Presence Of Additional Mutations Research Articles

A retrospective review of myeloproliferative neoplasms and correlation of bone marrow morphological results with next generation sequencing profiles

Background: Classification of Philadelphia negative (Ph-) myeloproliferative neoplasms (MPN) relies on the integration of peripheral blood counts, demonstration of driver mutations where possible and the interpretation of bone marrow aspirate and trephine morphology. Of these disorders, the distinction between essential thrombocythemia and prefibrotic myelofibrosis can be challenging. Objectives and methods: To assess reproducibility of the WHO classification for this group of MPNs and to assess the potential of utilising next generation sequencing (NGS) to assist with the diagnosis. This retrospective study was conducted at Pathwest QEII, Western Australia. Twenty cases of MPNs from 2017–2021 with an initial diagnosis or differential diagnosis of essential thrombocythaemia (ET), prefibrotic myelofibrosis (prePMF) and primary myelofibrosis (PMF) were centrally reviewed using a standardised proforma for the FBP parameters and defined morphologic criteria based on the WHO classification. NGS was performed to assess the variant allele frequencies (VAF) of driver mutations and the presence of additional mutations. Results: Fifteen cases were concordant with the initial morphological review. ET cases had a lower JAK2V617F VAF compared to PMF and fewer number of additional mutations. NGS was of limited use in the differentiation between ET and prePMF in the other five cases that were difficult to classify on morphological grounds. Conclusions: This study is limited by the small sample size but reflects practice in a diagnostic laboratory. In this study, the application of NGS was of limited use in further assisting the diagnosis of ET or prePMF where the strict WHO criteria are applied.

Genetic landscape of chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm caused by the BCR::ABL1 fusion gene, which aberrantly activates ABL1 kinase and promotes the overproduction of leukemic cells. CML typically develops in the chronic phase (CP) and progresses to a blast crisis (BC) after years without effective treatment. Although prognosis has substantially improved after the development of tyrosine kinase inhibitors (TKIs) targeting the BCR::ABL1 oncoprotein, some patients still experience TKI resistance and poor prognosis. One of the mechanisms of TKI resistance is ABL1 kinase domain mutations, which are found in approximately half of the cases, newly acquired during treatment. Moreover, genetic studies have revealed that CML patients carry additional mutations that are also observed in other myeloid neoplasms. ASXL1 mutations are often found in both CP and BC, whereas other mutations, such as those in RUNX1, IKZF1, and TP53, are preferentially found in BC. The presence of additional mutations, such as ASXL1 mutations, is a potential biomarker for predicting therapeutic efficacy. The mechanisms by which these additional mutations affect disease subtypes, drug resistance, and prognosis need to be elucidated. In this review, we have summarized and discussed the landscape and clinical impact of genetic abnormalities in CML.

Natural History of Patients with Clonal Hematopoiesis Seen in Hematology Clinics

Background: The International Consensus Classification (ICC) recently recognized clonal hematopoiesis of indeterminate potential (CHIP) as a clinically defined pre-malignant state. A subgroup of patients with clonal hematopoiesis (CH), herein termed progressors, ultimately develop to a frank clinical diagnosis, while others, herein termed non-progressors, may have CH indefinitely with variable risk of progression. Although eight years have passed since the large-scale descriptions of CHIP as an age-related phenomenon, the risk factors for progression and management guidelines are still not well-defined. Moreover, while many quaternary care centers have developed CHIP databases, little is known about the epidemiology of CHIP from tertiary care centers that draw patients from the community setting. Methods: Patients with CH were identified via concurrent morphologic, cytogenetic, and molecular diagnostics of bone marrow aspirates reviewed by UMass Hematopathology between 2013 and 2022. CH was defined here as per the 2022 ICC: presence of a somatic mutation with variant allele frequency (VAF) ≥ 2% on molecular diagnostics or non-myelodysplastic syndrome (MDS)-defining clonal cytogenetic aberration in a patient without morphologic evidence of myeloid neoplasm. Patients with germline mutations were excluded. Independent review of all bone marrow aspirates was performed by at least two pathologists. This study included a pre-selected group of patients who had an indication for bone marrow evaluation; reasons for performing bone marrow evaluation included presentation with pancytopenia (41.7%), anemia (14.6%), leukocytosis (12.5%), thrombocytopenia (6.3%), leukopenia (4.2%), bicytopenia (4.2%), staging workup for a lymphoma (4.2%), erythrocytosis (4.2%), thrombocytosis (4.2%), abnormal radiographic marrow signal (2.1%), eosinophilia (2.1%). A total of 48 patients with CH were identified. Results: A total of 102 aberrations were found among 48 patients with CH. The mean, median, mode, and range for CH aberrations were 2.15, 1.5, 1, and 5, respectively. The mean age at detection of CH for non-progressors was 69.2 ± 1.8 years vs. 63.1 ± 3.5 years for progressors (p = 0.072). The most prevalent somatic variants in CH were: TET2 (29.1%), DNMT3A (14.5%), and gross structural abnormalities (7.7%) (Panel A, outer circle). Progressors had a distinct profile (Panel A, inner circle). Zero of 48 patients with CH carried TP53, IDH1, or EZH2 aberrations. The most prevalent genomic disruptions were missense mutations (45.6%), nonsense mutations (15.6%), and frameshift deletions (13.3%). We assessed the clinical trajectory of patients with CH (Panel B). Nine (18.8%) of 48 patients progressed to neoplasm (confirmed by bone marrow evaluation). Median overall survival (OS) at the time of analysis was 6.85 years, with longest follow up of 9.58 years. For non-progressors, median OS was not reached. For progressors, median OS was 6.85 years (from time of identification of CH). Median time from identification of CH to frank neoplasm was 1.05 years. For progressors, median time from diagnosis of frank neoplasm to death was 1.76 years. Of the 14 patients harboring DNMT3A, TET2, or ASXL1 (DTA) mutations alone, without mutations in other biological clusters, none (0%) progressed (Panel B,blue bars). Conclusion: In contrast to the original reports of CH, which studied unselected patients from multiple consortia, the patients in our study were pre-selected for suspicion of an underlying hematologic condition, reflecting real-world data. CH spans multiple functional clusters that are recurrently mutated in MDS and/or acute myeloid leukemia (AML), with the notable exception of TP53, IDH1, and EZH2. This may be due to more rapid progression to malignancy for those mutations, with only a transient, if any, CH state. All patients who progressed with DTA mutations did so only in the presence of additional mutations - at least a second mutation was required for DTA cases to progress. There was a trend toward significance for younger mean age at CH detection for progressors vs. non-progressors. Lead-time bias may skew OS results for patients found to have CH at an early point; and CH detected at a younger age may be more likely progress, leading to shorter OS. Systematic studies are needed to more comprehensively profile CH and develop formalized risk-adapted management strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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).

Single-cell analysis reveals selection of TP53-mutated clones after MDM2 inhibition

AbstractThe mechanisms of transformation of chronic myeloproliferative neoplasms (MPN) to leukemia are largely unknown, but TP53 mutations acquisition is considered a key event in this process. p53 is a main tumor suppressor, but mutations in this protein per se do not confer a proliferative advantage to the cells, and a selection process is needed for the expansion of mutant clones. MDM2 inhibitors may rescue normal p53 from degradation and have been evaluated in a variety of cancers with promising results. However, the impact of these drugs on TP53-mutated cells is underexplored. We report herein evidence of a direct effect of MDM2 inhibition on the selection of MPN patients' cells harboring TP53 mutations. To decipher whether these mutations can arise in a specific molecular context, we used a DNA single-cell approach to determine the clonal architecture of TP53-mutated cells. We observed that TP53 mutations are late events in MPN, mainly occurring in the driver clone, whereas clonal evolution frequently consists of sequential branching instead of linear consecutive acquisition of mutations in the same clone. At the single-cell level, the presence of additional mutations does not influence the selection of TP53 mutant cells by MDM2 inhibitor treatment. Also, we describe an in vitro test allowing to predict the emergence of TP53 mutated clones. Altogether, this is the first demonstration that a drug treatment can directly favor the emergence of TP53-mutated subclones in MPN.

Clinical Features and Long-Term Outcomes in a Molecularly-Annotated Cohort of Adolescents and Young Adults with Myeloproliferative Neoplasms

▪Background:Myeloproliferative neoplasms (MPNs) are a heterogenous group of chronic hematologic malignancies that lead to morbidity and early mortality primarily due to thrombotic complications and progression to acute leukemia. Clinical and mutational risk factors have been demonstrated to predict outcomes in patients with MPNs, and are used commonly to guide therapeutic decisions (including allogenic stem cell transplant) in myelofibrosis. Adolescents and young adults (AYA, age ≤45 years) comprise less than 10% of all MPN patients, and have unique clinical and therapeutic considerations. The prevalence and clinical impact of somatic mutations implicated in myeloid disease has not been extensively reported in this population.Methods:We conducted a retrospective review of patients evaluated at the Princess Margaret Cancer Centre (PMCC) between 1/1/2000 and 30/6/2021 for an MPN diagnosed at ≤45 years of age. Diagnoses were defined per 2016 WHO criteria using available information on chart review. Targeted mutational profiling of clinically relevant myeloid genes (49- or 54-gene panel) was previously performed on peripheral blood or bone marrow samples. Pathologic variants in ASXL1, EZH2, IDH1/2, SRSF2, and U2AF1 Q157 were categorized as high-molecular risk (HMR). Categorical variables were compared using the χ2 test. Kaplan-Meier method was used for overall survival (OS) and time-to-event analysis; and were compared using the log-rank test.Results:A total of 237 patients with initial MPN diagnosis ≤45 years were included in the study, with median age of diagnosis 35 (range 12-45) years. MPN diagnosis included: essential thrombocythemia (ET, n=100), polycythemia vera (PV, n=75), prefibrotic primary myelofibrosis (Pre-PMF, N=29), overt PMF (n=24), and MPN-unclassifiable (MPN-u, n=9). Driver mutation data, available for 230 (97%) patients, were: JAK2 in 134 (56%), CALR in 70 (30%), MPL in 6 (2.5%), and triple-negative in 20 (8.4%) patients.Median follow-up was 10.2 (range 0.3-45.3) years, and median OS was the shortest for those with overt PMF (21.4 years, P=0.001) compared with PV (33.0 years), ET (31.4 years), and Pre-PMF (median OS not reached). No difference in OS based on driver mutation was observed. Progression to a secondary MF (SMF) was observed in 84 patients (47% of ET, 48% of PV) after median time of 19.7 years; with no difference in time to progression due to diagnosis or driver mutation. Progression to an accelerated/blast phase (AP/BP) was observed in 26 patients and was associated with diagnosis of overt PMF (P=0.04) and triple-negative for driver mutation (P=0.04).Thrombosis was observed in 61 (26%) patients: 34 (14%) patients with thrombosis prior to/concurrent with MPN diagnosis, and 34 (14%) patients with thrombosis after diagnosis. Splanchnic circulation thrombosis was found in 29 (12%) patients and 12 (5%) patients had Budd-Chiari syndrome. Portal hypertension was reported in 37 (16%) patients. CALR type-2 mutations were associated with the lowest frequency of total thrombosis (4%, P=0.02); while JAK2 had the greatest frequency of splanchnic circulation clot (17%, P=0.03).Targeted mutational profile was obtained for 202 (85%) of patients: 135 samples obtained during the initial disease phase, 51 samples during a secondary MF phase, and 16 during an AP/BP. For the patients with mutation analysis in the initial phase, 24 pathogenic mutations were observed in 16 (12%) patients including 3 patients with HMR mutations. The most frequent mutations observed were in TET2 (n=12, 9%), and ASXL1 (n=3, 2%). Presence of additional mutations did not predict OS, AP/BP progression, or thrombosis; though TET2 mutation was associated with shorter time to SMF progression in patients with PV/ET (P=0.002). Additional mutations were found in 34/51 (67%) patients in whom mutational analysis was first performed during a SMF disease phase.Conclusions:A long-term follow-up study of a large molecularly annotated cohort of AYA patients with MPN demonstrated excellent long-term survival for these patients. Overt PMF is associated with the lower OS and higher risk of AP/BP transformation. Thrombotic complications including splanchnic circulation thrombosis are frequent complications. Mutations aside from JAK2/MPL/CALR are uncommon and HMR mutations rare in the initial phase of MPN in the AYA population. [Display omitted] DisclosuresGupta: Roche: Consultancy; Pfizer: Consultancy; AbbVie: Consultancy, Honoraria; BMS-Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sierra Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Research Funding; Constellation Pharma: Consultancy, Honoraria. Maze: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene-BMS: Honoraria; Takeda: Research Funding; PharmaEssentia: Research Funding; Kronos Bio: Research Funding.

Visualizing Clonal Hematopoiesis Associated with gata2 Deficiency in Zebrafish Using Color-Barcoding

Myeloid neoplasms, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), arise from abnormal hematopoietic stem and progenitor cells (HSPCs) with acquired somatic mutations. Germline GATA2 haploinsufficiency is associated with an autosomal dominant familial MDS/AML syndrome. Leukemic transformation in GATA2 haploinsufficiency occurs with acquisition of secondary mutations or cytogenetic abnormalities. We generated gata2 heterozygous zebrafish to model this germline predisoposition syndrome, and evaluate clonal architecture and dynamics over time at steady-state and in the presence of additional mutations. We hypothesized that clinically benign clonal hematopoiesis precedes the establishment of myeloid neoplasms in GATA2 haploinsufficiency. We used a lineage tracing system of zebrafish Brainbow, Zebrabow, to track the hematopoietic output of individually colored HSPC clones, with the goal to detect abnormal expansion of single color clones in the early stages of leukemogenesis, indicating clonal hematopoiesis. A heterozygous line of GATA2 zebrafish ortholog gata2b was generated in the Zebrabow background. We introduced combinations of genetic mutations described in GATA2 associated MDS and AML into Zebrabow embryos with CRISPR/Cas9 technology. Zebrafish embryos, either wild-type or heterozygous for gata2b, were injected with Cas9 protein and guide RNAs targeting zebrafish orthologs of ASXL1 and STAG2. Zebrabow color recombination was induced to color-label stem cells prior to their emergence from the dorsal aorta. At this time roughly 20 stem cell clones exist in the zebrafish embryo. The animals were then raised to adulthood. Kidney marrow cells of juvenile zebrafish were analyzed at various time points in between 2-4 months post-fertilization (mpf) by flow cytometry and morphology on cytospins. At steady-state, juvenile gata2b+/- zebrafish had a decreased myeloid compartment in the kidney marrow. A third of gata2b+/- zebrafish also had an expansion of single color clones in the kidney marrow by 3 mpf, contributing to ≥30% of granulocytes, while no wild-type clutchmate demonstrated single color expansion to this degree at steady-state. Mosaic combinations of mutations in the presence of gata2b deficiency resulted in an increase of precursor and a more pronounced decrease of myeloid populations in the kidney marrow at 3 mpf, compared to wild-type zebrafish injected with same mutations. No changes were observed in the lymphoid compartment. Single color expansion was observed in gata2b+/- zebrafish three times more frequently than in wild-type zebrafish. In summary, gata2b deficiency sensitizes juvenile zebrafish to developing myelocytopenia at steady-state, reminiscent of well-characterized monocytopenia in GATA2 deficiency. We found that myelocytopenia is more profound and associated with an increase in early precursors upon additional somatic mutations associated with MDS/AML, possibly due to a differentiation block in myelopoiesis. Single color dominance observed in the hematopoeitic compartment of juvenile gata2b+/- zebrafish using the Zebrabow color-barcoding system presents a potential mechanism underlying the predisposition to myeloid neoplasms associated with GATA2 deficiency, and provides a tool for visualizing asymptomatic premalignant state in disease pathogenesis. DisclosuresZon:Scholar Rock, Inc: Consultancy, Equity Ownership; Marauder, Inc.: Consultancy, Equity Ownership; Stemgent: Consultancy; Fate, Inc.: Consultancy, Equity Ownership.

Expanding the Nude SCID/CID Phenotype Associated with FOXN1 Homozygous, Compound Heterozygous, or Heterozygous Mutations

Human nude SCID is a rare autosomal recessive inborn error of immunity (IEI) characterized by congenital athymia, alopecia, and nail dystrophy. Few cases have been reported to date. However, the recent introduction of newborn screening for IEIs and high-throughput sequencing has led to the identification of novel and atypical cases. Moreover, immunological alterations have been recently described in patients carrying heterozygous mutations. The aim of this paper is to describe the extended phenotype associated with FOXN1 homozygous, compound heterozygous, or heterozygous mutations. We collected clinical and laboratory information of a cohort of 11 homozygous, 2 compound heterozygous, and 5 heterozygous patients with recurrent severe infections. All, except one heterozygous patient, had signs of CID or SCID. Nail dystrophy and alopecia, that represent the hallmarks of the syndrome, were not always present, while almost 50% of the patients developed Omenn syndrome. One patient with hypomorphic compound heterozygous mutations had a late-onset atypical phenotype. A SCID-like phenotype was observed in 4 heterozygous patients coming from the same family. A spectrum of clinical manifestations may be associated with different mutations. The severity of the clinical phenotype likely depends on the amount of residual activity of the gene product, as previously observed for other SCID-related genes. The severity of the manifestations in this heterozygous family may suggest a mechanism of negative dominance of the specific mutation or the presence of additional mutations in noncoding regions.

Abstract 4715: Characterization of potential co-drivers of pathogenesis in ALK positive ALCL

Abstract ALK positive Anaplastic large cell lymphoma (ALCL) is an aggressive non-Hodgkin lymphoma characterized by expression of the NPM/ALK fusion tyrosine kinase essential for neoplastic cells growth. When resistant or relapsed to front-line chemotherapy, ALK+ ALCL prognosis is very poor. Although ALK+ ALCL cells are mainly driven by the constitutively active NPM/ALK kinase, presence of additional mutations might affect the disease course and response to treatment, leading to different outcomes in different patients. We employed whole exome sequencing to characterize the co-mutational burden in ALK+ ALCL samples at diagnosis. Diagnosis biopsy and matched healthy control DNA samples from 24 ALK+ ALCL patients were sequenced. Somatic variants present at high frequency within the tumor (at least 25% variant allele frequency), which can be viewed as potential co-drivers, were annotated; the identified mutations were weighed using in silico tools to predict their oncogenic potential and their effects on protein function. All identified variants were confirmed by Sanger sequencing. Gene ontology and pathway analyses identified cell adhesion and actin cytoskeleton among the most enriched biological functions. Using the above-mentioned criteria, we found recurrent somatic variants in 7 genes including PPP1R9A, RUNX1T1, FAT4 and RBBP8. Here we report the initial characterization of the role of FAT4 in ALCL cells. FAT4 is a member of the cadherin superfamily with tumor suppressor functions. In our cohort, 3/24 patients (13%) were found to carry FAT4 mutations, including nonsense mutations and missense changes predicted to be deleterious, suggesting a loss of function effect. To characterize the biological consequences of these mutations, we generated FAT4 knockdown ALCL cell lines. FAT4-silenced cells grew faster and showed increased migratory properties compared to parental cells. They also differed from parental cells in terms of morphology, having bigger size and modulated F-actin rearrangement. Tumor suppressing activity of FAT4 has been linked to Wnt/β-catenin pathway; we found that FAT4-silenced cells had decreased levels of serine/threonine phosphorylated β-catenin and higher nuclear localization, which insinuates activation of Wnt/β-catenin signalling and henceforth, increased cellular proliferation. To explore therapeutic implications of FAT4 mutations, we analyzed the sensitivity of FAT4 silenced cells to chemotherapeutic agents. FAT4 knockdown cells were less sensitive to a CHOP combination treatment as compared to parental cells. No difference was seen in terms of sensitivity to crizotinib. Finally, we investigated the effects of RUNX1T1 somatic variants found in our study. Preliminary luciferase assay results showed that the variants significantly increase the transcriptional repression ability of RUNX1T1. Altogether, our data provides information about genes other than NPM-ALK that might play a role in the pathogenesis of ALCL. Citation Format: Luca Mologni, Geeta G. Sharma, Matteo Villa, Mario Mauri, Giulia Arosio, Cosimo Lobello, Hugo Larose, Alessandra Pirola, Silvia Bombelli, Nicoletta Cordani, Luca Massimino, Šárka Pospíšilová, Suzanne D. Turner, Giorgio Inghirami, Fabio Pagni, Rocco Piazza, Roberto Perego, Roberto Chiarle, Carlo Gambacorti-Passerini. Characterization of potential co-drivers of pathogenesis in ALK positive ALCL [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4715.

Molecular profiling and risk classification of patients with myeloproliferative neoplasms and splanchnic vein thromboses

AbstractMyeloproliferative neoplasms (MPNs) are the most frequent underlying causes of splanchnic vein thromboses (SVTs). MPN patients with SVTs (MPN-SVT) often have a unique presentation including younger age, female predominance, and low Janus kinase 2 (JAK2) mutation allele burden. This study aimed at identifying risk factors for adverse hematologic outcomes in MPN-SVT patients. We performed a retrospective study of a fully characterized cohort of MPN-SVT patients. The primary outcome was the incidence of evolution to myelofibrosis, acute leukemia, or death. Eighty patients were included in the testing cohort. Median follow-up was 11 years. Most of the patients were women with a mean age of 42 years and a diagnosis of polycythemia vera. The primary outcome was met in 13% of the patients and was associated with a JAK2V617F allele burden ≥50% (odds ratio [OR], 14.7) and presence of additional mutations in genes affecting chromatin/spliceosome (OR, 9). We identified high-risk patients (29% of the cohort) as those harboring at least 1 molecular risk factor: JAK2-mutant allele burden ≥50%, presence of chromatin/spliceosome/TP53 mutation. High-risk patients had worse event-free survival (81% vs 100%; P = .001) and overall survival at 10 years (89% vs 100%; P = .01) than low-risk patients. These results were confirmed in an independent validation cohort of 30 MPN-SVT patients. In conclusion, molecular profiling identified MPN-SVT patients with dismal outcome. In this high-risk population, a disease-modifying therapy should be taken into consideration to minimize the probability of transformation.

Venetoclax and hypomethylating agents in TP53‐mutated acute myeloid leukaemia

Venetoclax and hypomethylating agents in <i>TP53</i>‐mutated acute myeloid leukaemia

Targeted re-sequencing of F8, F9 and VWF: Characterization of Ion Torrent data and clinical implications for mutation screening.

Mutations are not identified in ~5% of hemophilia A and 10–35% of type 1 VWD patients. The bleeding tendency also varies among patients carrying the same causative mutation, potentially indicating variants in additional genes modifying the phenotype that cannot be identified by routine single-gene analysis. The F8, F9 and VWF genes were analyzed in parallel using an AmpliSeq strategy and Ion Torrent sequencing. Targeting all exonic positions showed an average read depth of >2000X and coverage close to 100% in 24 male patients with known disease-causing mutations. Discrimination between reference alleles and alternative/indel alleles was adequate at a 25% frequency threshold. In F8, F9 and VWF there was an absolute majority of all reference alleles at allele frequencies >95% and the average alternative allele and indel frequencies never reached above 10% and 15%, respectively. In VWF, 4–5 regions showed lower reference allele frequencies; in two regions covered by the pseudogene close to the 25% cut-off for reference alleles. All known mutations, including indels, gross deletions and substitutions, were identified. Additional VWF variants were identified in three hemophilia patients. The presence of additional mutations in 2 out of 16 (12%) randomly selected hemophilia patients indicates a potential mutational contribution that may affect the disease phenotype and counseling in these patients. Parallel identification of disease-causing mutations in all three genes not only confirms the deficiency, but differentiates phenotypic overlaps and allows for correct genetic counseling.

A new role for Escherichia coli Dam DNA methylase in prevention of aberrant chromosomal replication.

The Dam DNA methylase of Escherichia coli is required for methyl-directed mismatch repair, regulation of chromosomal DNA replication initiation from oriC (which is DnaA-dependent), and regulation of gene expression. Here, we show that Dam suppresses aberrant oriC-independent chromosomal replication (also called constitutive stable DNA replication, or cSDR). Dam deficiency conferred cSDR and, in presence of additional mutations (Δtus, rpoB*35) that facilitate retrograde replication fork progression, rescued the lethality of ΔdnaA mutants. The DinG helicase was required for rescue of ΔdnaA inviability during cSDR. Viability of ΔdnaA dam derivatives was dependent on the mismatch repair proteins, since such viability was lost upon introduction of deletions in mutS, mutH or mutL; thus generation of double strand ends (DSEs) by MutHLS action appears to be required for cSDR in the dam mutant. On the other hand, another DSE-generating agent phleomycin was unable to rescue ΔdnaA lethality in dam+ derivatives (mutS+ or ΔmutS), but it could do so in the dam ΔmutS strain. These results point to a second role for Dam deficiency in cSDR. We propose that in Dam-deficient strains, there is an increased likelihood of reverse replication restart (towards oriC) following recombinational repair of DSEs on the chromosome.

The assessment of minimal residual disease versus that of somatic mutations for predicting the outcome of acute myeloid leukemia patients

BackgroundIn addition to morphological and cytogenetic features, acute myeloid leukemias are characterized by mutations that can be used for target-therapy; also the minimal/measurable residual disease (MRD) could be an important prognostic factor. The purpose of this retrospective study was to investigate if somatic mutations could represent an additional prognostic value in respect of MRD alone.MethodAt baseline, 98 patients were tested for NPM1, FLT3, and for WT1 expression; 31 for ASXL1, TET2, IDH1, IDH2, N-RAS, WT1, c-KIT, RUNX1, and DNMT3A. The same genes have been also tested after induction and consolidation.ResultsOverall, 60.2% of our patients resulted mutated: 24.5% carried mutations of FLT3-ITD, 38.7% of NPM1, 48.4% of c-KIT, 25.8% of N-RAS and 19.3% of IDH2. The probability of achieving a complete response (CR) was higher for younger patients, with low ELN risk score, NPM1-mutated, with low WT1 levels, and without FLT3. The presence of additional mutations represented a poor predictive factor: only 19% of these cases achieved CR in comparison to 43% of subjects without any of it. Concerning survival, it was conditioned by a lower ELN risk score, younger age, reduction > 1 log of the NPM1 mutational burden, disappearance of FLT3 mutations and lower WT1 expression. Regarding the role of the additional mutations, they impaired the outcome of 20% of the already MRD-negative patients. Concerning the possibility of predicting relapse, we observed an increase of the NPM1 mutational burden at the time-point immediately preceding the relapse (about 2 months earlier) in 50% of subjects. Similarly concerning WT1, an increase of its expression anticipated disease recurrence in 64% of cases.ConclusionsWe demonstrated that additional somatic mutations are able to impair outcome of the already MRD-negative subjects. About MRD, we suggest a prognostic role also for the WT1 expression. Finally, we considered as relevant the assessment of NPM1 quantity clearance instead of the presence/absence of mutations alone. Still remains in doubt the utility in terms of long-term prognosis of a baseline more complex mutational screening; we could hypothesize that it would be useful for those patients where other markers are not available or who reached the MRD negativity.

Prognostic Value of Genetic Mutations in Patients with Acute Myeloid Leukemias: Results of a Cooperative Study of Hematology Clinics of Saint Petersburg (Russia) and Charite Clinic (Germany)

Aim. To analyze the effect on prognosis of mutations that are typical of acute myeloid leukemia (AML) patients. Materials &amp; Methods. The study included 620 AML patients surveyed at Hematology Clinics of Saint Petersburg (Russia) and Charite Clinic (Berlin, Germany). G-banding of chromosomes was employed for cytogenetic testing. Aberration screening in DNMT3A, IDH1/2 genes was based on real-time polymerase chain reaction (PCR) with subsequent analysis of melting and sequencing profiles. Mutations in FLT3, NPM1 genes were revealed by PCR. Results. Mutations were identified in 343 (55.3 %) out of 620 patients. Significantly more often mutations were discovered in patients with normal karyotype (NK) (p = 0.001). FLT3-ITD mutation was associated with reduced medians of overall survival (OS) and disease-free (DFS) survival: 11.3 vs. 15.8 months with FLT3-ITD- (p = 0.005) and 10.0 vs. 13.3 months with FLT3-ITD+ (p = 0.009), respectively. The relation of FLT3-ITD allele burden to OS duration was also assessed. In the ITD&lt;sup&gt;low&lt;/sup&gt;/ITD- group the OS median was considerably longer than in the ITD&lt;sup&gt;high&lt;/sup&gt; group (p = 0.028). In the group of patients with 1 mutation in NPM1 gene OS and DFS were much better in comparison with other patients (medians of 27.4 and 13.9 months, respectively, p = 0.040; 19.3 and 12.0 months, p = 0.049). Negative impact of mutations in DNM-T3A gene was noticed while assessing OS median: 12 (DN-MT3A+) and 15 months (DNMT3A-), respectively (p = 0.112). Mutations in IDH1 gene correlated with a better OS than in the group without mutations (p = 0.092). The rs11554137 polymorphism in IDH1 gene was associated with worse OS in the group of patients with NK (p = 0.186). In 144 patients various mutation combinations (from 2 to 5) were identified. It was demonstrated that mutations in FLT3 (FLT3-ITD), NPM1, DNMT3A, and IDH2 were identified significantly more often in combinations with other mutations (p = 0.001): NPM1+/ FLT3-ITD+ (20.8 %), NPM1+/FLT3-ITD+/DNMT3A+ (8.3 %), and FLT3-ITD+/DNMT3A+ (8.3 %). Patients with 1 mutation had a noticeably longer OS median compared with patients with 2 mutations (18.1 and 12.2 months; p = 0.003). In patients with NPM1+ according to their OS the most unfavorable additional mutation was FLT3-ITD (median 27.4 vs. 9.2 months; p = 0.019) and the combination of NPM1+/FLT3-ITD+/DNM-T3A+ (median 27.4 vs. 14.6 months; p = 0.141). OS of patients with DNMT3A+ showed a downward trend if FLT3-ITD additional mutation was identified (17.3 vs. 7.1 months; p = 0.074). Conclusion. Mutations in FLT3, DNMT3A, IDH1/2, NPM1 genes frequently occur in AML intermediate-risk patients, i.e. they determine the intermediate prognosis group in AML. The studied mutations considerably impact prognosis. It is important to take into consideration mutation type, its allele burden, and the presence of additional mutations. A patient with 2 mutations has a considerably worse OS compared with a patient with 1 mutation. The studied group of patients with the combination of NPM1+/FLT3-ITD+, NPM1+/ FLT3-ITD+/DNMT3A+, DNMT3A+/FLT3-ITD+ mutations has the poorest prognosis. Comprehensive analysis of genetic damages in AML patients allows to most accurately predict the course and prognosis of the disease and to plan targeted therapy.

Number of Mutations and Type of Prior Myeloproliferative Neoplasm Are Prognostic Factors in Acute Myeloid Leukemia Post Myeloproliferative Neoplasms

BACKGROUNDPost myeloproliferative neoplasms (MPN) acute myeloid leukemia (AML) occurs respectively in 1.5%, 7.0% and 11% of patients with essential thrombocytosis (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). This subgroup of AML has very poor prognosis and are often excluded from clinical trials. Therefore, only few cohorts including molecular data are available.MATERIAL AND METHODSWe retrospectively collected data from 111 patients treated in four centers in France for post MPN-AML. Clinical, molecular and treatment information was available for all patients at AML and MPN stages. DNA was extracted from samples at diagnosis of MPN chronic phase, at diagnosis of AML phase and after induction treatment. JAK2-V617F mutations were identified by qPCR (Ipsogen® MutaQuant kit, Qiagen, Germany), MPL-W515L/K mutations were identified by PCR (Ipsogen® MutaScreen kit, Qiagen, Germany) and CALR mutations were identified by conventional sequencing (Applied Biosystems, 3500Genetic Analyzer). NGS on 36 genes using Ampliseq librairy and Ion Proton sequencing (Thermofisher, Waltham, MA, USA) were performed in 96/111 patients. Overall response rate (ORR) was defined by complete remission (CR), CR with incomplete hematologic recovery (CRi), partial remission (PR) and stable disease (SD). Overall survival (OS) was calculated from the date of AML diagnosis to the date of death or last follow-up. All statistical analyses were performed using SPSS v.22 software (IBM SPSS Statistics).RESULTS111 patients treated for post MPN-AML were retrospectively included in this study. Sex ratio M/F was 54%/46%. Median age at AML diagnosis was 66 years (28-89, range). Cytogenetic categories were favorable, intermediate and adverse in 2 (2%), 51 (46%) and 47 (42%) patients, respectively. 25/111 (23%) patients had a monosomal karyotype (MK). Median number of additional mutations excluding from JAK2/MPL/CALR mutations was 2 (0-6, range). The most frequent additional mutations were TP53 (23%), ASXL1 (17%), TET2 (13%), SRSF2 (10%), DNMT3A (8%), SF3B1 (8%) and RUNX1 (8%). Only 2 patients were mutated for NPM1 and 2 and 4 patients were FLT3-ITD and FLT3-TKD, respectively. Prior MPN were PV, ET and PMF in 20%, 34% and 46% of patients, respectively. First line treatment was intensive chemotherapy (IC) for 61 (55%) patients, hypomethylating agents (HMA) for 10 (9%) or other treatments including best supportive care, cytoreduction for the other ones. 24/111 (22%) underwent to ASCT. ORR was 54% (with 30/71 (42%) in CR/CRi) in patients treated by IC or HMA.We did not identify factors predicting a higher rate of CR/CRi. OS was 12 months [6-18] and was not influenced by transplant, cytogenetic categories or by the type and allele frequencies of JAK2/CALR/MPL mutations. OS was significantly longer in the group treated with HMA as compared to IC (10 versus 46 months, respectively, p=0.006); in patients with prior PV as compared to ET or MF (26 months [0-57] versus 10 months [7-13] versus 10 months [4-16] respectively, p=0.07) and in patients with presence of additional mutations other than JAK2/CALR/MPL (5 months [0-12] versus 46 months [32-60] in 38 patients without mutation versus 58 patients with presence of at least one mutation, respectively, p=0.04). By multivariate analysis, only presence of additional mutations was predictive for OS with a hazard ratio (HR) = 0.42 [0.18-0.97] (p=0.04). Finally, we followed the VAFs of JAK2 in seven patients before and after IC. We observed in 2 patients an increase of JAK2 clone correlated with CR whereas no variation of VAFs was associated with absence of CR.CONCLUSIONSIn conclusion, we confirmed the poor prognosis of post MPN AML. Classical AML prognostic factors were not validated in our cohort. We identified the presence of mutations other than JAK2/MPL/CALR as the main prognostic factor whereas post-PV AML appeared to do better than post-ET and post-PMF AML. The very poor result of IC with or without ASCT highlights the need to develop specific clinical trials in this subgroup of AML. DisclosuresKuykendall:Celgene: Honoraria; Janssen: Consultancy. Sallman:Celgene: Research Funding, Speakers Bureau. Cluzeau:CELGENE: Consultancy; MENARINI: Consultancy; JAZZ PHARMA: Consultancy.

Utility of Next Generation Sequencing in Prognostication and Therapeutic Decision Making in Cytogenetically Normal AML with DNMT3A Mutations

Background: The prognostication of cytogenetically normal AML (CN-AML) continues to evolve with the use of NGS-based risk stratification. Emerging data indicate that the presence of additional mutations in good- and intermediate-risk patients (as defined by conventional cytogenetic and molecular analyses) changes the behavior of their disease, suggesting that more personalized treatment approaches are needed.Methods: We analyzed the mutational profile of newly diagnosed AML patients with DNMT3A mutations (N = 48) seen at our center and described their clinical characteristics and associated mutations. These patients were identified as part of the Advanced Genomics in Leukemia (AGILE) clinical project currently underway at the Princess Margaret Cancer Centre, Toronto. NGS molecular profiling was performed using the TruSight Myeloid Sequencing Panel (TMSP; Illumina) on the MiSeq benchtop genome sequencer (Illumina). This process permitted profiling of 54 genes (39 in the hotspot region; 15 complete coding region coverage) using amplicon-based library preparation and sequencing by synthesis. 160 patients with newly diagnosed AML were evaluated for this project from February 2015 to February 2016. All were analyzed in parallel by the standard cytogenetic and molecular (NPM1, FLT3-ITD/TKD) diagnostic algorithm.Results: 48 unique patients were identified bearing mutated DNMT3A. Of these, 31 patients had a normal karyotype. Their clinical characteristics are depicted in Table 1.The R882X DNMT3A variant was detected in 12 patients. A total of 100 additional mutations were identified on sequencing (Figure1). The most common associated mutations were in NPM1 (38.7%) followed by IDH1 (29%), RUNX1 (25.8%) and TET2 (22.6%). PTPN11 mutations were identified in 6 patients, 75% of which also had mutated NPM1. Two patients were found to have biallelic CEBPA mutations. Potential gene-gene interactions were also examined and led to the identification of subgroups such as NPM1-FLT3-DNMT3A mutated (n=4), DNMT3A-IDH2R140 (n=3) and DNMT3A-IDH2R172 (n=3) based on recent data by Papaemmanuil, et al (New England Journal of Medicine, 2016) defining these subgroups as being prognostically relevant.( Patients >60 years of age had more frequent mutations in NRAS, BCOR, BCORL1 and TET2. NRAS and BCOR mutations were mutually exclusive with NPM1. RUNX1, IDH2, SRSF2 and U2AF1 mutations were also seen exclusively in the NPM1 negative group.Eligible patients (n=25) received induction therapy with daunorubicin and cytarabine leading to CR1 in 18 patients (72%). Primary induction failure occurred in 6 cases (24%). All 6 cases were NPM1 negative and had missense mutations in DNMT3A including 3 R882X variants. (Figure 2) Additional mutations were identified in IDH1, RUNX1 and/or TET2 in all 6 patients. During the median follow up of 8 months (range 1 - 15 months), 4 patients relapsed, 2 of which had mutated NPM1 with wild type FLT3-ITD. Sixteen patients are alive at this point and 12 are in CR, six having received allogeneic stem cell transplants. One patient with relapsed disease entered a clinical trial of an IDH1 inhibitorConclusion: Genomic analysis is increasingly recognized as a vital adjunct to conventional diagnostic and prognostic approaches. With ongoing advancements in technology leading to increasing cost effectiveness and decreased turnaround times, the use of NGS is likely to become an up-front investigation resulting in a more personalized approach to therapy. Also, unique patient subgroups defined by gene-gene interactions can be identified to further predict clinical behavior and potentially identify druggable targets for therapy. [Display omitted] [Display omitted] [Display omitted] DisclosuresGupta:Novartis: Consultancy, Honoraria, Research Funding; Incyte Corporation: Consultancy, Research Funding. Schimmer:Novartis: Honoraria. Yee:Novartis Canada: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kamel-Reid:BMS: Research Funding. Schuh:Amgen: Membership on an entity's Board of Directors or advisory committees.

Role of Non-Driver Mutations and JAK2V617F Allele Burden in Myelofibrotic and Acute Myeloid Transformation of Patients with Polycythemia Vera and Essential Thrombocythemia

Background:Non-driver mutations and JAK2V617F allele burden have been involved in progression to myelofibrosis (MF) or acute myeloid leukemia (AML) in patients with polycythemia vera (PV) and essential thrombocythemia (ET). It is unknown if both mechanisms play a different role in disease transformation and if they are useful in routine clinical practice.Methods:JAK2V617F allele burden was monitored in 208 patients (PV n=106, ET n=102) for a median of 6.5 years (range: 1-13). Quantification of JAK2V617F allele burden was assessed on the first available sample and every year thereafter. The evolutionary pattern of JAK2V617F allele burden was categorized as persistently low (<50%), persistently high (>50%), progressive increase (> 25% from baseline) or unexplained decrease (not therapy related).Next generation sequencing (NGS) analysis of 51 myeloid-related genes was performed in 100 patients with a median molecular follow-up of 10 years including all cases with transformation to AML or MF. Detected mutations by NGS in the last sample were studied in first paired sample obtained in the chronic phase (median time from diagnosis: 1.6 years).Time to myelofibrosis and time to AML were calculated according to the presence of non-driver mutations or the JAK2V617F evolutionary pattern. Multivariate analysis was performed by Cox regression.Results:With a median follow-up of 13 years (range: 1-30) 32 patients died whereas 24 and 12 patients progressed to MF and AML, respectively. Median age at diagnosis was 63 years (range: 20-94), 115 were women (55%) and 173 (83%) received cytoreduction. A persistently low JAK2V617F allele burden was observed in 62% of patients whereas the remainder presented a persistently high (25%), a progressive increase (11%) or a non-therapy-related decrease of JAK2V617F allele burden (2%).Non-driver mutations were detected in last sample in 48% of patients. Median number of mutations was 1 (range: 1-5). Mutational frequencies were: TET2 12%, DNMT3A 12%, TP53 9%, ASXL1 7%, RUNX1 4%, SF3B1 4%, SRSF2 4%, IDH1/2 4%, SH2B3 3 % and <2% for EZH2, ZRSR2, SETBP1, FLT3, NPM, BCOR, CBL, PTPN11, KAT6A, NF1, U2AF1, GNAS, PHF6, KTM2A, JAK2, BCORL1, NOTCH1, MPL and PRPF40B.Mutations were detected in first sample in 28% of patients (58% of those with mutations in last sample). Frequencies of mutations in first sample were: TET2 10%, DNMT3A 5%, TP53 5%, ASXL1 4%, SRSF2 4%, IDH1/2 3%, and < 2% for SF3B1, SH2B3, KMT2A and ZRSR2. The evolutionary pattern of JAK2V617F allele burden was not associated with the presence of mutations in first or last sample.Twelve patients progressed to AML (post-PV n=7, post-ET n=5), nine of them presented mutations in first sample. AML transformation at 15 years was 27% and 6.8% for patients with and without additional mutations in first sample, respectively (p=0.001). Mutated genes associated with a higher probability of AML transformation were DNMT3A (p<0.0001), SRSF2 (p<0.0001) and IDH (p<0.0001). Other variables associated with a higher probability of AML were age > 65 years (p=0.012) and exposure to busulfan (p=0.003). Evolutionary JAK2V617F pattern was not associated with the probability of AML (p=0.667). In multivariate analysis, an increased risk of AML transformation was observed for patients with additional mutations in the chronic phase (HR: 6.3; 95%CI 1.6-24.7, p=0.008) after adjusting for initial diagnosis, age and exposure to busulfan.Twenty-four MF transformations were documented (post-PV n= 18, post-ET n=6). Presence of additional mutations was not associated with the probability of MF (p=0.189). Patients with persistently high or a progressive increase of the JAK2V617Fallele burden showed a higher probability of MF transformation (24% versus 1.5% at 10 years, p<0.0001) that persisted in multivariate analysis after correction for age and initial diagnosis (HR 13.9, 95%CI: 2.9-65.6, p=0.001).Conclusion:Non-driver mutations are involved in the progression of PV and ET to AML but not to MF. NGS could be useful for identifying patients with PV or ET at risk of AML transformation.Acknowledgment:This work was supported by grants from the Instituto de Salud Carlos III, Spanish Health Ministry, FISPI13/00557, FISPI1300393, RD012/0036/0004, 2014 SGR567. Alicia Senín received a grant from Sociedad Española de Hematología y Hemoterapia. DisclosuresNo relevant conflicts of interest to declare.

Clinical and molecular response to interferon-α therapy in essential thrombocythemia patients with CALR mutations

AbstractMyeloproliferative neoplasms are clonal disorders characterized by the presence of several gene mutations associated with particular hematologic parameters, clinical evolution, and prognosis. Few therapeutic options are available, among which interferon α (IFNα) presents interesting properties like the ability to induce hematologic responses (HRs) and molecular responses (MRs) in patients with JAK2 mutation. We report on the response to IFNα therapy in a cohort of 31 essential thrombocythemia (ET) patients with CALR mutations (mean follow-up of 11.8 years). HR was achieved in all patients. Median CALR mutant allelic burden (%CALR) significantly decreased from 41% at baseline to 26% after treatment, and 2 patients even achieved complete MR. In contrast, %CALR was not significantly modified in ET patients treated with hydroxyurea or aspirin only. Next-generation sequencing identified additional mutations in 6 patients (affecting TET2, ASXL1, IDH2, and TP53 genes). The presence of additional mutations was associated with poorer MR on CALR mutant clones, with only minor or no MRs in this subset of patients. Analysis of the evolution of the different variant allele frequencies showed that the mutated clones had a differential sensitivity to IFNα in a given patient, but no new mutation emerged during treatment. In all, this study shows that IFNα induces high rates of HRs and MRs in CALR-mutated ET, and that the presence of additional nondriver mutations may influence the MR to therapy.

Importance of Additional Mutation Analysis in Chorionic Villous Sample for Prenatal Diagnosis of Thalassemia

Introduction:β thalassaemia is a genetic disorder of β globiin gene synthesis. Its presentation ranges from transfusion dependent to non transfusion dependent thalassaemia. Non transfusion dependent thalassaemia can have normal life with proper care whereas only curative option for transfusion dependent thalassaemia is bone marrow transplantation which is costly as well as requiring technical expertise. Carrier screening before marriage/conception, antenatal screening and prenatal diagnosis are the only way to prevent birth of a child with thalassaemia. Objective of prenatal diagnosis is to identify homozygous or double heterozygous baby by mutation study. However clinical course after birth may get altered depending on associated other mutation. Till date these associated mutations are not considered in cases of prenatal diagnosis. However, this approach may avoid termination of the baby. This study was carried out retrospectively in the prenatal samples to detect presence of phenotype-modifying mutations.Method and Material:The study was conducted at the Institute of Haematology and Transfusion Medicine, Medical College, Kolkata. Study population consisted of 145 CVS samples and samples of carrier mother and father. Carrier status was detected by HPLC and confirmed by ARMS- PCR for β thalassaemia mutation. Co- inheritance of α- thalassaemia was assessed by GAP- PCR and polymorphism with haplotype assessment was done by RFLP-PCR. 36 parent samples were analyzed for mutation for the carrier status and also for presence of α and Xmn 1 polymorphism.Result:Out of 145 CVS, 8.96% (13 samples) were found to be homozygous, 12.41% (18 samples) double heterozygous, 51.72% (75 samples) heterozygous, 23.44% (34 samples) normal and 3.44% (5 samples) with uncharacterized for β mutation. Our results designated that IVS 1-5 was the most common beta mutation in the state of West Bengal population (Table 1). Co-inheritance of α 3.7 deletion was found in only three affected CVS. Xmn1 polymorphism was detected in 9 CVS samples. Out of these, 3 were homozygous and 6 were heterozygous. Both co-inheritance of α mutation and xmn1 polymorphism were detected in 22 parents sample and 18 affected CVS sample ( Table: 2). Out of 31 affected sample, 18 samples showed presence of Xmn 1 and 4 for α deletion. Additional disease modifying mutations were detected in 6 samples (19.35%). In cases where the 18 parents' sample were positive for Xmn1 polymorphism and 3 for α deletion in CVS sample, either homozygous or double heterozygous for parents' mutation.Table 1Common β globin gene mutation of CVS samplesBeta genotypeHomozygoteHeterozygoteCompound HeterozygoteNormalUncommonIVS 1-5 (G→ C)748-----CD26(G→A) HbE1135----CD15(G→A)89-----CD30 (G→ C)5--------CD41/42(-CTTT)23----CD8/9(+G)3--------CD6 (A → T)22----Total137553418Table 2Affected CVS with additional mutations:Additional mutationsHomozygous CVSDouble heterozygous CVSParentsα deletion1--7Homozygous Xmn1 +/+113Heterozygous Xmn 1 -/+9312α deletion + Homozygous Xmn1 +/+--1--α deletion + Homozygous Xmn1 -/+--2--Discussion:Common thalassaemia subtypes prevalent in West Bengal are HbE beta and Beta thalassaemia . In cases where both the parents are carrier, beta mutation or mutation for HbE was first analyzed and subsequent decision for termination of baby was taken on the presence of both these mutations in the CVS sample. But phenotype is determined by presence of additional mutations like co-inheritance of α mutation and Xmn 1 polymorphism. From our study it became evident that around 19.35% cases the baby was expected to have intermedia or NTDT phenotype. Complete genotype analysis of CVS samples in prenatal diagnosis can save a life and that give a baby to the parents. However, further exploration with larger number of patients and post natal follow up is necessary to reach a definite conclusion.Conclusion:Alpha deletion and Polymorphism detection should be employed for prenatal diagnosis. While developing such program in the population, we save of multiple neonates. DisclosuresNo relevant conflicts of interest to declare.