Myeloid Mutations Research Articles

Survival and Outcomes after Relapse in Newly Diagnosed Acute Myeloid Leukemia (AML) Patients Treated with Venetoclax-Based Chemotherapy

Background Venetoclax (bcl-2 inhibitor) was first approved in 2018 for use in combination with hypomethylating agents (HMA) or low-dose cytarabine (LDAC) for treatment of newly diagnosed acute myeloid leukemia (AML) in older patients, or those who have comorbidities that preclude use of intensive induction chemotherapy. However, AML is also known to evolve and undergo clonal selection when treated with venetoclax, leading to treatment failure. Mechanisms for adaptive resistance include kinase activation (e.g. FLT3-ITD), clones that acquire biallelic silencing of TP53, and monocytic AML cells that upregulate MCL1 to evade BCL2 blockade. Other issues with venetoclax-based regimens include prolonged cytopenias and increased risk of infection. Our aim was to investigate survival after relapse, subsequent responses to second- line agents, and pattern of infections in patients treated with first-line venetoclax-based combination therapies. Methods This was a retrospective study of patients who received venetoclax-based regimens for untreated AML or myelodysplastic syndrome with excess blasts (MDS-EB) at LAC+USC Medical Center and Norris Cancer Center in Los Angeles, CA from January 1, 2018 to June 30, 2022. The electronic medical record was reviewed for demographics, disease characteristics, clinical history, transplant status, and survival. Results 61 patients received frontline venetoclax-based regimens for untreated AML or MDS-EB with a mean age of 65 (range 22-84); 36% were female, 44% non-Hispanic white and 31% were Hispanic/Latino. 41% had AML-MRC (myelodysplasia-related changes), 36% had De Novo AML, 16% had therapy-related AML, and 7% had MDS-EB. The majority (70%) were adverse risk group per ELN 2017 and 46% had complex cytogenetics. 58 patients had available myeloid mutation panel at time of diagnosis. The most common mutations (median allele frequencies) were: TP53=33% (0.45), ASXL1=17% (0.37), NPM1=16% (0.39), IDH2=9% (0.42), IDH1=7% (0.29), FLT3-ITD=5% (0.51). 70% received venetoclax with decitabine, 21% received venetoclax with azacitidine, and 2% received venetoclax with LDAC. Median number of cycles was 3 (1-16). 10 patients underwent allogeneic stem cell transplant. Out of 61 patients, 12 (20%) had primary refractory disease and 23 (38%) relapsed on frontline venetoclax. 49% of these relapsed/refractory patients received salvage chemotherapy. The median time to relapse and number of cycles prior to relapse was 6.3 months (1.6-17.4) and 4 (1-12) respectively. There was no correlation between cytogenetics and genetic variables and risk of relapse. Only 3 (19%) out of 16 patients and 3 (27%) out of 11 patients who received second- and third-line treatment achieved CRi respectively. Myeloid mutation panel was available for 12 patients at time of relapse. Most common mutations were FLT3-ITD/TKD (n=5), TP53 (n=3) and NPM1 (n=3). Six patients acquired new mutations at the time of relapse: FLT3-ITD (n=2), TP53 (n=1), WT1 (n=2) and ETV6 (n=1). Median time from relapse to death was 3 months (1-23). Median overall survival was 10.2 months. Median survival in responder compared to non-responders after cycle 1 and cycle 2 was 16.8 vs. 5.3 months (p <0.001) and 19.4 months vs. 7.4 months (p=0.031) respectively. Median survival in patients with relapse compared to non-relapse was 12.1 vs 6 months (p=0.187). 40 (66%) patients died; major causes of death were disease progression (50%), bleeding (10%) and infection (25%). 28% patients died in intensive care unit. 9 patients had fungal infections; most common were aspergillosis (n=5) and mucormycosis (n=2). Conclusions Venetoclax-based regimens are effective options for untreated AML in elderly patients. Response to first and second cycle of combination treatment predict survival. Although we observed clonal evolution in relapsed patients, it did not affect their survival. FLT3-ITD was a commonly acquired mutation at time of relapse, thus patients received salvage with targeted agents. Also, as patients relapse, the chance of attaining CR/CRi with subsequent lines of treatment is less than 30% and risk of invasive fungal infection was approximately 15%. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Impact of Allelic State on Overall Survival in TP53-mutant Acute Myeloid Leukemia (AML) and Higher Risk Myelodysplastic Syndromes (HR-MDS)

Introduction: Mutations in TP53 confer an adverse prognosis in patients (pts) with AML and HR-MDS. There are conflicting results regarding the prognostic impact of mono-allelic vs bi-allelic and mono-hit vs multi-hit TP53 mutation status on prognosis in AML and HR-MDS. Additionally, assessment of loss-of-heterozygosity (LOH) status is not routinely available in many settings limiting the clinical implementation of TP53 mutational status. Methods: We took advantage of a large, randomized study with extensive clinical and molecular annotation to analyze 61 patients (pts) with TP53-mutated (TP53-mut) disease and to compare them to 144 TP53 wild-type pts. All pts received azacitidine and half were randomized to receive the anti-PDL1 antibody durvalumab, which did not improve clinical outcomes (Zeidan et al. Blood Advances 2022). A 38 gene-targeted myeloid mutation analysis from screening bone marrow was performed at Munich Leukemia Laboratory. Only pathogenic TP53 variants with variant allele frequency (VAF) ≥2% were included. Pts with a common SNP or VUS in the TP53 gene were classified as TP53-WT in the analysis. Multi-hit TP53 mutations were defined as previously published by the International Consensus Conference (ICC; Arber et al. Blood 2022) as ≥2 distinct TP53 mutations (VAF ≥10%) or a single TP53 mutation associated with either: 1) cytogenetic deletion involving chromosome 17p (e.g., del(17p) or monosomy 17); 2) a VAF of >50%; or 3) with any complex karyotype. We also defined multi-hit TP53 mutations as proposed by Grob et al. (Blood 2022) as (I) ≥2 TP53 gene variants irrespective of VAF, (II) ≥1 TP53 gene variant co-occurred with a cytogenetic deletion involving chromosome 17p, or (III) TP53 mutations with VAF >55%. Based on multiple prior studies (Grob et al. Blood 2022, Weinberg et al. Blood Advances 2022) patients with AML and HR-MDS with TP53 mutations were analyzed as one disease entity. Results: 37 had TP53-mut AML, 89 were TP53-WT AML, 24 had TP53-mut HR-MDS, and 55 were TP53-WT HR-MDS pts. The average VAF for TP53 mutations was 37%, and 90% had ≥10% VAF. 93% of TP53 mutations occurred in the p53 DNA-binding domain. TP53-mut pts had fewer co-occurring mutations compared with TP53-WT pts (mean number of mutations: 1.8 [SD: 0.77] for TP53-mut pts (including TP53) vs 2.9 [SD: 1.4] for TP53-WT pts; Wilcoxon P <0.001) with distinct patterns in the distribution of co-occurring mutations. In terms of TP53 allelic status, 22 (11.0%) were mono-hit and 41 (20.5%) pts were multi-hit as defined by Grob et al, while 7 (3.4%) and 56 (28.0%) pts were classified as mono-hit and multi-hit per ICC definition, respectively. In the AML cohort, TP53-mut pts were more likely to have poor-risk cytogenetics (59.5% vs 11.2%; p<0.001), therapy-related myeloid neoplasm (10.8% vs 1.1%; p=0.042), and lower bone marrow blast percentage (27.5% vs 35.5%; p=0.001) compared to TP53-WT. Among MDS pts, TP53-mut pts were more likely to have secondary MDS (20.8% vs 1.8%; p=0.013), very poor risk cytogenetics by IPSS-R (75% vs 14.6%; p<0.001), and very high risk IPSS-R score (62.5% vs 34.6%; p=0.039). There were no statistically significant differences in terms of baseline demographics and other clinical characteristics. There was no difference in overall response rate (ORR) between TP53-mut and TP53-WT pts (AML: ORR: 35.1% [95% CI: 20.7% - 52.6%] vs. 33.7% [95% CI: 24.2% - 44.6%]; p=1.000; MDS: ORR: 41.7% [95% CI: 22.8% - 63.1%] vs. 60.0% [95% CI: 45.9% - 72.7%]; p=0.208). TP53 allelic status did not influence OS using either definition (Grob et al: mono-hit: median OS 7.6 months [95% CI:5.4-10.6 months]; multi-hit 10.6 months [95% CI: 9.1-13.0 months] (Figure A); ICC definition: 8.0 months (95% CI: 6.1-20+ months) and 9.5 months [95% CI: 7.7-10.9 months], (Figure B)) and was inferior to TP53-WT (median OS: 18.9 months; 95% CI: 15.5-22.9 months). Conclusion: Outcomes of AML and MDS pts with TP53 mutations are worse compared to TP53-WT without differences between mono-hit vs multi-hit status as defined by the ICC and Grob et al. This suggests that either the negative prognostic impact of a multi-hit TP53 allelic status can be overcome by treatment with azacitidine or that a distinction by TP53 allelic status in newly diagnosed AML/HR-MDS pts treated with azacitidine does not provide additional prognostic information. Whether incorporating TP53 LOH status is necessary to further refine risk assessment warrants additional studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Blood Count Parameters As Early Predictors of Clonal Cytopenia of Undetermined Significance (CCUS) Using a Diverse Ethnoracial Data Set

Background: Hematologic malignancies are a spectrum of disease that frequently have an insidious onset and may be difficult to diagnose. Improved screening using basic complete blood count parameters are needed to enhance early detection of these processes. Aging results in an expansion of hematopoietic clones that may predispose individuals to subsequent development of a hematologic malignancy. Recent studies with next generation sequencing (NGS) and whole-exome sequencing have identified certain somatic mutations known as DTA mutations that are associated with aging (e.g., DNMT3A, TET2, and ASXL1). These mutations, although more commonly seen in myeloid malignancies, are initially benign but may develop into overt hematologic malignancy and are collectively termed clonal hematopoiesis of indeterminate potential (CHIP). Similarly, individuals with cytopenia and clonal mutations are designated as clonal cytopenia of undetermined significance (CCUS). However, these mutations carry a risk of progression to hematologic malignancy. Animal models have shown that clonal hematopoiesis can accelerate cardiovascular mortality due to increased atherogenesis by causing inflammation and endothelial injury via upregulation of inflammasome pathways. Also, it has already been shown that clonal hematopoiesis with increasing number of clonal mutations, higher variant allele frequency and mutations in known driver genes have a higher risk of progression to hematopoietic malignancy (acute myeloid leukemia, AML). Certain acute phase reactants, like C-reactive protein (CRP) or erythrocyte sedimentation rate (ESR) have been used in prior studies as markers of inflammation and are strongly associated with variations in red cell size as determined by the red cell distribution width (RDW), reflecting chimeric clonal origins. We therefore propose that RDW and mean corpuscular volume (MCV) with or without other blood count parameters (red cell count, white blood cell count and platelet count) might be used as important predictors of progression of myeloid neoplasms, especially myelodysplastic syndromes (MDS), clonal cytopenias (CCUS) and acute myeloid leukemias (AML). Methods: Utilizing Epic's self-service reporting tool SlicerDicer, cohort discovery was performed on the Caboodle Patients data model. Cohort criteria included individuals with the cytopenia diagnostic grouper that had undergone blood or bone marrow myeloid panel next generation sequencing, that also had concurrent or historic complete blood count (CBC) results. Patients with known acute leukemias were excluded. Once the cohort was identified, a data pull was performed that captured CBC values, age, gender, race, and ethnicity, in addition to the NGS results. The NGS results were ranked into 3 classes from 0 to 2. Class 0 included results with no apparent disease, while class 2 results included high grade myeloid mutations (i.e. mutations in NRAS, KRAS, TP53, RUNX1 or ASKL1). Class 1 mutations were considered low grade and featured mutations not seen in class 2. The collated data was analyzed using SAS software. Results: Overall 72 patients met inclusion criteria. Patients ranged in age from 23 to 92 years, 48% identified as female and 52% identified as male with a mixture of race and ethnicity (Table 1). Abnormalities in several CBC components including MCH, MCHC, RBC count, and hemoglobin concentration were associated with increased odds of identifying those with malignant myeloid processes (Figure 1). It is notable that no mutations in FLT3 or KIT were observed in this cohort. Discussion: Based on our preliminary data, these cost-effective parameters can be added to an algorithm for risk assessment of hematologic neoplasms and better predict which patients will further benefit from targeted gene testing. Also, additional data will be helpful and applying this data to modern machine learning techniques will further the clinical application of these findings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MDS-287 Genetic Landscape of Somatic Myeloid Mutations in the Presence of Rare TERT Variants and Their Relation to Myeloid Neoplasia

Germline variants in the TERT gene may lead to impaired telomere maintenance implicated in the pathogenesis of myelodysplastic syndromes (MDS) by unknown mechanisms. To explore the clinical and genetic associations of germline TERT variants in patients with suspicion of myeloid neoplasm. We selected patients tested through our institutional amplicon-based sequencing panel (42 myeloid genes, including exons 2 to 16 of TERT, read depth >250X) that carried a TERT variant with allele frequency between 35% and 65%. Variants were classified into those with CADD score >20 and <20 to identify the top 1% according to predicted deleteriousness. Results were correlated with MDS diagnosis and clinical and survival information. Division of Hematology (Mayo Clinic). Research protocol approved by Institutional Review Board. Unselected cohort of patients screened with a myeloid mutation gene panel due to clinical suspicion of myeloid neoplasia. 55 different TERT variants (46 missense, 3 splice sites, 4 synonymous, and 2 in-frame deletions) were identified in 148 individuals from April 2016 to November 2021. None of these patients were diagnosed with a telomere biology disorder and no telomere length measurement was available. All variants were clinically classified as uncertain significance. 17 (12%) patients carried CADD>20 TERT variants of which, 59% (10 patients) were diagnosed with MDS. [5 with <5% bone marrow blasts (4 with normal karyotype) and 5 with 5-19% blasts (all with complex karyotype)] This percentage was higher compared to CADD<20 TERT carriers (34%, 49 patients, p=0.048 by Chi-square test). No major differences were found in age at diagnosis (median 70 vs 73-year-old, p=0.2305), AML-free survival (median 10 vs 15.5 months since diagnosis, p=0.4674), or percentage of carriers of additional variants in myeloid-related genes (60% vs 64%). The most frequently mutated genes in CADD>20 carriers were TP53 (22%), TET2 (18%) and RUNX1 (9%) while in CADD<20 carriers were TET2 (14%), ASXL1 (11%), and SRSF2 (9%). TP53 variants were significantly enriched in CADD>20 TERT carriers. (22.7% vs 7.4%, p=0.0169) Conclusions: CADD>20 TERT variant carriers were more likely to be associated with a diagnosis of MDS and with somatic TP53 mutations than CADD<20 carriers.

ANALYTICAL PERFORMANCE OF A DNA/RNA-BASED NEXT-GENERATION SEQUENCING (NGS) PANEL FOR MYELOID NEOPLASMS

Myeloid neoplasms (MNs) are a heterogeneous group of neoplasms including acute myeloid leukemia, myelodysplastic syndrome, myeloproliferative neoplasms and myelodysplastic syndrome/myeloproliferative neoplasms. MNs show a complex genetic basis, and the number of alterations to be analyzed for risk diagnostic, stratification and management of patients increases continuously. Consequently, there is a demand for multiple molecular and cytogenetic techniques with the aim of detecting a wide variety of genetic abnormalities. Since conventional techniques are insufficient to study all such abnormalities, the NGS panel has become a useful tool for broad characterization of the spectrum of mutations in MNs. However, before NGS panels can be introduced in the laboratory routine, their performance should be evaluated. In this context, we describe the analytical performance of the Oncomine™Myeloid Research Assay (OMA) (Thermo Fisher), a DNA/RNA-based amplicon sequencing panel, that evaluated simultaneously 40 genes (17 full genes and 23 hotspots genes) and 29 fusions drivers genes (over 600 partners) known to be recurrently mutated in MNs. Commercial controls Seraseq Myeloid Mutation DNA and the Seraseq Myeloid Fusion RNA were used for sensitivity, specificity, accuracy, repeatability, reproducibility and limit of detection (LoD) testing. Prospective sequencing was performed for samples from 34 patients presenting with suspected or confirmed MN. Libraries was prepared by manual method and sequenced by the Ion S5 Sequencer. Data obtained were analysed on Ion Reporter 5.18 software. The OMA assay showed good performance in terms of depth of coverage, on-target reads, and uniformity. The mean reads lenght ranged from 223 to 231 bp for DNA sample and from 88 to 124 bp for RNA, within the expected standard for the panel. The panel achieved 98,8% and 100% concordance with DNA and RNA controls, reaching analytical sensitivity of 99% and 100% for DNA and RNA, respectively. Regarding the type of DNA mutation: single nucleotide variants (SNP), indels or indels hot spots, the sensitivity found was 100%, 96% and 95%, respectively. The accuracy and analytical specificity was 100% for DNA panel. The LoD was 5% variant allele fraction (VAF) for DNA (SNP and indel) and 1-log reduction from initial number of reads counts for RNA fusion genes. Repeatability and reproducibility were 98% for DNA and 100% for RNA. However, the coefficient of variation (CV) within-run and between-run in VAF was >20% for large indels in CALR, FLT3 (ITD) and SRSF2 . The vertical coverage of each sample was inspected with a coverage threshold of 100X. Some amplicons in the assay failed repeatedly (ASXL1_1.875, BCOR_8.202477, BRAF_18.30633, CEBPA_1.1.86676, PRPF8_12.173121, SH2B3_1.85663). However, the OMA is unable to detect specific variants in ASXL1 (c.1900_1922del23 and c.1934_1935insG) and CEBPA (c.68_69insC). This is a known limitation of the Ion Torrent chemistry for accurate sequencing of large homopolymers and high GC content regions. Of the 34 patient samples, 28 (87.5%) had at least one reportable variant. A total of 58 DNA variants and six gene fusions were detected. Some variants were confirmed by orthogonal techniques. In conclusion, OMA is a highly accurate and reproducible NGS panel for the detection of common genetic abnormalities in MNs.

Mutational Landscape of Patients Referred for Elevated Hemoglobin Level.

Background: Since the identification of JAK2 V617F and exon 12 mutations as driver mutations in polycythemia vera (PV) in 2005, molecular testing of these mutations for patients with erythrocytosis has become a routine clinical practice. However, the incidence of myeloid mutations other than the common JAK2 V617F mutation in unselected patients referred for elevated hemoglobin is not well studied. This study aimed to characterize the mutational landscape in a real-world population of patients referred for erythrocytosis using a targeted next-generation sequencing (NGS)-based assay. Method: A total of 529 patients (hemoglobin levels >160 g/L in females or >165 g/L in males) were assessed between January 2018 and May 2021 for genetic variants using the Oncomine Myeloid Research Assay (ThermoFisher Scientific, Waltham, MA, USA) targeting 40 key genes with diagnostic and prognostic implications in hematological conditions (17 full genes and 23 genes with clinically relevant “hotspot” regions) and a panel of 29 fusion driver genes (>600 fusion partners). Results: JAK2 mutations were detected in 10.9% (58/529) of patients, with 57 patients positive for JAK2 V617F, while one patient had a JAK2 exon 12 mutation. Additional mutations were detected in 34.5% (20/58) of JAK2-positive patients: TET2 (11; 19%), DNMT3A (2;3.4%), ASXL1 (2; 3.4%), SRSF2 (2; 3.4%), BCOR (1; 1.7%), TP53 (1; 1.7%), and ZRSR2 (1; 1.7%). Diagnosis of PV was suspected in 2 JAK2-negative patients based on the 2016 World Health Organization (WHO) diagnostic criteria. Notably, one patient carried mutations in the SRSF2 and TET2 genes, and the other patient carried mutations in the SRSF2, IDH2, and ASXL1 genes. Three JAK2-negative patients with elevated hemoglobin who tested positive for BCR/ABL1 fusion were diagnosed with chronic myeloid leukemia (CML) and excluded from further analysis. The remaining 466 JAK2-negative patients were diagnosed with secondary erythrocytosis and mutations were found in 6% (28/466) of these cases. Conclusion: Mutations other than JAK2 mutations were frequently identified in patients referred for erythrocytosis, with mutations in the TET2, DNMT3A, and ASXL1 genes being detected in 34.5% of JAK2-positive PV patients. The presence of additional mutations, such as ASXL1 mutations, in this population has implications for prognosis. Both the incidence and mutation type identified in patients with secondary erythrocytosis likely reflects incidental, age-associated clonal hematopoiesis of indeterminate potential (CHIP).

Germline ERCC excision repair 6 like 2 (ERCC6L2) mutations lead to impaired erythropoiesis and reshaping of the bone marrow microenvironment.

Despite the inclusion of inherited myeloid malignancies as a separate entity in the World Health Organization Classification, many established predisposing loci continue to lack functional characterization. While germline mutations in the DNA repair factor ERCC excision repair 6 like 2 (ERCC6L2) give rise to bone marrow failure and acute myeloid leukaemia, their consequences on normal haematopoiesis remain unclear. To functionally characterise the dual impact of germline ERCC6L2 loss on human primary haematopoietic stem/progenitor cells (HSPCs) and mesenchymal stromal cells (MSCs), we challenged ERCC6L2-silenced and patient-derived cells ex vivo. Here, we show for the first time that ERCC6L2-deficiency in HSPCs significantly impedes their clonogenic potential and leads to delayed erythroid differentiation. This observation was confirmed by CIBERSORTx RNA-sequencing deconvolution performed on ERCC6L2-silenced erythroid-committed cells, which demonstrated higher proportions of polychromatic erythroblasts and reduced orthochromatic erythroblasts versus controls. In parallel, we demonstrate that the consequences of ERCC6L2-deficiency are not limited to HSPCs, as we observe a striking phenotype in patient-derived and ERCC6L2-silenced MSCs, which exhibit enhanced osteogenesis and suppressed adipogenesis. Altogether, our study introduces a valuable surrogate model to study the impact of inherited myeloid mutations and highlights the importance of accounting for the influence of germline mutations in HSPCs and their microenvironment.

P730: GENETIC LANDSCAPE OF SOMATIC MYELOID MUTATIONS IN THE PRESENCE OF RARE TERT VARIANTS AND THEIR RELATION WITH MYELOID NEOPLASIA

Background: Genetic variants in TERT, the gene that encodes telomerase reverse transcriptase, can result in accelerated telomere attrition. Impaired telomere maintenance is implicated in the pathogenesis of myelodysplastic syndromes (MDS), with mechanisms remaining to be elucidated. Aims: To explore the clinical and genetic associations of TERT variants in an unselected cohort of patients screened with a myeloid mutation next generation sequencing panel due to clinical suspicion of a myeloid neoplasia. Methods: Using a research protocol approved by the Mayo Clinic Institutional Review Board, we performed a retrospective chart review to identify a cohort of unselected individuals within all historical data at our institution that had undergone somatic mutational analysis for suspected myeloid neoplasia using our insitutional amplicon based next generation sequencing panel (42 genes, including exons 2 to 16 of TERT, with a read depth of at least 250X). TERT variants were classified into those with CADD score >20 and <20 in line with previous literature to identify variants within the top 1% according to their predicted deleteriousness. Additionally, we identified within our cohort individuals that received a diagnosis of MDS, clinical and survival information. Results: We identified 55 different TERT variants (46 missense, 3 in canonical splice sites, 4 synonymous and 2 small inframe deletions) in 148 individuals tested from April of 2016 to November of 2021 (Fig. 1A&B). The five most recurrent variants were c.1234C>T (48 patients), c.1323_1325del (29 patients), c.3164C>T (5 patients), c.604G>A (4 patients) and c.969G>A (4 patients), all with CADD<20. None of these patients were diagnosed with a bona fide telomere biology disorder and no clinical information about telomere length status was available. All variants were considered germline since their allele frequency was near 50%, although no germline confirmation was performed. All variants were clinically classified as uncertain significance according to the American College of Medical Genetics guidelines. Of the 148 total patients, 17 (12%) carried TERT variants with CADD>20 that were distributed thorough all the gene domains with no clustering or presence of hotspots (Fig.1A&B). 59% (10 patients) of CADD>20 variants carriers were diagnosed with MDS: 5 patients with <5% bone marrow blasts (4 with normal karyotype) and 5 patients with 5-19% blasts, all with complex karyotype. This percentage was significantly higher compared to CADD<20 variant carriers (34%, 49 patients, p=0.048 by Chi-square test, Fig. 1C). However, no major differences were found between the two groups in terms of age at diagnosis (median 70 vs 73 years old, p = 0.2305) or AML-free survival (median 10 vs 15.5 months since diagnosis, p=0.4674). Similarly, the percentage of patients presenting additional somatic variants in other myeloid-related genes was similar between CADD<20 and CADD>20 TERT variant carriers (60% vs 64%). The two most frequenly mutated genes in CADD>20 carriers were TP53 (22%), TET2 (18%), RUNX1 (9%), U2AF1 (9%) and BCOR (9%) while in the CADD<20 carriers were TET2 (14%), ASXL1 (11%), SRSF2 (9%), TP53 (7%) and JAK2 (6%). Our data showed that TP53 pathogenic variants were significantly enriched in CADD>20 TERT variant carriers compared to CADD<20 carriers. (22.7% vs 7.4%, p=0.0169, Fig. 1D). Image:Summary/Conclusion: Carriers of TERT variants with CADD score of >20 were more likely to be associated with a diagnosis of MDS and with somatic TP53 mutations in comparison to those with CADD score <20, findings that we plan to validate functionally and prospectively.

Abstract 6257: Mutations in the ASXL1 and ZRSR2 genes are associated with the response to the combination of alvocidib and 5-azacytidine in higher-risk myelodysplastic syndromes

Abstract The hypomethylating agent 5-azacytidine (5-Aza) is a standard-of-care for patients with higher-risk myelodysplastic syndromes (MDS). Although an initial response is induced in approximately 50% of 5-Aza treated patients, subsequent relapse is almost certain. Recently, inhibitors of anti-apoptotic BCL-2 protein family members have shown therapeutic potential in acute myeloid leukemia (AML) and higher-risk MDS. Alvocidib (Alv), a CDK9 inhibitor and indirect transcriptional repressor of the anti-apoptotic factor MCL-1, has shown anti-leukemic effects in a phase 1 study of patients with AML (Lee DJ et al, Expert Opin Investig Drugs 2019; Zeidner JF et al, Leuk Res 2015). A phase 1b/2 study with Alv and 5-Aza or decitabine in higher-risk patients with MDS was recently completed (NCT03593915); however, biomarkers for response to the Alv and 5-Aza combination are not well characterized. To identify potential biomarkers of response, we performed a comprehensive in vitro assessment of Alv and 5-Aza combination using a clinically well-characterized cohort of n=45 higher-risk patients with MDS and n=11 healthy controls (HC). CD34+ cells were purified from bone marrow (BM) aspirates using positive selection with MACS beads. CD34+ cells of HC were obtained from femur head replacement surgery bone specimens. After 4 days of expansion in SFEM II medium containing StemSpan Myeloid Expansion Supplement, cells were treated with 5-Aza for 48h, Alv for 24h or their combination (5-Aza for 48h followed by Alv for 24h). Cell viability was determined using CellTiter-Glo (CTG) and Annexin-V apoptosis assays. MCL-1 dependency of MDS samples was assessed using MS1 peptide-based assay. Recurrent myeloid neoplasia mutations in 67 genes were assessed in BM mononuclear cells using NGS panel deep sequencing. The combination of 5-Aza+Alv had an additive cytotoxic effect on CD34+ MDS cells in CTG assay (median cell viability = 74%, 73.8% and 55% for 5-Aza, Alv and combination respectively, p&amp;lt;0.0001). In Annexin-V apoptosis assay, MDS samples were more sensitive to the combination treatment compared to HC (median % of apoptotic and dead cells = 36.6% for MDS vs 25.6% for HC, p=0.0288). MCL-1 dependency inversely and not significantly correlated with CD34+ cell viability in CTG assays (Spearman r=-0.37, p=0.1119). In contrast, we found significant associations between ASXL1 and ZRSR2 mutations and higher sensitivity of MDS samples to 5-Aza+Alv combination (p=0.008 and p=0.0005 in univariable analysis respectively). ZRSR2 mutations also retained an independent impact on cell viability in multivariable analysis (p=0.035). Overall, we provide pre-clinical support for the use of 5-Aza+Alv combination for higher-risk MDS and identified ASXL1 and ZRSR2 mutations as potential genetic biomarkers of augmented response. Citation Format: Vladimir Ryabov, Nanni Schmitt, Qingyu Xu, Alexander Streuer, Johann-Christoph Jann, Alina Wein, Eva Altrock, Verena Nowak, Nadine Weimer, Julia Obländer, Iris Palme, Ahmed Jawhar, Ali Darwich, Patrick Wuchter, Christel Weiss, Georgia Metzgeroth, Jason M. Foulks, Laurenz Steiner, Mohamad Jawhar, Wolf-Karsten Hofmann, Daniel Nowak. Mutations in the ASXL1 and ZRSR2 genes are associated with the response to the combination of alvocidib and 5-azacytidine in higher-risk myelodysplastic syndromes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6257.

Abstract 764: Fully automated sample-to-report NGS workflow for comprehensive genomic profiling for myeloid neoplasms

Abstract Introduction: Myeloid malignancies are associated with a broad and diverse set of genomic alterations, including SNVs, insertions, deletions and gene fusions. Comprehensive characterization of genetic mutations in hematological disorders currently requires a variety of diagnostic tests and takes multiple days to complete. We developed a fully automated NGS Myeloid Assay that offers an easy to use sample-to-report workflow and the capability for processing up to 8 samples per day. Methods: The Genexus System is comprised of two software linked instruments, the Genexus Purification System and the Genexus Integrated Sequencer. The Genexus Purification System was used to isolate the DNA and RNA from blood or bone marrow samples from precharacterized myeloid samples representative of Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS) and from the blood of healthy donors. The Genexus Integrated Sequencer was used to dilute the nucleic acids to optimal concentration and to sequence the samples in replicates with Oncomine Myeloid Genexus v2 Assay. Six DNA and RNA samples were sequenced per run per day along with commercially available analytical controls and a No Template Control. The report was generated by the Genexus Software analysis pipeline optimized to detect different variant types with high sensitivity and specificity. Results: The purification workflows were tested with blood input of 50-400uL for DNA and 50-150uL for RNA. Genexus Purification System extracted and quantified nucleic acids showed input dependent yields. DNA and RNA yields obtained with 50uL sample inputs consistently met the 27.5ng DNA and 15ng RNA minimum requirements for Genexus Integrated Sequencer workflows. DNA libraries had &amp;gt;97% Uniformity of Amplicon Coverage and &amp;gt;95% Target Base Coverage at 350x. The percentage ratio of Mapped Reads for DNA and RNA libraries was approximately 80:20. Detected genetic variations included key hotspots in CEBPA, FLT3, IDH1/2, NPM1, NRAS, RUNX1, and U2AF1 genes that are prevalent in AML and MDS. Genexus Variant Calling results showed high reproducibility and high concordance to the Ion GeneStudio S5 sequencing platform (&amp;gt;95%). The analytical controls, AcroMetrix Oncology Hotspot Control, Seraseq Myeloid Mutation DNA Mix and Seraseq Myeloid Fusion RNA Mix, were sequenced with Sensitivity and PPV &amp;gt;95%. Conclusion: The Genexus System offers an automated sample-to-report workflow with minimal hands-on-time and run results in 30 hours which allows an easy to use solution for next day turnaround time. When used with the Oncomine Myeloid GX v2 Assay, it provides accurate and comprehensive information on diverse mutations including fusions that are relevant to the study of myeloid cancers. For research use only. Not for use in diagnostic procedures. Citation Format: Marina Sedova, Alexy Ongpin, Jennifer Burke, Collyn Seeger, Sarah Brozio, Janice Au-Young, Jiajie Huang, Thilanka Jayaweera, Iris Casuga, Milton Huynh, Fiona Hyland. Fully automated sample-to-report NGS workflow for comprehensive genomic profiling for myeloid neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 764.

Standards of Genetic Testing in the Diagnosis and Prognostication of Systemic Mastocytosis in 2022: Recommendations of the EU-US Cooperative Group

Mastocytosis comprises rare heterogeneous diseases characterized by an increased accumulation of abnormal mast cells in various organs/tissues. The pathogenesis of mastocytosis is strongly linked to the presence of KIT-activating mutations. In systemic mastocytosis (SM), the most frequent mutation encountered is KIT p.D816V, whose presence constitutes one of the minor diagnostic criteria. Different techniques are used to search and quantify the KIT p.D816V mutant; however, allele-specific quantitative PCR and droplet digital PCR are today the most sensitive. The analysis of the KIT p.D816V allele burden has undeniable interest for diagnostic, prognostic, and therapeutic monitoring. The analysis of non-mast cell hematological compartments in SM is similarly important because KIT p.D816V multilineage involvement is associated with a worse prognosis. In addition, in advanced forms of SM, mutations in genes other than KIT are frequently identified and affect negatively disease outcome and response to therapy. Thus, combined quantitative and sensitive analysis of KIT mutations and next-generation sequencing of other recurrently involved myeloid genes make it possible to better characterize the extent of the affected cellular compartments and additional molecular aberrations, providing a more detailed overview of the complex mutational landscape of SM, in relation with the clinical heterogeneity of the disease. In this article, we report the latest recommendations of the EU-US Cooperative Group presented in September 2020 in Vienna during an international working conference, on the techniques we consider standard to detect and quantify the KIT p.D816V mutant in SM and additional myeloid mutations found in SM subtypes.

Which FLT3 Inhibitor for Treatment of AML?

Treatment options in acute myeloid leukemia (AML) have improved significantly over the last decade with better understanding of disease biology and availability of a multitude of targeted therapies. The use of FLT3 inhibitors (FLT3i) in FLT3-mutated (FLT3mut) AML is one such development; however, the clinical decisions that govern their use and dictate the choice of the FLT3i are evolving. Midostaurin and gilteritinib are FDA-approved in specific situations; however, available data from clinical trials also shed light on the utility of sorafenib maintenance post-allogeneic stem cell transplantation (allo-SCT) and quizartinib as part of combination therapy in FLT3mut AML. The knowledge of the patient's concurrent myeloid mutations, type of FLT3 mutation, prior FLT3i use, and eligibility for allo-SCT helps to refine the choice of FLT3i. Data from ongoing studies will further precisely define their use and help in making more informed choices. Despite improvements in FLT3i therapy, the definitive aim is to enable the eligible patient with FLT3mut AML (esp. ITD) to proceed to allo-SCT with regimens containing FLT3i incorporated prior to SCT and as maintenance after SCT.

Pathology Trainees Gain Clinical Pathology Experience as Lab Consultants Through Auditing Myeloid Mutation Panel Send-Out Tests: Hitting Two Birds With One Stone?

Inappropriate laboratory testing and the threat it poses to patient care and rising health care costs has become an important focus in the medical literature. Pathology residents, as physicians with an intimate knowledge of laboratory testing, may be uniquely equipped with the tools to intervene in situations of inappropriate testing and also benefit from lab use experience as part of their clinical pathology training. To employ a resident-driven initiative aimed at incorporating pathology residents as consultants for appropriate ordering of high-volume, send-out myeloid mutation panel testing. During a 6-month study period, all myeloid mutation panel send-out tests were screened by senior pathology residents on their clinical chemistry rotation prior to approval at an academic medical center. A retrospective review of myeloid mutation panels from the prior 6 months was conducted with the same criteria to determine effectiveness of the intervention. Of the 234 tests ordered during the study period, screening resulted in cancellation of 17% (n = 39), with proportional cost savings. The number of inappropriate orders successfully cancelled was significant compared with the preintervention period (control, 0%; intervention, 76.5%; P < .001, Fisher exact test). There was no significant difference in the proportion of inappropriate tests before and after intervention. Although test ordering patterns did not substantially change during the intervention period, pathology residents effectively reduced inappropriate myeloid mutation panel testing through prospective send-out auditing, leading to significant cost savings. Moreover, assessment of test use and appropriateness provided critical clinical pathology training within the areas of hematology, molecular genetics, and laboratory management.

Genomic studies controvert the existence of the CUX1 p75 isoform

CUX1, encoding a homeodomain-containing transcription factor, is recurrently deleted or mutated in multiple tumor types. In myeloid neoplasms, CUX1 deletion or mutation carries a poor prognosis. We have previously established that CUX1 functions as a tumor suppressor in hematopoietic cells across multiple organisms. Others, however, have described oncogenic functions of CUX1 in solid tumors, often attributed to truncated CUX1 isoforms, p75 and p110, generated by an alternative transcriptional start site or post-translational cleavage, respectively. Given the clinical relevance, it is imperative to clarify these discrepant activities. Herein, we sought to determine the CUX1 isoforms expressed in hematopoietic cells and find that they express the full-length p200 isoform. Through the course of this analysis, we found no evidence of the p75 alternative transcript in any cell type examined. Using an array of orthogonal approaches, including biochemistry, proteomics, CRISPR/Cas9 genomic editing, and analysis of functional genomics datasets across a spectrum of normal and malignant tissue types, we found no data to support the existence of the CUX1 p75 isoform as previously described. Based on these results, prior studies of p75 require reevaluation, including the interpretation of oncogenic roles attributed to CUX1.

Spectrum of Myeloid Mutations in Patients with Elevated Hemoglobin

▪Background: JAK2 V617F and exon 12 mutations are the characteristic driver mutations in polycythemia vera (PV), identified in more than 95% of patients. In addition, other genetic mutations have previously been described in JAK2-positive PV that appear to have prognostic significance (Tefferi et al., Blood 2016). The incidence of other driver mutations in unselected patients referred for elevated hemoglobin is less well studied. This study aims to characterize the genetic mutational landscape in a real-world population of patients referred for elevated hemoglobin using a targeted Next-Generation Sequencing (NGS)-based assay.Methods: We reviewed all patients referred for elevated hemoglobin levels (>160 g/L in females or >165 g/L in males) between 2018 and 2020 to hematology clinics at London Health Sciences Centre in Southwestern Ontario, Canada who underwent testing for genetic variants using the NGS-based Oncomine Myeloid Research Assay (ThermoFisher Scientific, MA, USA). This assay targets 40 key genes with diagnostic and prognostic implications in several myeloid malignancies (17 full genes and 23 genes with clinically relevant “hotspot” regions) and a panel of 29 fusion driver genes (>600 fusion partners). Patient demographics, laboratory data and final diagnosis were extracted from the electronic medical record. For all patients with genetic mutations, clinical diagnosis was confirmed by three independent reviewers.Results: A total of 529 patients underwent genetic testing for elevated hemoglobin levels: 389 (73.5%) were males (mean age 58; range 18-95) and 140 (26.5%) were female (mean age 60; range 24-85). JAK2 mutations were detected in 10.9% (58/529) of patients and a diagnosis of PV was confirmed. The majority of JAK2-mutated PV patients (n=57) were positive for JAK2 V617F, while one patient had an exon 12 mutation. Additional single myeloid mutations were detected in 34.5% (20/58) of JAK2-positive patients and involved the following genes: TET2 (11; 19%), DNMT3A (2; 3.4%), ASXL1 (2; 3.4%), SRSF2 (2; 3.4%), BCOR (1; 1.7%), TP53 (1; 1.7%) and ZRSR2 (1; 1.7%) (Figure 1A). JAK2 mutations were not detected in 89.0% (471/529) of our cohort. A diagnosis of PV was confirmed in 2 JAK2-negative patients based on clinical features and myeloid mutations were detected in both: SRSF2 and TET2 gene mutations in 1 patient and SRSF2, IDH2, ASXL1 gene mutations in the other patient. Three JAK2-negative patients tested positive for the BCR-ABL fusion and were diagnosed with chronic myeloid leukemia. The remaining 466 JAK2-negative patients were diagnosed with secondary erythrocytosis and myeloid mutations were found in 6% (28/466) of these cases. Mutations were detected in DNMT3A (12; 2.6%), TET2 (5; 1.1%), ASXL1 (5; 1.1%), TP53 (2; 0.4%), NF1 (2; 0.4%), KIT (1; 0.2%), U2AF1 (1; 0.2%) (Figure 1B). All patients with JAK2-negative secondary erythrocytosis had only one myeloid gene mutation detected.Conclusion: Additional myeloid mutations other than JAK2 mutations are frequently identified in patients referred for erythrocytosis, with the highest frequencies observed in the TET2, DNMT3A and ASXL1 genes. The spectrum of myeloid mutations and overall incidence in JAK2-negative patients with secondary erythrocytosis is similar to the reported incidence of Clonal Hematopoiesis of Indeterminate Potential (CHIP) (Jaiswal et al., NEJM 2014), and suggests that these may represent incidental age-related mutations. By contrast, among the JAK2-positive patients, 34.5% had at least one additional myeloid mutation supporting a pathogenic role in these patients with myeloproliferative neoplasms. While concomitant myeloid mutations in patients with PV are well-described, further research is required to elucidate the significance of variants identified in JAK2-negative patients classified as secondary erythrocytosis in order to determine whether these mutations contribute to clinical phenotype or represent background CHIP. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Updated Results from a Phase II Study of Mini-Hyper-CVD Plus Inotuzumab Ozogamicin, with or without Blinatumomab, in Older Adults with Newly Diagnosed Philadelphia Chromosome-Negative B-Cell Acute Lymphoblastic Leukemia

Updated Results from a Phase II Study of Mini-Hyper-CVD Plus Inotuzumab Ozogamicin, with or without Blinatumomab, in Older Adults with Newly Diagnosed Philadelphia Chromosome-Negative B-Cell Acute Lymphoblastic Leukemia

The Impact of IDH1c.315C>T Single Nucleotide Polymorphism on Outcomes in Acute Myeloid Leukemia: A Propensity Score-Adjusted Cohort Study

IntroductionMutations have become essential in determining risk stratification for acute myeloid leukemia (AML) and are often used to guide treatment. Variants of unknown significance (VUS) can be classified as pathogenic or benign once their roles are better understood. Therefore, studying VUS in mutated genes may improve overall risk stratification and prognostication for AML patients.Mutations in isocitrate dehydrogenases, IDH1 or IDH2, occur in approximately 20% of AML. R132 (in IDH1) and R140/R172 (in IDH2) mutations lead to the production of oncometabolite 2-hydroxyglutarate and have been targeted with selective oral inhibitors; however, the clinical impact of the synonymous single nucleotide polymorphism (SNP) in codon 105 in exon 4 of the IDH1 gene (c.315C>T (p.Gly105Gly), s11554137) is poorly understood. This VUS is not commonly screened for in myeloid mutation panels. Here, we aimed to investigate the prognostic significance of the c.315C>T SNP in AML patients.MethodsWe conducted a single-site retrospective study to compare overall survival (OS), event-free survival (EFS), complete remission (CR) and complete remission with incomplete hematologic recovery (CRi) rates in adults with AML with and without the IDH1:c.315C>T (p.Gly105=) between 2013 and 2020. The IDH1 c.315C>T SNP was identified using Sanger's technique. Data collected included age, gender, ethnicity, ECOG performance status, baseline comorbidities, AML category, cytogenetics, myeloid mutations including IDH1/IDH2/FLT3, treatments received, and survival outcomes. Data were analyzed using propensity score-weighting to obtain adjusted estimands. CR+CRi rates in patients with and without c.315C>T IDH1 mutation were compared using Fisher's exact test or Chi-square test. Survival outcomes were compared using the Gehan-Breslow-Wilcoxon rank test and adjusted Cox proportional hazards regression.ResultsWe included 228 patients (median age 65.2 years [IQR: 54-75]). The IDH1 c.315C>T SNP was identified in 21 (9%) patients. Table 1 shows unadjusted baseline characteristics. The presence of IDH1 c.315C>T SNP was not mutually exclusive with conventional IDH mutations. The CR+CRi adjusted rates were 60% and 51% with first-line treatment in patients with and without the mutation (P=0.42). The adjusted-median OS was 26.1 (95 CI: 9.8-NA) and 17 (95 CI: 12.6-21.8) months (P=0.69). The adjusted hazard ratio for mortality was 1.4 higher in the unmutated category (95 CI: 0.997-1.98, P=0.05). The adjusted-median EFS was 9.53 (95 CI: 4.17-NA) and 5.57 (95 CI: 4.43-7.87) months (P=0.89). The adjusted hazard ratio for mortality or relapse was 1.1 higher in the unmutated category (95 CI: 0.81-1.47, P=0.56). Figures 1 and 2 show OS and EFS survival curves.ConclusionsUnlike in previous studies, the presence of IDH1 c.315C>T SNP was not associated with inferior OS, PFS or CR+CRi rates compared with its absence. [Display omitted] DisclosuresEmadi: Secura Bio.: Consultancy; Servier: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Servier: Research Funding; NewLink Genetics: Research Funding; Jazz Pharmaceuticals: Research Funding; KinaRx, Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder.

Single Centre Analysis of JAK2 V617F and Exon 12 Negative Idiopathic Erythrocytosis

▪IntroductionThe molecular categorisation of myeloproliferative neoplasms (MPNs) has changed the landscape of diagnosis and treatment. Polycythaemia vera (PV) is characterised by red blood cell proliferation and JAK2 V617F or Exon 12 mutations in up to 98% of patients 1. However, some patients without such mutations have an arduous diagnostic course with varying management and prognostic outcomes 2. We present our experience in managing this challenging cohort and aim to illuminate a potential diagnostic pathway for patients.MethodWe searched electronic records of patients attending haematology clinics over the last 20 years at University College Hospital with a prior diagnosis of PV / erythrocytosis (presenting with raised Haemoglobin (Hb) &/ Haematocrit (Hct)) with no evidence of JAK2 exon 12 or 14 mutations on bone marrow or peripheral blood molecular analysis (multiplex PCR sensitivity 0.1%). We reviewed their diagnostic workup, which included full blood count & where available, bone marrow myeloid (Illumina TruSight Myeloid) & erythroid next generation sequencing (NGS) panels.Results37 patients with JAK2 V617F & Exon 12 mutation negative erythrocytosis were identified. Patients were categorised in to 3 groups 2; idiopathic erythrocytosis (IE), secondary polycythaemia (SP) & high affinity haemoglobinopathies (HAH); patient characteristics are summarised in Table 1. The median age of IE & HAH was younger, their presenting Hb/Hct levels was higher compared to SP, with a male predominance. Constitutional symptoms were only reported in the IE cohort. Erythropoietin (EPO) was elevated in HAH & IE patients but within normal range in SP. Thrombotic events occurred in all cohorts, most frequently in IE. Splenomegaly was reported in 4/21 IE, 1/13 SP, but was not a feature in HAH patients. When performed, IE red cell mass (RCM) studies were raised but within normal range in SP patients. Table 2 details IE cohort erythroid mutations. Myeloid NGS only identified MPL and BCOR mutations of pathogenic significance and multiple single nucleotide polymorphisms of no known significance. No abnormalities were demonstrated in 15% of SP patients that underwent bone marrow myeloid mutational analysis. Venesections (VS) were instigated in 95% of the IE cohort, antiplatelets (AP) in 52%, anticoagulation (AC) in 14% and cytoreductive therapy (CT) in 19% due to intolerance/failure of VS. VS programme was instigated in 46% of SP patients, AP in 7% and AC in 47%.DiscussionThe median age of our IE cohort was 48 yrs with a 19% incidence of thrombosis. Where performed, bone marrow histology demonstrated hypercellularity but was not consistent with MPN diagnostic criteria. Myeloid NGS panel mutations such as BCOR may represent clonal haematopoieis of indeterminate potential. Heterozygous VHL C598T & C376A mutations, in keeping with Chuvash polycythaemia, were demonstrated. Mutations in EGLN1 &BPGM,were detected in our patients, however there was an absence of correlating haematological parameters or family history to support a diagnosis of congenital erythrocytosis (2). Variants of unknown significance were also detected in SH2B3, BMP6 &EGLN3 gene duplication. SP patients were older (median age 68 years) and where performed had normal RCM and no myeloid mutations identified.The initial approach adopted at our centre for diagnosing and managing JAK2 V617F & Exon 12 negative erythocytosis begins with clinical evaluation for secondary causes. This is followed by assessment of EPO level, RCM study, extended molecular mutational analysis involving screening for high affinity haemoglobins, congenital erythrocytosis and bone marrow histology. Where patients are symptomatic or considered at high risk for thrombosis, we venesect to a personalised target and patients are offered AP and/or AC. CT is instigated where VS is not tolerated or ineffective, however this approach is not corroborated in the literature. Our experience highlights the clinical heterogeneity of JAK2 negative erythrocytosis and the need to develop a robust and systematic diagnostic and treatment algorithm with further clarification of the role of molecular profiling.1. William W, Kralovics R. Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms. Blood. 2017 Feb; 129(6):667-6792. McMullin MF. Idiopathic erythrocytosis: a disappearing entity. Hematology Am Soc Educ Program. 2009; 2009(1):629-635 [Display omitted] DisclosuresSekhar: Novartis: Consultancy, Research Funding.

PIEZO1 Mutation May Determine Early Onset of Clinical Manifestation of Anemia of Myelodysplastic Syndromes

BACKGROUNDAnemia is the most frequent cytopenia in myelodysplastic syndromes (MDS), neoplastic diseases characterized by ineffective hematopoiesis and bone marrow dysplasia. MDS usually affect elderly individuals (median age in Italy 74 years), and quite rarely concern younger patients. It is of increasing interest to verify whether “young”, not therapy-related MDS patients carry germline (GM) predisposition, in order to optimize therapeutic and transplant choices. The best approach is to perform Whole Exome Sequencing (WES) instead of targeted next-generation sequencing (t-NGS) panel in cases in which MDS patients have no syndromic signs or any indicative sign of predisposing alterations. AIMWe considered a cohort of 21 consecutive MDS cases with age at diagnosis < 60 years and anemia, referred to our MDS Unit for a second opinion. METHODSAfter bone marrow (BM) re-evaluation and morphological confirmation of dysplasia compatible with MDS, we screened the 21 MDS patients by WES on BM-DNA or peripheral blood DNA (PB-DNA). For DNA sequencing, DNA libraries were sequenced using the NextSeq 550 Illumina System. Variant calling was carried out using GATK caller. T-NGS myeloid panel was in some cases applied to confirm somatic nature of mutations. Samples of 5/21 MDS patients and their relatives were further analyzed after WES by applying high-coverage t-NGS 86-gene custom panel for hereditary anemias on PB-DNA. WES of DNA extracted from saliva (S-DNA) samples was performed to provide a GM confirmation. In order to confirm pathogenic role in red blood cell (RBC) membrane defects of identified variants, we also performed the ektacytometry analysis that evaluates the erythrocyte deformability and the hydration status by subjecting them to an increasing osmotic gradient with constant shear stress. RESULTSFive of 21 MDS cases (23.8%) with symptomatic anemia (3 MDS-MLD, 1 MDS-SLD, 1 MDS with isolated del5q) were identified as carriers of variants suspected responsible of RBC membrane defects in both BM/PB-DNA and S-DNA samples, where no other germline myeloid predisposing alterations were demonstrated. MDS-associated somatic variants were present in 4/5 patients (KRAS, TET2, CBL, SRSF2, U2AF1, IDH2, with VAF >25 %). High coverage t-NGS analysis confirmed the presence of a heterozygous rare missense variant in PIEZO1 in probands 1 and 5; a heterozygous rare missense variant in PIEZO1 and ANK1 in proband 2; a heterozygous rare missense variant in PIEZO1 and SPTA1 in proband 3; a complex mode of inheritance in proband 4 with causative variants in G6PD, KCCN4, SPTB, and ABCB6 genes. PIEZO1 or KCNN4 gene variants result in erythrocyte dehydration and determine dehydrated hereditary stomatocytosis (DHS). Ektacytometry analysis showed a significant difference in the osmolarity curve of these patients, confirming the pathogenic role of the variants identified. Among these MDS suspected cases, only proband 4 was diagnostically re-categorized because of lack of acquired recurrent somatic mutations and presence of multiple alterations in erythroid genes causing dysplasia of erythroid precursors, masquerading as MDS. None of the patients presented a history of unexplained anemia before the onset of MDS, consistent with what shown for DHS1 patients with well-compensated hemolysis. Inheritance pattern of the identified variants according the study of the families confirmed the segregation of the pathogenic variants. CONCLUSIONOnset of de novo MDS in unusually young age should always prompt investigation of predisposing conditions. We suggest here that co-existence of inherited RBC membrane defects with MDS may determine earlier clinical manifestation of the clonal disease. Hereditary anemias, in particular xerocytosis, may mimic MDS morphology. In our study, among the 5 “young” MDS with concomitant inherited RBC membrane defect, one case did not finally confirm MDS diagnosis by karyotype or NGS myeloid mutation analysis. This case fulfilled the diagnostic criteria for combined hereditary hemolytic anemia: DHS2, G6PD deficiency, spherocytosis, familial pseudohyperkalemia 2, with multi-locus mode of inheritance that justifies the clinical condition (splenomegaly, hemolytic crisis, ascites). Addition of genetic testing for hereditary anemias beside assessment of germline predisposing variants may allow a more precise diagnosis in controversial cases of “young” MDS. DisclosuresSanna: Janssen: Consultancy; Abbvie: Consultancy; Astra Zeneca: Consultancy. Iolascon: Celgene: Other: Advisory Board; Bluebird Bio: Other: Advisory Board. Santini: Menarini: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Geron: Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astex: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees.

A Novel Machine Learning-Derived Molecular Classification Scheme with Prognostic Significance

The gold standard for the diagnosis of MDS relies on morphologic alterations hampered by a great deal of subjectivity. Cytogenetic and clinical features allow for clinical classifications predictive of survival. However, with a few exceptions (SF3B1MT, del5q, and certain balanced translocations), neither classic histo-morphology nor prognostic scoring systems (e.g., IPSS-R) are reflective of pathogenic underpinnings. To date supervised analyses of mutational data did not succeed to produce profiles specific or predictive of traditional disease sub-entities. Large cohorts with clinical annotation and a sufficient follow-up allow for innovative biostatistical approaches to subgroup patients according to molecular profiles. Objective operator-independent subcategorization may be congruent with common pathogenic links, rational applications of targeted therapeutics and better prognostications.We hypothesized that machine-learning (ML) strategies used for analysis of mutational/cytogenetic profiles will enable recognition of invariant disease subcategories according to their molecular configurations. Herein, we compiled a meta-analytic database (our cohorts and publicly available sources) of 3,011 MDS (median age 71yrs) and 6,788 pAML/sAML. Results of deep targeted sequencing of a panel of 55 myeloid mutations were collected together with cytogenetics. We then performed unsupervised analysis of MDS and AML patients using Bayesian Latent Class Analysis (BLCA). A consensus matrix was then clustered using Ward's criteria to generate final cluster assignment based on the highest silhouette value. To identify genomic signatures, we used Random Forest classification and extracted mutations with highest global importance indicated by mean decrease in accuracy.Using BLCA we identified 5 unique genomic clusters (GCs) with 3 distinct prognostic outcomes [low risk (LR), intermediate risk (Int), and high risk (HR)] that were validated by survival analysis (Fig.1A,B). The LR group included GC-1 and was characterized by the highest prevalence of normal cytogenetics (100%) and SF3B1 MT (25%) with co-occurring DNMT3A MT (14%), and absence of ASXL1 MT, ETV6 MT, STAG2 MT, TP53 MT, and complex/abnormal cytogenetics. Int group included GC-2 and GC-4. GC-2 was characterized by a higher percentage of abnormal cytogenetics cases than LR group and absence of STAG2 MT, SRSF2 MT, ASXL1 MT, TP53 MT, and normal/complex cytogenetics. GC-4 had the highest frequency of SRSF2 MT (52%) with co-occurring ASXL1 MT (59%), TET2 MT (40%), normal karyotype, and absence of complex/abnormal cytogenetics. Finally, HR included GC-3 and GC-5. GC-3 included ASXL1 MT (67%) with co-occurring SRSF2 MT (47%), TET2 MT (37%), STAG2 MT (22%), and absence of normal cytogenetics. GC- 5 had the highest frequency of -5/del(5q) (50%), -7/del(7q) (43%), -17/del(17p) (16%) and the highest odds of complex karyotype (92%) as well as TP53 MT (48%). Paralleling the genomic ML-based clustering, the clinical relevance of these subgroups was reflected in significantly different survivals [median (95% CI)]: i) GC-1 [69 (59-80)], ii) GC-2 [35 (29-41)], iii) GC-3 [12 (10-16)], GC-4 [27 (22-34)], and GC-5 [9 (7-11)] months (Fig.1C). We then classified the MDS cohort according to the recently published and validated AML GCs (Awada et al Blood 2021) to investigate overlapping genomic features. Overall, 90% of MDS GC-1 and 67% of MDS GC-2 had the same molecular architecture of AML GC-2 and 70% of MDS GC-5 had the same molecular features of AML GC-4. In addition, 98% of MDS GC-3 and 92% of MDS GC-4 had the same features of AML GC-3 (Fig.1D).In sum, we propose a novel objective molecular classification of MDS and related diseases that allows subgrouping of patients according to shared pathogenesis for a better prognostic resolution without errors derived from subjectivity. The model was then internally and externally validated using a cohort of 200 cases. Results of a validation cohort and online URL site of molecular clustering will be presented at the meeting. [Display omitted] DisclosuresBalasubramanian: Servier Pharmaceuticals: Research Funding. Patel: Alexion: Consultancy, Other: educational talks, Speakers Bureau; Apellis: Consultancy, Other: educational talks, Speakers Bureau. Carraway: Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astex: Other: Independent review committee; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Other: Independent review committee; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Other: Independent review committee; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Regeneron: Consultancy; Novartis: Consultancy; Alexion: Consultancy; Bristol Myers Squibb/Celgene: Consultancy.