognostic information. In the AML cohort CBA and WGS detected 795 CNA concordantly. In addition WGS called 54 CNA with size 1-5 MB (below the detection limit of CBA), i.e. 3 BCOR deletions in inv(3)(q21q26) cases and 67 CNA with size > 5 MB, which were missed by CBA. 35 CNA were missed by WGS due to small clone sizes (median 6% as determined by FISH). WGS detected copy neutral loss of heterozygosity (CN-LOH) in AML most frequently on 21q (n=17), 4q (n=15), 13q (n=15), 11q (n=13) and in T-ALL on 9p (n=19), mostly encompassing CDKN2A/B deletions. Expression profiling provided additional diagnostic information for 57 ALL cases (41 BCR-ABL1-like, 16 DUX4 rearranged) that can only insufficiently be obtained by WGS or CBA. WGS reliably detected all gene mutations with a VAF > 15% (n = 647) identified by TPS encompassing especially all mutations in genes relevant for WHO diagnosis and prognostication. 26/171 mutations with a VAF < 15% were missed by WGS. Evaluation of WGS data for 121 genes recurrently mutated in hematologic neoplasms revealed an additional 2 mutations per sample on average (range: 0-9) which might qualify as targets for therapy.Conclusions: WGS and WTS provide all necessary genetic information to accurately determine the diagnostic and prognostic subgroup according to WHO and ELN guidelines in AML and ALL. Compared to today's gold standards, these novel methods provide a comprehensive genome wide characterization with higher resolution that directly identifies genes of impact, offering the basis for targeted treatment selection and monitoring of residual disease. Both can be implemented with automated analysis pipelines, consequently reducing time and error rates. [Display omitted] DisclosuresHaferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.

Assessment of Next Generation Sequencing Approaches for the Cytogenetic and Molecular Evaluation of Acute Myeloid Leukemia

Background:Genomic evaluation of structural and molecular alterations is essential for the classification and risk stratification of patients with acute myeloid leukemia (AML). Cytogenetics (CG) has traditionally been used for the identification of large structural or copy number changes and molecular studies for small sequence changes. Rapid technical advances and decreased sequencing (seq) costs have made whole-genome sequencing (WGS) more feasible and recent efforts have suggested it can match the analytic performance of traditional CG while providing additional prognostic information. However, the use of WGS in the clinic is still in its infancy and questions remain regarding the utility of many factors including seq depth, structural events not identifiable by traditional CG, normal samples, AML enrichment, paired RNA-seq, and single-cell DNA-seq.Methods:Baseline bone marrow (BM) samples from 10 AML patients underwent evaluation using standard clinical CG and targeted next generation seq tests. Additionally, samples were extensively characterized using: 200X WGS (bulk and enriched BM), 50X germline WGS (buccal swab and remission blood), 50X RNA-seq, targeted error-corrected (EC) DNA-seq, and single-cell DNA-seq with oligonucleotide-conjugated antibodies (scDNA-seq+ab).Results:The AML patients analyzed had a mean age of 71 (30-81) and a range of BM blast percentage by clinical flow cytometry (0.4-33%). Standard clinical assessment revealed 14 CG abnormalities and 20 mutations across all patients. 50X WGS of the bulk BM utilizing previously published conditions (PMID:33704937) successfully identified 89% of the alterations. Increasing the WGS depth to 100X recovered 2 additional abnormalities, increasing the capture rate to 94%.Enrichment of AML using the primary cell surface marker prior to WGS achieved a 94% capture rate at only 50X depth, with a depth of only 10X required for the CG abnormalities. Integration of 50X WGS on buccal swab samples reduced the large number of structural variant calls and private mutations without the need for aggressive filtering conditions required for tumor-only samples. However, this also resulted in the removal of a few true mutations due to buccal contamination with immune cells. 50X WGS on remission blood samples from 2 patients mitigated this problem.Next, we sought to determine what additional information WGS could provide that was missed by standard CG (>5Mb) and seq (>5% variant allele fraction, VAF) analyses. Firstly, 3 of the deletions reported by CG were found to be complex translocation events, resulting in different losses and gains than anticipated. Most importantly, decreasing the CNV size threshold (<5Mb) and utilizing SNP data to identify copy neutral loss of heterozygosity (LOH) identified 7 new events in bulk BM and 2 new events in enriched BM. This information provided important prognostic information, including 3 newly identified structural alternations in a patient with a CG-reported normal karyotype, multiple small deletions in critical regions (e.g. within 5q, 7q), and LOH which in combination with molecular data revealed a biallelic loss of important genes (including TP53, EZH2).Targeted EC DNA-seq was used to detect of variants <5% VAF and identified an additional 23 variants beyond clinical capture sequencing. 50X RNA-seq with integrated WGS DNA/RNA calling was able to validate the mutations and translocation fusion genes reported by 50X WGS, while also capturing an additional 4 variants found by EC DNA-seq.Finally, personalized scDNA-seq+ab provided further resolution of each patient's AML through confirmation of new structural and mutational findings, identification of mutations associated with clonal hematopoiesis versus AML, and definition of AML clonal structure (Figure 1).Conclusions:We confirmed that 50X WGS can recapitulate standard clinical CG and mutational profiling in AML patients while providing improved resolution. Deeper seq, AML pre-enrichment, and integrated RNA-seq further aided in identifying low level events. We also demonstrated that WGS can identify additional important structural events missed by CG. Finally, personalized scDNA-seq+ab was able to define clonal architecture important for prognostic and residual disease tracking. Together, these results provide a framework for future integration of WGS into the clinic and personalized patient care. [Display omitted] DisclosuresSciambi: Mission Bio Inc.: Current Employment. Durruthy-Durruthy: Mission Bio Inc.: Current Employment. Hourigan: Sellas: Research Funding.

Molecular genetic evidence supporting diverse histogenic origins of germ cell tumors.

Germ cell tumors (GCTs) originate during the histogenesis of primordial germ cells to mature gametes. Previous studies identified five histogenic mechanisms in ovarian mature teratomas (type I: failure of meiosis I; type II: failure of meiosis II; type III: duplication of the genome of a mature gamete; type IV: no meiosis; and type V: fusion of two different ova), but those of other GCTs remain elusive. In this study, we analyzed 84 GCTs of various pathologic types to identify the histogenesis using single-nucleotide polymorphism array by analyzing copy-neutral loss of heterozygosity (CN-LOH) and copy number alterations (CNAs). We detected types I and II in ovarian teratomas, type III in ovarian teratomas and yolk sac tumors (YSTs), and type IV in all GCT types. The GCTs with multiple-type histogenesis (I-IV) (ovarian mature/immature teratomas and YST) show meiotic CN-LOH with scant CNAs. Type IV-only GCTs are either with mitotic CN-LOH and abundant CNAs (seminoma, dysgerminoma, testicular mixed GCTs) or with scant CNAs and no CN-LOH (pediatric testicular and mediastinal teratomas). The development sequences of CN-LOH and CNA are different between the multiple type (I-IV) GCTs and type IV-only GCTs. We analyzed two different histologic areas in eight GCTs (one mature teratoma with a mucin-secreting adenoma, two immature teratomas, and five mixed GCTs). We found that GCTs (mature teratoma, immature teratoma, and mixed GCT) showed different genomic alterations between histologic areas, suggesting that genomic differences within a GCT could accompany histologic differentiation. Of note, we found evidence for collision tumors in a mixed GCT. Our data indicate that GCTs may have various histogenesis and intratumoral genomic differences, which might provide important information for the identification of GCTs, especially for those with different histologic areas. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Copy Neutral LOH Affecting the Entire Chromosome 6 Is a Frequent Mechanism of HLA Class I Alterations in Cancer.

Simple SummaryLoss of antigen presentation due to the altered expression of tumor HLA class I (HLA-I) molecules is a common mechanism of cancer immune escape. Loss of HLA-I haplotype, locus or a single allele due to genetic and chromosomal aberrations, may result in reduced antigen presentation and, thus, facilitate immune evasion. Here, we demonstrate the prevalence of the copy neutral loss of heterozygosity (CN-LOH) involving HLA-I heavy chain and B2M gene in various human tumor cell lines and tissues. We discuss the impact of the LOH in HLA genes on tumor immune rejection, clonal expansion and association with the cancer recurrence in the immunotherapy settings. It represents a genetic barrier for effective treatment and can be considered as a potential genetic biomarker of cancer immune escape.Total or partial loss of HLA class I antigens reduce the recognition of specific tumor peptides by cytotoxic T lymphocytes favoring cancer immune escape during natural tumor evolution. These alterations can be caused by genomic defects, such as loss of heterozygosity at chromosomes 6 and 15 (LOH-6 and LOH-15), where HLA class I genes are located. There is growing evidence indicating that LOH in HLA contributes to the immune selection of HLA loss variants and influences the resistance to immunotherapy. Nevertheless, the incidence and the mechanism of this chromosomal aberration involving HLA genes has not been systematically assessed in different types of tumors and often remains underestimated. Here, we used SNP arrays to investigate the incidence and patterns of LOH-6 and LOH-15 in a number of human cancer cell lines and tissues of different histological types. We observed that LOH in HLA is a common event in cancer samples with a prevalence of a copy neutral type of LOH (CN-LOH) that affects entire chromosome 6 or 15 and involves chromosomal duplications. LOH-6 was observed more often and was associated with homozygous HLA genotype and partial HLA loss of expression. We also discuss the immunologic and clinical implications of LOH in HLA on tumor clonal expansion and association with the cancer recurrence after treatment.

Pretransplant HLA mistyping in diagnostic sample of a T-ALL patient due to loss of heterozygosity in the major histocompatibility complex

PurposeThe degree of HLA compatibility between donor and recipient in hematopoietic stem cell transplantation is critical. In this report, we describe an acute lymphoblastic leukemia case with loss of heterozygosity (LOH) encompassing the entire HLA. 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-B,-C,-DR, and -DP loci. A BS sample demonstrated heterozygosity at the above loci. A subsequent PB sample drawn after count recovery confirmed heterozygosity. The CMA performed on PB samples collected during blast crisis revealed a large terminal region of copy-neutral LOH involving chromosome region 6p25.3p21.31, spanning approximately 33.32 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.

CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells

CRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers.