Chromosome Banding Analysis Research Articles

Optical Genome Mapping for Cytogenetic Diagnostics in AML

The classification and risk stratification of acute myeloid leukemia (AML) is based on reliable genetic diagnostics. A broad and expanding variety of relevant aberrations are structural variants beyond single-nucleotide variants. Optical Genome Mapping is an unbiased, genome-wide, amplification-free method for the detection of structural variants. In this review, the current knowledge of Optical Genome Mapping (OGM) with regard to diagnostics in hematological malignancies in general, and AML in specific, is summarized. Furthermore, this review focuses on the ability of OGM to expand the use of cytogenetic diagnostics in AML and perhaps even replace older techniques such as chromosomal-banding analysis, fluorescence in situ hybridization, or copy number variation microarrays. Finally, OGM is compared to amplification-based techniques and a brief outlook for future directions is given.

Association of ABCB1, ABCG2 drug transporter polymorphisms and smoking with disease risk and cytogenetic response to imatinib in chronic myeloid leukemia patients

BackgroundDespite acceptable results of imatinib in the treatment of chronic myeloid leukemia (CML), some patients fail to acquire a complete cytogenetic response (CCyR), which may be caused by polymorphisms in the pharmacogenetic genes. The study aimed to evaluate the association of two polymorphisms in the ABCB1 and ABCG2 genes with cytogenetic response to imatinib and the risk of CML development. MethodsWe genotyped ABCB1 (c .2677G/T/A) and ABCG2 (c .421C/A) polymorphisms by PCR-RFLP, T-ARMS-PCR methods in 111 patients with CML and 102 sex- and age-matched healthy subjects. CCyR was determined by standard chromosome banding analysis (CBA). ResultsAnalysis of polymorphisms showed significant association of ABCG2 c.421CA genotype (p < 0.0001; OR = 0. 17), and ABCG2c.421A allele (p < 0.0001; OR = 0.31) with decreased risk of CML. Moreover, ABCB1c.2677GT- ABCG2c.421CC combined genotype (p = 0.017; OR = 4.20) was associated with increased risk of CML. Analysis of the joint effect of SNP-smoking combination showed that smoker subjects with the ABCB1c.2677GG/GT (p = 0.001; OR = 15.96, p = 0.001; OR = 8.13, respectively) or ABCG2c.421CC genotypes (p = 0.001; OR = 5.82) had the increased risk of CML, while the risk of the CML in non-smokers carrying the ABCG2c.421CA (p < 0.0001; OR = 0. 18) genotype was strongly decreased compared with reference group. Regarding drug response, ABCG2c.421 CC/CA genotypes in the smoker patients were associated with an increased risk of resistance to imatinib (p < 0.0001; OR = 7.02, p = 0.018; OR = 4.67, respectively). ConclusionOur results suggest the impact of ABCG2c .421C/A polymorphism on CML development, and smoking may have a synergistic role in the risk of CML and resistance to imatinib.

Cytogenetic peripheral blood monitoring in azacitidine treated patients with high-risk MDS/sAML: A monocentric real-world experience

In this single center retrospective analysis 76 patients with high-risk (HR) myelodysplastic syndrome (MDS) treated with azacitidine (AZA) were reviewed for response, especially cytogenetic response (cyR) using repeated chromosome banding analyses (CBA) of bone marrow (bm) metaphases and frequent sequential Fluorescence-in-situ Hybridization (FISH) analyses of immunomagnetically enriched CD34 + circulating peripheral blood cells (CD34 +pb-FISH). In total, 526 CD34 +pb-FISH analyses and 236 CBA were examined. Median observation time was 8.45 months, median number of AZA cycles applied was 8, median overall survival (OS) was 14.9 months, 42.1 % of patients responded to therapy according to IWG criteria: 5 complete response (CR), 0 partial response (PR), 12 bmCR, 15 stable disease with hematologic improvement (HI). HI was reached in 36.8 % of patients, 31.5 % became transfusion-independent. By CBA or CD34 +pb-FISH 20.4 % and 31.6 % of patients showed cyR, respectively. HI rate was significantly higher in cytogenetic responders than in non-responders, but there was no impact on OS or leukemia-free-survival. Cytogenetic responders showed significantly better OS than non-responders. Patients with ≥ 6 AZA cycles had significantly better OS than patients with < 6 cycles applied. Karyotype evolution (KE) as a manifestation of cytogenetic progression was diagnosed in 29.5 % and 17.1 % of patients by CBA and CD34 +pb-FISH, respectively. KE was associated with significantly poorer OS and leukemia-free-survival.

Myeloid Neoplasms with MYC-Positive Double Minutes, a Specific Subgroup?

Background: Double minutes (dmin) are a rare cytogenetic finding in hematologic malignancies, mostly associated with acute myeloid leukemias. The acentric chromosomal structures usually represent the cytogenetic equivalent of the amplification of an oncogene, such as MYC. Due to their small size dmin are difficult to detect in chromosome banding analysis (CBA) and FISH analysis is required to identify the corresponding gene amplification. In literature several case reports described an association of the presence of MYC-positive dmin with an APL-like cytomorphology, however, larger studies comprehensively characterizing these cases are missing so far. Aim: Characterization of 68 cases of myeloid neoplasms harboring MYC dmin with respect to cytogenetics, morphology, gene expression (GE) and gene mutations. Patients and methods: The study enrolled 68 patients (pts) with myeloid neoplasia and MYC dmin. The diagnosis was established following WHO guidelines. Dmin and MYC amplification were assessed via CBA and FISH. Cases were screened for morphological features indicating a similarity to APL and/or suspected diagnosis of APL (APL-like). NGS of a 72 gene panel and WTS (50 Mio reads) were performed on NovaSeq instruments. Variant calling was performed with Pisces (minimum sensitivity: 3 %). Results: The majority of pts were diagnosed with AML (52/68, 77 %, 7 of these secondary AML) or MDS/AML (3/68, 4 %). Other diagnoses comprised CMML (4/68, 6 %), MPN in acceleration or blast crisis, MDS/MPN overlap or MDS (3/68, 4 % each). APL-like cases amounted to 17/68 (25 %). Auer rods were detected in 24/68 pts (35 %) with a higher frequency in APL-like cases (p=0.007). Myeloperoxidase level was >75 % in 46/54 pts (85 %). CBA revealed a complex karyotype (ck, ≥3 aberrations in addition to MYC dmin) in 21/68 pts (31 %), whereas 19/68 pts (28 %) presented with MYC dmin as the only cytogenetic aberration (MYC dmin only). Most common additional chromosomal gains were +4 (11/68, 16 %), +6 (9/68, 13 %) and +22 (8/68, 12 %), while most frequent chromosomal losses comprised 5q deletions (19/68, 28 %), 17p deletions (18/68, 27 %) and 9q deletions or -9 (16/68, 24 %). Gene mutations (mut) most frequently occurred in TET2 (48/68, 71 %), TP53 (22/67, 33 %), U2AF1 (17/67, 25 %) and NRAS (12/66, 18 %). Almost all pts (66/68, 97 %) harbored at least one mutation in TET2, TP53 or U2AF1. TP53 mut were associated with ck (p<0.001) and were not detected in MYC dmin only pts (p<0.001). EZH2 mut were exclusively observed in cases with non-ck and 5/7 mut occurred in MYC dmin only pts (p=0.014). TET2 mut were observed more frequently in cases with non-ck (89 % vs. 29 %, p<0.001). Similarly, U2AF1 mut co-occurred with ck only in one case (p=0.013). In addition, U2AF1 showed significantly higher mutation frequency in APL-like cases (47 % vs. 18 %, p=0.025). A comparison of the mutation frequencies of all MYC dmin cases to a cohort of AML from routine diagnostics (707 pts) revealed overrepresentation of mut in TP53 (33 % vs. 11 %, p=0.003), TET2 (71 % vs. 19 %, p<0.001), U2AF1 (25 % vs. 4 %, p<0.001) and EZH2 (11 % vs. 2 %, p<0.001) in the cohort of MYC dmin cases, whereas CEBPA, NPM1, FLT3, FLT3-ITD and WT1 mut were only detected in the AML cohort (10 %, p=0.004; 22 %, p<0.001; 8 %, p=0.011, 16 %, p<0.001 %; 8 %, p=0.017 respectively) and IDH2 mut occurred less frequently in MYC dmin cases (2 % vs. 9 %, p=0.022). GE analysis of 40 MYC dmin pts revealed high expression of MPO compared to various AML subtypes, similar to APL (Figure (A)). A tSNE plot based on the top 5 % most variable genes demonstrated similar GE profiles within the MYC dmin cases compared to other AML cases (857 pts) (Figure (B)). A comparison of GE profiles within the MYC dmin cohort revealed differential GE between APL-like and all other MYC dmin cases (46 differentially expressed genes (DEG), adjusted p-value <0.05), as well as between ck and non-ck MYC dmin cases (106 DEG, adjusted p-value <0.05). Conclusions:MYC dmin cases often show an APL-like cytomorphology, Auer rods and high levels of MPO expression. They are characterized by a high frequency of TET2, TP53 and U2AF1 mut as well as a relatively uniform GE profile. Mutation patterns and GE profiles allow further differentiation within MYC dmin cases. In cases with suspected APL after exclusion of a PML::RARA or other RARA fusions CBA and FISH for the detection of MYC dmin is helpful to identify this rare myeloid subtype which is described to be associated with an unfavorable prognosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Detecting the Unusual without Compromising Diagnostic Accuracy - a Prospective WGS/Wts Pilot Study in Acute Leukemias Provided Additional Information for Diagnosis, Prognosis and Treatment

Background: Following WHO and ELN guidelines, standard diagnostics and risk stratification for acute leukemias (AML and ALL) are based on genetic information gathered by a combination of cytogenetics and targeted molecular profiling. Today, comprehensive techniques, i.e WGS and WTS (whole genome and transcriptome sequencing) and the accompanying clinical bioinformatics workflows may even refine and individualize diagnostics. Aim: To evaluate the clinical utility and added value of WGTS in a real-world setting in comparison to standard of care genetic diagnostics. Patients and Methods: The study enrolled 113 patients, 97 with AML and 16 with ALL, diagnosed according to WHO criteria based on cytomorphology, immunophenotyping, cyto- and molecular genetics. WGS (100x) and WTS (50 Mio reads) were performed on NovaSeq instruments. For selected AML cases CD3+ MAC sorted cells were used as normal controls (30x). Variants were called with Strelka2, Manta and GATK using a tumor w/o normal pipeline, fusions with Arriba, STAR-Fusion and Manta. Results: All patients were analyzed for structural variants (SV), copy number variations (CNV) and copy-neutral loss of heterozygosity (cnLOH) based on WGS data and the presence of fusion transcripts and expression patterns based on WTS data. Samples without a normal control (n = 74) were analyzed for 121 genes recurrently mutated in myeloid/lymphoid neoplasms, whereas for 39 AML cases with CD3+ normal controls the whole exome was assessed. Overall, 308/333 (92%) gene mutations were consistently identified by targeted sequencing and WGS. Due to the higher read depth, 25/333 (8%) sub-clonal mutations were only detected by targeted sequencing, with 23/25 showing a variant allele fraction <10%. However, 40 gene mutations were identified exclusively by WGS due to the unrestricted analytical scope (e.g. PTPN11, DDX54). Every patient carried on average 3.3 gene mutations within the 121 genes of interest. The number increased to 63 variants (mean; range: 46-116) when analyzing the exome. 52/63 variants are most likely germline variants based on occurrence in CD3+ control sample. Hence, the CD3+ control samples reduced the number of somatic variants in the exome to 11 (mean; range: 1-23) mostly patient-specific mutations that can be explored for personalized treatment options. Addressing cytogenetics with relevance to risk stratification and assignment to AML/ALL genetic subgroups, WTS identified all entity defining fusions and WGS and chromosome banding analysis (CBA) showed a very high concordance. In 10 patients WGS missed subclonal aberrations due to small clone size (median clone size detected by IP-FISH analysis: 6%). However, WGS provided additional information in 40 patients, mostly SV, CNV and cnLOH of unknown significance, but also alterations of biological interest and prognostic and/or therapeutic importance: in one AML case WGTS revealed a KMT2A::MLLT10 rearrangement that was cytogenetically cryptic and not detectable by FISH (KMT2A break apart probe), assigning the patient to the adverse risk group. WGS also identified a cytogenetically cryptic SV leading to an ETV6::ABL1 rearrangement in an ALL patient with complex karyotype. Retrospective analysis showed that the aberration had already been present when the patient presented with an MPN and a normal karyotype (CBA) two years earlier. Hence, WGS led to the diagnosis of an ETV6::ABL1-positive neoplasm in blast phase (poor prognosis), which might be treated with tyrosine kinase inhibitors. Of note, in 3 AML patients a 7q deletion or complex SV leading to a 7q deletion resulted in a CDK6::NOM1 fusion transcript and high MNX1 expression. In addition, WGS revealed in one patient with ETP-ALL a cytogenetically cryptic translocation t(7;14)(q36;q32) affecting the IGH locus and the NOM1 gene, accompanied by high MNX1 expression. Elevated MNX1 expression as a result of t(7;12)(q36;p13) is a very rare event in pediatric AML, but our findings might suggest a more important role of MNX1 expression in adult acute leukemia than previously known. Conclusions: This prospective study in a real-world setting 1) demonstrated the diagnostic power of WGTS with an accuracy comparable to gold standard techniques, 2) added important prognostic information, 3) identified potential new therapeutic targets and 4) gave new insights into disease biology. WGTS significantly will improve and refine diagnostics in the future.

High Karyotypic Complexity and Translocations Are Adverse Prognostic Features in Patients with Chronic Lymphocytic Leukemia without TP53 Aberrations Treated with Venetoclax-Based Time-Limited Combinations

Background: The presence of ≥3 (complex karyotype, CKT) and specifically ≥5 (high-CKT, hCKT) chromosomal aberrations (CA) is associated with a poor prognosis in patients (pts) with chronic lymphocytic leukemia (CLL) treated with chemoimmunotherapy (CIT). Studies on the prognostic impact of CKT with targeted agents are not only heterogeneous in size and methodologies but also conflicting, with reports on both negative impact (venetoclax [ven] + rituximab [RVe] and ibrutinib monotherapy) and no impact (ven + obinutuzumab [GVe], ven + ibrutinib, idelalisib + rituximab) on progression-free (PFS) and overall survival (OS). Here, we prospectively evaluate the impact of CKT in a large cohort of pts treated with ven-based time-limited combinations within the GAIA/CLL13 trial. Methods: The phase 3 GAIA/CLL13 trial compared 3 different time-limited ven-based combinations against CIT in fit, treatment-naïve pts with CLL. Pts with TP53 aberrations were excluded. Pts were randomized to CIT (FCR in pts ≤65y; BR in pts >65y), RVe, GVe, or GVe plus ibrutinib (GIVe). Chromosome banding analyses were performed following IL-2/CpG-stimulation, karyotypes were analyzed according to ISCN 2020. No CKT (nCKT) was defined as ≤2, CKT as ≥3, intermediate CKT (iCKT) as 3-4 and hCKT as ≥5 CA. Undetectable MRD (uMRD) was defined as less than 1 CLL cell/10 000 leukocytes measured by 4-color flow cytometry. Results: Chromosome analysis was successfully performed at baseline in 895 of 926 pts. Of these, 672 (RVe: 231; GVe: 218; GIVe: 223) were in the pooled ven population (pooled ven) and 223 in the CIT arm. nCKT was present in 79.4%, 81.0%, 83.5%, and 87.9%, iCKT in 13.5%, 14.7%, 11.5%, 9.4% and hCKT in 7.2%, 4.3%, 5.0% and 2.7% of pts treated with CIT, RVe, GVe and GIVe, respectively. Male gender, del(11q), del(6q) and trisomy 12 were associated with CKT. No correlation was observed between IGHV status or CLL-IPI and CKT. With CIT, uMRD rates in peripheral blood at month 15 were lower in pts with CKT compared with nCKT (37.0% vs 57.1%; OR 0.44, p=0.016). In none of the ven arms, uMRD rates were significantly different between pts with CKT and nCKT (RVe: 52.3% vs 56.7%, GVe: 86.1% vs 86.3%, GIVe: 92.6% vs 92.3%). Across all treatment arms, pts with ≥5 CA had significantly shorter PFS than pts with <5 CA (Figure 1). With CIT, also iCKT was associated with shorter PFS (vs nCKT: HR 2.49, p=0.003), while in none of the ven arms there was a significant PFS difference between iCKT and nCKT (RVe: HR 1.35, p=0.376; GVe: HR 0.94, p=0.912; GIVe: HR 0.38, p=0.341). Pts harboring translocations (TL) (CIT: n=52 [23.4%], pooled ven: n=160 [24.1%]) had inferior PFS compared to pts without TL irrespective of the treatment arm (Figure 2). Focusing on the ven-treated population, this difference was mostly driven by unbalanced TL (vs no TL: HR 3.83, p<0.001) however balanced TL still conferred inferior PFS compared to pts without any TL (HR 1.66, p=0.035). Pts with CKT but without TL had comparable PFS to nCKT (HR 0.53, p=0.217) whereas pts with CKT and TL had significantly shorter PFS (HR 2.79, p<0.001). In pooled ven, particularly pts with TL involving 8q (n=22) or 18q (n=29) showed significantly shorter PFS (HR t(8q;x) 3.94, p<0.001; HR t(18q;x) 2.48, p=0.006), while with CIT numbers were too small for reliable conclusions (n=5 and n=6, respectively). In a multivariate analysis, CKT was identified as an independent prognostic factor for PFS with CIT, while in pooled ven, only hCKT and TL were identified as independent adverse factors for PFS. OS was shorter for CKT vs nCKT with CIT (HR 3.25, p=0.044), but not in the ven arms. At progression (PD), karyotyping data was available in 88 of 161 (54.7%) pts. With CIT, 7 of 20 (35.0%) pts with nCKT at baseline acquired CKT at PD, in the pooled ven group this fraction was substantially smaller (6/45, 13.3%). With CIT, the number of CA increased between baseline and PD (mean, 2.0 to 3.4) while it remained stable after ven (2.1 to 2.0). Conclusions: In pts lacking TP53 aberrations, hCKT (≥5 CA) but not iCKT (3-4 CA) was associated with shorter PFS following treatment with time-limited ven combinations. While this study confirms the adverse impact of CKT with CIT, it identifies hCKT as an adverse prognostic factor in the context of ven-based combination treatment in CLL. Presence of translocations was associated with inferior PFS in all treatment arms. Karyotyping at relapse shows increased genomic complexity after CIT as opposed to ven-based treatments. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MDS Classification - Do We Still Have to Count Blasts?

Background: Since the definition of MDS their classification relied on cytomorphology determining dysplasia, cytopenia and blast count. The revised 4th edition of the WHO classification encompasses only one genetically defined entity (MDS with isolated del(5q)). In the 5th edition of the WHO classification 2 additional entities are defined by genetics: MDS with low blasts and SF3B1 mutation and MDS with biallelic TP53 inactivation. All other are classified still based on morphology into i) hypoplastic MDS, ii) MDS with low blasts and iii) MDS with increased blasts. Aim: As blast counting is subjective and the boundary between low and elevated blasts is arbitrary, we set out to propose a classification for MDS based solely on genetic abnormalities obviating the need for blast counting. Patients and Methods: The cohort comprised 735 de novo MDS. The diagnosis was established following WHO classifications. In all cases chromosome banding analysis and WGS (100x, 2x151bp, NovaSeq) were performed. Variants were called with Strelka2, Manta and GATK using a tumor w/o normal pipeline. For all cases overall survival data was available (median follow-up: 9.3 years). Results: We categorized our cohort 1) according to WHO revised 4th edition, 2) 5th edition, 3) according to blast count (<5%, 5 to <10%, 10 to <20%), and 4) according to numbers of mutations. For all 4 categorizations prognostic differences between subgroups was confirmed. Next, we performed cluster analyses to identify genetic abnormalities associated with blast count. While SF3B1 mutations and isolated del(5q) were associated with low blast count, mutations in ASXL1 and RUNX1 were associated with higher blast count (for all p<0.0001). Further, we evaluated the genomic landscape for associations of genetic parameters. RUNX1 mutations and ASXL1 mutations were highly associated with spliceosome mutations. 49/62 (79%) RUNX1mut MDS and 97/153 (63%) ASXL1mut MDS harbored also spliceosome mutations. On the other hand RUNX1mut, ASXL1mut and spliceosome mutations rarely co-occurred with biallelic TP53 inactivation or complex karyotype (only 5/41 cases and 19/78 cases). 102/735 (14%) cases harbored neither a del(5q), a complex karyotype, biallelic TP53 inactivation nor a mutation in SF3B1, SRSF2, U2AF1, ZRSR2, DNMT3A, TET2, RUNX1 or ASXL1. In 20 of these at least one mutation was found in 21 additional genes known to be mutated in MDS. Based on these data 9 genetically defined non-overlapping hierarchical subgroups (definitions of the respective prior subgroups are exclusion criteria for the following ones) are proposed: 1. biallelic TP53 inactivation 2. complex karyotype, 3. RUNX1 mutated, 4. ASXL1 mutated, 5. del(5q), 6. SF3B1 mutated, 7. mutated DNMT3A, TET2, U2AF1, SRSF2, and/or ZRSR2, 8. presence of at least one mutation in one of 21 other genes recurrently mutated in MDS, 9. none of the genetic markers present that define any of the previous entities. Survival analyses revealed marked differences between these biologically defined entities (figure 1). In univariate cox regression analyses including all 9 genetic categories and blast count the categories biallelic TP53 inactivation, complex karyotype, RUNX1mut, ASXL1mut, SF3B1mut, none of the genetic markers and blast count <5% vs ≥5% and <10% vs ≥10% were significantly associated with OS (for all p<0.002). In multivariate analyses both blast count cut offs did not show a significant impact, while the categories biallelic TP53 inactivation, complex karyotype, RUNX1mut, ASXL1mut, SF3B1mut were independently associated with OS (for all p<0.05). In figure 2 the relationship of classification according to the 4th revised, the 5th edition of the WHO and the purely genetically based classification as proposed here is depicted. Conclusions: Based on karyotype and mutation status of 9 genes complemented by 21 genes in 10-15% of cases, MDS can be separated into 9 biologically distinct subgroups, reflecting biology better than blast counts. These are associated with significant differences in overall survival. RUNX1 and ASXL1 define distinct subgroups within spliceosome mutated MDS potentially driving progression. Given the poor survival of the MDS subgroups with biallelic TP53 inactivation, complex karyotype and RUNX1 mutation it has to be further evaluated whether or not these MDS subtypes should be combined with AML harboring the respective genetic abnormalities for treatment approaches. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High-resolution structural variant profiling of myelodysplastic syndromes by optical genome mapping uncovers cryptic aberrations of prognostic and therapeutic significance

Chromosome banding analysis (CBA) remains the standard-of-care for structural variant (SV) assessment in MDS. Optical genome mapping (OGM) is a novel, non-sequencing-based technique for high-resolution genome-wide SV profiling (SVP). We explored the clinical value of SVP by OGM in 101 consecutive, newly diagnosed MDS patients from a single-center, who underwent standard-of-care cytogenetic and targeted NGS studies. OGM detected 383 clinically significant, recurrent and novel SVs. Of these, 224 (51%) SVs, seen across 34% of patients, were cryptic by CBA (included rearrangements involving MECOM, NUP98::PRRX2, KMT2A partial tandem duplications among others). SVP decreased the proportion of normal karyotype by 16%, identified complex genomes (17%), chromothripsis (6%) and generated informative results in both patients with insufficient metaphases. Precise gene/exon-level mapping allowed assessment of clinically relevant biomarkers (TP53 allele status, KMT2A-PTD) without additional testing. SV data was complementary to NGS. When applied in retrospect, OGM results changed the comprehensive cytogenetic scoring system (CCSS) and R-IPSS risk-groups in 21% and 17% patients respectively with an improved prediction of prognosis. By multivariate analysis, CCSS by OGM only (not CBA), TP53 mutation and BM blasts independently predicted survival. This is the first and largest study reporting the value of combined SVP and NGS for MDS prognostication.

Optical Genome Mapping: A Promising New Tool to Assess Genomic Complexity in Chronic Lymphocytic Leukemia (CLL).

Simple SummaryGenome complexity, detected by chromosome banding analysis or chromosomal microarray analysis, is a poor prognostic factor for chronic lymphocytic leukemia (CLL). Herein, we aimed to assess the performance of optical genome mapping (OGM) for the cytogenomic characterization of CLL patients, with a special focus on risk stratification based on genomic complexity. A cohort of 42 patients enriched in complex karyotypes was assessed by OGM, and the results were compared with those obtained from current methods. Moreover, clinical–biological characteristics and time to first treatment were analyzed according to the OGM-defined complexity. Globally, OGM identified 90% of the known alterations and provided novel structural information about these aberrations in 55% of patients. Regarding genomic complexity, OGM allowed us to identify a complex group (≥10 alterations) displaying enrichment of TP53 abnormalities and poorer evolution. Altogether, we confirmed that OGM is a valuable tool for the cytogenomic assessment and prognostic stratification of CLL patients.Novel treatments in chronic lymphocytic leukemia (CLL) have generated interest regarding the clinical impact of genomic complexity, currently assessed by chromosome banding analysis (CBA) and chromosomal microarray analysis (CMA). Optical genome mapping (OGM), a novel technique based on imaging of long DNA molecules labeled at specific sites, allows the identification of multiple cytogenetic abnormalities in a single test. We aimed to determine whether OGM is a suitable alternative to cytogenomic assessment in CLL, especially focused on genomic complexity. Cytogenomic OGM aberrations from 42 patients were compared with CBA, FISH, and CMA information. Clinical–biological characteristics and time to first treatment (TTFT) were analyzed according to the complexity detected by OGM. Globally, OGM identified 90.3% of the known alterations (279/309). Discordances were mainly found in (peri-)centromeric or telomeric regions or subclonal aberrations (<15–20%). OGM underscored additional abnormalities, providing novel structural information on known aberrations in 55% of patients. Regarding genomic complexity, the number of OGM abnormalities had better accuracy in predicting TTFT than current methods (C-index: 0.696, 0.602, 0.661 by OGM, CBA, and CMA, respectively). A cut-off of ≥10 alterations defined a complex OGM group (C-OGM, n = 12), which included 11/14 patients with ≥5 abnormalities by CBA/CMA and one patient with chromothripsis (Kappa index = 0.778; p < 0.001). Moreover, C-OGM displayed enrichment of TP53 abnormalities (58.3% vs. 3.3%, p < 0.001) and a significantly shorter TTFT (median: 2 vs. 43 months, p = 0.014). OGM is a robust technology for implementation in the routine management of CLL patients, although further studies are required to define standard genomic complexity criteria.

P624: CHROMOTHRIPSIS IN PATIENTS WITH CLL AND COMPLEX KARYOTYPE: PATTERNS OF ABERRATIONS AND PROGNOSTIC VALUE

Background: Chromothripsis is a unique catastrophic event found in 1-5% of chronic lymphocytic leukemia (CLL) patients, associated with poor prognostic factors and short survival (Edelmann et al, 2012; Puente et al, 2015; Leeksma et al, 2021). It is characterized by the presence of multiple genomic rearrangements in one chromosome, resulting in oscillating switches between 2-3 copy number states by chromosomal microarrays (CMA) or next-generation sequencing (Stephens et al, 2011). Optical genome mapping (OGM) is a new method based on the imaging of long DNA molecules (>250kb) labelled at specific motifs, generating a unique pattern. This allows mapping of the genomic location of each molecule and detection of structural and numerical chromosomal abnormalities with high resolution and sensitivity. Aims: 1. To analyze the genome complexity associated with chromothripsis and the patterns of rearrangements detected by OGM; 2. To determine the prognostic impact of chromothripsis in a cohort of CLL patients enriched in complex karyotypes (CK). Methods: A total of 33 patients with chromothripsis detected by CMA were included: 11 with chromothripsis-like patterns (≥7 switches), 22 with classical chromothripsis (≥10 switches). Nine of the cases were studied with OGM (Bionano Genomics). Clinico-biological features and time to first treatment (TTFT) were analyzed. Results were compared with a cohort of 129 patients showing CK without chromothripsis (control group) (Ramos-Campoy et al, 2021). Results: All patients with chromothripsis showed a high genomic complexity (30/33 with CK by chromosome banding analysis and 3/33 classified as complex by CMA [range: 10-24 CNV, being at least three of them >5Mb (range: 3-6)]. Forty-six chromothriptic events were identified. These affected 1-4 chr/patient, but a single chromosome was involved in 25/33 patients (76%). Chromothripsis was distributed throughout all chromosomes, most frequently affecting 3 (n=5), 6 (n=5) and 13 (n=5). The events were mainly multiple losses (25/46) or alternated gains and losses (19/46). Size and coordinates of CNVs detected by OGM and CMA were highly concordant. OGM detected multiple rearrangements cryptic by CMA (median: 9/chromothriptic event). Two patterns of chromothripsis-related rearrangements were identified: one clustered in the chromothriptic region (3/9) and the other involving both chromothriptic and non-chromothriptic chromosomes (range: 1-6 chr) (6/9). One patient showed 36 rearrangements throughout the genome, suggesting the coexistence of chromothripsis and chromoplexy, characterized by the presence of multiple chained translocations (Figure). Patients with chromothripsis showed a higher frequency of TP53del/mut (70% vs 39%; p=0.001) and a shorter TTFT (15m vs 2m; p=0.013) than the control group. No differences were observed for IGHV and del(11q) status. When stratifying patients based on TP53 status, the presence of chromothripsis was not associated with shorter TTFT. In the multivariate analysis including TP53, genomic complexity by CMA and chromothripsis, only TP53 was statistically significant (HR=1.6; p=0.029). Image:Summary/Conclusion: 1. Chromothriptic events involve multiple loci in CLL patients; 2. OGM allows not only the detection of CNV but also the identification of two rearrangement patterns associated with chromothripsis (clustered or involving non-chromothriptic chromosomes) whose clinical impact should be further explored. 3. In CLL patients with CK, the adverse prognosis of chromothripsis is associated with a higher frequency of TP53del/mut.

PB1891: VARIANT PHILADELPHIA CHROMOSOME IN CML

Background: Chronic myeloid leukemia is a clonal malignant disorder of a pluripotent hematopoietic stem cell characterized by the Philadelphia chromosome and the reciprocal translocation t(9:22)(q34;q11). Almost 5 to 10 percent of chronic myeloid leukemia cases demonstrate variant Philadelphia translocations. Aims: To contribute to the description of patients’ clinical characteristics and prognosis with variant Ph chromosome-positive leukemia, we report a Ph chromosome-positive CML in the chronic phase characterized by an unusual variant Ph chromosome. Methods: An elderly male Libyan patient was suspected to have chronic myeloid leukemia. Cytogenetic and molecular exploration were conducted using bone marrow and blood samples. Cytogenetic analysis was performed on lymphocytes and bone marrow cells after short-term culture (48 hours). The cells were treated with colchicine and hypotonic KCl solution. The pellet was fixed and washed in methanol-acetic acid (3:1). The cells were resuspended in fixative and dropped onto slides. Chromosome-banding analysis was performed using RHG and GTG banding techniques. Three karyotypes and 25 metaphases were analyzed according to the International System for Human Cytogenetic Nomenclature. Multiplex reverse transcription-PCR (RT-PCR) was used to identify the BCR-ABL1 transcript. RT-PCR was performed according to European recommendations, as described previously. Results: Clinical assessment confirmed the first chronic phase of a chronic myeloid leukemia. Chromosomal formula showed an homogeneous structural translocation: t(1;9;12;22)(p35-36;q34;p13;q11) involving four chromosomal regions, 1p35-36, 9q34, 12p13 and 22q11.2. Molecular investigation confirmed the Ph nature of the chromosomal abnormality by showing the BCR-ABL1 transcript. The patient was treated by using Imatinib molecule with a favorable therapeutic response. Summary/Conclusion: A new variant Ph chromosome-positive CML in the chronic phase is reported. It is a four-way Ph translocation t(1;9;12;22)(p35-36;q34;p13;q11) involving four chromosomal regions, 1p35-36, 9q34, 12p13 and 22q11.2. Imatinib, the first-generation TKI treatment was efficient.

PB1912: MYELODYSPLASTIC SYNDROME WITH ISOLATED 5Q DELETION AND ASSOCIATED 5Q DELETION TO 13Q DELETION

Background: The 5q deletion is the classic chromosomal abnormality in myelodysplastic syndrome (MDS). Critical regions are 5q31 and 5q33, leading to gene haplodeficiency and other pathogeneic mechanisms. 13q deletion occurs in about 2% of MDS cases. This deletion involved band 13q14. Aims: Here we describe cytogenetic abnormalities in a Tunisian cohort of MDS patients and we shed the light on cases with 5q deletion. Methods: Cytogenetic analyses of bone marrow cells were performed according to standard procedures for 12 Tunisian patients presenting with MDS. Cytogenetic analysis was performed on lymphocytes and bone marrow cells. Cultured cells were treated with colchicine and hypotonic KCl solution. The pellet was fixed and washed in methanol-acetic acid (3:1). The cells were resuspended in fixative and dropped onto slides. Chromosome-banding analysis was performed using RHG and GTG banding techniques. Three karyotypes and 25 metaphases were analyzed according to the International System for Human Cytogenetic Nomenclature. Results: Cytogenetic abnormalities were detected in 9 cases and del5q was recorded in four patients (3 patients with refractory anemia (RA) and a fourth with RA and excess blasts-T (RAEB-T)). The del5q was isolated in two RA cases as a del (5) (q13 q33) in a new diagnosed and a treated patients. It was associated in two patients: a RA patient explored during the evolution, harboring a del (5) (q12 q33) associated to a del (13) (q12 q14) and the RAEB-T patient harbouring a del (5) (q21 q31) associated to a clonal hyperploidy. Summary/Conclusion: The diagnosis of MDS was based on cytomorphology and cytogenetics. However, nowadays, an increasing number of molecular aberrations have been discovered and included in the WHO revised new classification of myeloid neoplasms. The del13q described in MDS seems to be a marker of the evolution of the clonal cells harboring 5q deletion. However, the molecular basis and the implicated genes of the deletion of the long arm of chromosome 13 associated to the 5q deletion clone remain until now not well explored.

FISH improves risk stratification in acute leukemia by identifying KMT2A abnormal copy number and rearrangements

Most cases of acute leukemia (AL) with KMT2A rearrangement (KMT2A-r) have a dismal prognosis. Detection of this aberration in Chinese adult patients relies on reverse transcription polymerase chain reaction (RT-PCR) and chromosome banding analysis (CBA). The fluorescence in situ hybridization (FISH) probe for KMT2A detects KMT2A-r and copy number variation (CNV) but is not routinely used as a detection technique. This study investigated the potential value of FISH in the treatment of AL by performing FISH along with CBA and RT-PCR in 269 de novo cases of AL. The three detection techniques were compared in identification of KMT2A-r, and the applicability of FISH for detecting KMT2A CNV was evaluated. Twenty-three samples were identified as positive for KMT2A-r (20 using FISH, 15 using RT-PCR, 16 using CBA, and eight according to all three). FISH also identified 17 KMT2A CNV, 15 with gains and two with deletions. Ten patients with acute myeloid leukemia (AML) harboring KMT2A CNV had a complex karyotype, a negative prognostic factor in AML. Adding FISH of KMT2A to routine detection leads to more accurate detection of KMT2A-r and improved identification of KMT2A CNV, which would benefit patients by improving the risk stratification in AL.

Typical, atypical and cryptic t(15;17)(q24;q21) (PML::RARA) observed in acute promyelocytic leukemia: A retrospective review of 831 patients with concurrent chromosome and PML::RARA dual-color dual-fusion FISH studies.

The diagnosis of acute promyelocytic leukemia (APL) relies on the identification of PML::RARA fusion. While the majority of APL cases harbor a typical t(15;17)(q24;q21), atypical genetic mechanisms leading to the oncogenic PML::RARA fusion have been reported yet their frequency and scope remain poorly characterized. We assessed the genetic findings of 831 cases with APL investigated with concurrent chromosome banding analysis and dual-color dual-fusion fluorescence in situ hybridization (D-FISH) analysis at our institution over an 18.5-year timeframe. Seven hundred twenty-three (87%) cases had a typical balanced t(15;17) with both testing modalities. Atypical karyotypic results including complex translocations, unbalanced rearrangements and insertional events occurred in 50 (6%) cases, while 6 (0.7%) cases were cryptic by conventional chromosome studies despite PML::RARA fusion by D-FISH evaluation. Atypical FISH patterns were observed in 48 (6%) cases despite apparently balanced t(15;17) on chromosome banding analysis. Two hundred fifty (30%) cases displayed additional chromosome abnormalities of which trisomy/tetrasomy 8 (37%), del(7q)/add(7q) (12%), and del(9q) (7%) were most frequent. Complex and very complex karyotypes were observed in 81 (10%) and 34 (4%) cases, respectively. In addition, 4 (0.5%) cases presented as an apparently doubled, near-tetraploid stemline clone. This report provides the largest appraisal of cytogenetic findings in APL with conventional chromosome and PML::RARA D-FISH analysis. By characterizing the frequency and breadth of typical and atypical results through the lens of these cytogenetic testing modalities, this study serves as a pragmatic source of information for those involved in the investigation of APL in both the clinical and research laboratory settings.

IGH Translocations in Chinese Patients With Chronic Lymphocytic Leukemia: Clinicopathologic Characteristics and Genetic Profile.

Immunoglobulin heavy chain translocations (IGH-t) have occasionally been reported in Chinese patients with chronic lymphocytic leukemia (CLL). The objective of the present study was to identify the clinicopathologic features of patients with IGH-t CLL and compare them with those of patients with non-IGH-t CLL. We performed fluorescence in situ hybridization (FISH) based on a routine CLL prognostic FISH panel using IGH, IGH-BCL2, BCL3, IGH-CMYC, and BCL6 FISH probes. Furthermore, we retrospectively evaluated the clinical features of 138 newly diagnosed CLL patients via chromosome banding analysis (CBA), FISH, and targeted next-generation sequencing. IGH-t was identified in 25 patients (18.1%). Patients with IGH-t CLL had lower flow scores than those with non-IGH-t CLL. The most frequent translocation was t(14;18) (10 patients), followed by t(14;19) (3 patients), and t(2;14)(p13;q32), t(7;14)(q21.2;q12), t(9;14)(p13;q32) (3 patients). The remaining nine patients included three with abnormal karyotypes without translocation involving 14q32, four with a normal karyotype, and two who failed CBA. The most frequently concomitant FISH-detected aberrations were 13q deletion, followed by +12 and TP53 deletion, while one case involved ATM deletion. Complex karyotypes were detected in five patients with IGH-t CLL, in whom all partner genes were non-BCL2. Available mutational information indicated that KMT2D mutation was the most frequent mutation among tested 70 patients, while TP53 mutation was the most frequent mutation in the IGH-t group. Moreover, the IGH-t group had higher FBXW7 (P=0.014) and ATM (P=0.004) mutations than the non-IGH-t group, and this difference was statistically significant. Our study demonstrates that IGH-t is not uncommon among Chinese CLL patients, and that its partner genes are multiple. The gene mutational profile of the IGH-t group was distinct from that of the non-IGH-t group, and the concomitant chromosomal abnormalities within the IGH-t CLL group differed. Thus, identification of IGH-t and its partner genes in CLL patients may help further refine risk stratification and strengthen the accurate management in CLL patients.

Cytogenetics in Chronic Lymphocytic Leukemia: ERIC Perspectives and Recommendations.

Mounting evidence underscores the clinical value of cytogenetic analysis in chronic lymphocytic leukemia (CLL), particularly as it allows the identification of complex karyotype, that has recently emerged as a prognostic and potentially predictive biomarker. That said, explicit recommendations regarding the methodology and clinical interpretation of either chromosome banding analysis (CBA) or chromosome microarray analysis (CMA) are still lacking. We herein present the consensus of the Cytogenetic Steering Scientific Committee of ERIC, the European Research Initiative on CLL, regarding methodological issues as well as clinical interpretation of CBA/CMA and discuss their relevance in CLL. ERIC considers CBA standardized and feasible for CLL on the condition that standards are met, extending from the use of novel mitogens to the accurate interpretation of the findings. On the other hand, CMA, is also standardized, however, robust data on its clinical utility are still scarce. In conclusion, cytogenetic analysis is not yet mature enough to guide treatment choices in CLL. That notwithstanding, ERIC encourages the wide application of CBA, and potentially also CMA, in clinical trials in order to obtain robust evidence regarding the predictive value of specific cytogenetic profiles towards refining risk stratification and improving the management of patients with CLL.

Biology and Treatment of High-Risk CLL: Significance of Complex Karyotype.

Several reports highlight the clinical significance of cytogenetic complexity, namely, complex karyotype (CK) identified though the performance of chromosome banding analysis (CBA) in chronic lymphocytic leukemia. Indeed, apart from a number of studies underscoring the prognostic and predictive value of CK in the chemo(immune)therapy era, mounting evidence suggests that CK could serve as an independent prognosticator and predictor even in patients treated with novel agents. In the present review, we provide an overview of the current knowledge regarding the clinical impact of CK in CLL, touching upon open issues related to the incorporation of CK in the clinical setting.

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.

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.

Normal FISH CLL Represents a Heterogeneous Subgroup Where Prognosis Can be Refined with IGHV Mutational Status

Normal FISH CLL Represents a Heterogeneous Subgroup Where Prognosis Can be Refined with IGHV Mutational Status