Comprehensive Molecular Analyses of BCR-ABL1 Negative MPN Show That PMF Is Genetically Much More Heterogeneous Than ET and PV

Abstract Introduction: In the WHO classification (2008) JAK2 and MPL mutations are major criteria for the diagnosis of myeloproliferative neoplasms (MPN): polycythemia vera (PV), primary myelofibrosis (PMF), and essential thrombocythemia (ET). Cytogenetic aberrations are rare in these entities. Although the prognostic impact of JAK2 mutations beside some other gene mutations has been shown in PMF patients, the driving events for establishing accelerated phase or blast crises are unknown. In recent years, novel molecular markers such as ASXL1, SRSF2, and CALR were identified and PMF was investigated in several studies. However, comprehensive mutational analyses of MPN entities in comparison to each other are still rare. Aim: To identify gene mutations beyond JAK2, CALR, and MPL using a 28 gene panel, and to compare mutational data with clinical data and prognostic information in order to identify a risk profile. Patients and Methods: We in the first step investigated 56 patients (19 ET, 18 PMF, and 19 PV; 21 females, 35 males) diagnosed by cytomorphology following WHO criteria and accompanied by genetic studies. All patients underwent mutation analyses by a 28 gene panel containing: ASXL1, BCOR, BRAF, CALR, CBL, DNMT3A, ETV6, EZH2, FLT3-TKD, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, NRAS, KRAS, MPL, NPM1, PHF6, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, and WT1. The library was generated with the ThunderStorm (RainDance Technologies, Billerica, MA) and sequenced on MiSeq instruments (Illumina, San Diego, CA). BCR-ABL1 fusion transcripts were shown to be negative in all cases by PCR. Not yet described genetic variants (n=6) were excluded from statistical analyses. Cytogenetics was available in 55/56 cases and grouped in normal karyotype (n=45, 82%) or aberrant karyotype (n=10, 18%). Results: In the total cohort JAK2 (44/56, 79%) was the most frequently mutated gene, followed by TET2 (13/56, 23%), ASXL1 (11/56, 20%), SRSF2 (7/56, 13%), and CALR (6/56, 11%). All other analyzed genes showed mutation frequencies below 10% (10 genes) or even no mutation (13 genes). Analyzing the number of mutations per patient revealed that only 4 patients showed no mutation (4/56, 7%), the great majority had 1 mutation (19/56, 34%) and 2 mutations (23/56, 41%), while 5 patients showed 3 mutations (5/56, 9%), 4 patients had 4 (4/56, 7%) and 1 patient even 5 mutations (1/56, 2%). Accordingly, the mean number of mutations per patient was 1.9. Summing up the mutations in JAK2, CALR, and MPL resulted in 52/56 (93%) patients that had a mutation in at least 1 of these genes, indicating that most of the patients had just 1 or 2 additional gene mutations to one of the 3 known key player MPN genes (mean: 1.3 additional mutations). Cytogenetically there were no significant differences between the 3 entities in frequencies of normal (65-90%) and aberrant karyotypes (11-35%), although in the PMF cohort there were more aberrant karyotypes (6/17, 35%) in comparison to ET and PV (for each 2/19, 11%). Addressing the mutation patterns of these 3 MPN entities revealed similar frequencies of TET2 mutations. In contrast, as expected JAK2 was more often mutated in PV (18/19, 95%) compared to ET (12/19, 63%, p=0.042) and PMF (14/18, 78%) and CALR was more often mutated in ET (5/19, 26%) in comparison to PMF (1/18, 6%) and PV (0/19, 0%, p=0.046). In PMF ASXL1 (8/18, 44%) and SRSF2 (6/18, 33%) were more often mutated compared to ET (1/19, 5%, p=0.008; 1/19, 5%, p=0.042) and PV (2/19, 11%; p=0.029; 0/19, 0%; p=0.008), respectively. Investigating the numbers of mutated genes per patient resulted in a significantly different distribution within MPN entities: in the ET and PV cohorts patients carried mostly 1 or 2 mutations (36/38, 95%; mean: 1.5), while in PMF 9/18 (50%) patients carried >2 mutations (mean: 2.5; p=0.045). Looking at the affected genes besides JAK2 and CALR showed that in ET and PV 4 more genes were affected, while in PMF 11 different additional genes showed mutations, indicating that PMF is genetically much more heterogeneous than ET or PV. This nicely matches to the finding that PMF is also marked by the highest cytogenetic aberration rate of these 3 BCR-ABL1 negative MPN (24-42%). Conclusions: 1)JAK2 is the most and TET2 the second most frequently mutated gene in BCR-ABL1 negative MPN. 2) Most patients carry only 1 or 2 gene mutations. 3) However, PMF patients are genetically much more heterogeneous than ET and PV patients regarding both cytogenetic and molecular alterations. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment; Novartis: Research Funding. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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In CLL with Normal Karyotype By Conventional and Genomic Array Karyotyping the Prognostic Impact of an Unmutated IGHV Status Is Stronger Than the Impact of Gene Mutations

Blood ◽

10.1182/blood.v128.22.4357.4357 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4357-4357 ◽

Cited By ~ 1

Author(s):

Calogero Vetro ◽

Torsten Haferlach ◽

Manja Meggendorfer ◽

Sabine Jeromin ◽

Constance Regina Baer ◽

...

Keyword(s):

Gene Mutation ◽

Mutation Rate ◽

Gene Mutations ◽

Normal Karyotype ◽

Prognostic Impact ◽

Employment Equity ◽

Mutation Status ◽

Genomic Array ◽

Equity Ownership ◽

The Impact

Abstract Background: In 15-20% of CLL cases no aberrations are detected by chromosome banding analysis (CBA) and FISH due to limited resolution, lack of evaluable metaphases or presence of aberrations in loci not covered by standard-panel FISH probes. As reported in our previous study (Haferlach C. et al., ASH 2015, abs ID#79545), genomic arrays (GA) detected abnormalities in almost 20% of cases classified as normal by CBA and FISH and these showed an impact on time to first treatment (TTT) (Vetro C. et al., EHA 2016, abs ID# E1069). The CLL subgroup without abnormalities in CBA, FISH, and GA has not been characterised in detail, so far. Aims: 1) to describe CLL without abnormalities by CBA/FISH/GA by evaluating an extended gene panel, the IGHV mutation status and the B-cell receptor (BCR) stereotypy; 2) to determine prognostic impact of these factors. Patients and Methods: CLL diagnosis was based on cytomorphology and immunophenotyping according to standard guidelines. From a cohort of 1190 patients at diagnosis, 133 (11%) were selected based on normal karyotype by CBA, no abnormalities by interphase FISH with probes for 17p13 (TP53), 13q14 (D13S25, D13S319, DLEU), 11q22 (ATM), centromeric region of chromosome 12 and t(11;14)(q13;q32) (IGH-CCND1) and no abnormalities by GA (SurePrint G3 ISCA CGH+SNP Microarray, Agilent, Waldbronn, Germany). IGHV mutation status and BCR stereotypy were determined according to Agathangelidis et al., Blood 2012, and DNA sequencing was performed for the following genes: ATM; SF3B1; TP53; KLHL6; KRAS; MYD88; NOTCH1; NRAS; POT1; FBXW7; HIST1H1E; XPO1; ITPKB; MAPK1; BIRC3; BRAF; DDX3X; EGR2; RIPK1; RPS15; CND2. Results: Median age was 66 years (range: 33-83). Median follow-up was 5.6 years, 33 patients (25%) received treatment since genetic analyses. 10-year overall survival (OS) was 76% and median TTT was 9.2 years. Mutations were observed in 53 patients (40%): SF3B1 (n=17; 13%); NOTCH1 (n=10; 8%); KLHL6 (n=6; 5%); TP53 (n=6; 5%); ATM (n=5; 4%); XPO1 (n=4; 3%); FBXW7 (n=3; 2%); MYD88 (n=3; 2%); DDX3X (n=2; 2%); POT1 (n=2; 1.5%); ITPKB (n=1; 1%); KRAS (n=1; 1%); NRAS (n=1; 1%); and no mutation in RPS15, CCND2, MAPK1, EGR2, BRAF, HIST1H1E, RIPK1, BIRC3. 6 patients had 2 simultaneous gene mutations and 1 patient had 3 (i.e. NOTCH1, ATM and TP53). A mutated IGHV status (IGHV-M) was present in 100 patients (75%) and an unmutated IGHV status (IGHV-U) in 33 patients (25%). IGHV-U was related to both the occurrence of any gene mutation (p<0.001) and the number of gene mutations (p=0.001). NOTCH1 was mutated in 7 out of the 33 IGHV-U patients (21%), but only in 3 out of 99 IGHV-M patients (3%) (p=0.001). XPO1 mutation occurred in 4 IGHV-U patients (12%) and none out of IGHV-M (p<0.001). Two IGHV-U patients showed POT1 mutation (6%), but no IGHV-M case (p=0.014). 9 patients out of 133 (7%) showed BCR-stereotypy. 2 were in cluster CLL#1 (both showing NOTCH1 mutation), 2 in cluster CLL#2 (both of them with SF3B1 mutation), 2 in CLL#4, 1 in CLL#8 (showing NOTCH1 and XPO1 mutations), 1 in CLL#201 (with KLHL6 mutation) and 1 in CLL#202 (with mutations in ATM, TP53 and NOTCH1 genes). In Kaplan-Meier analysis, IGHV-M patients did not reach a median TTT, while IGHV-U had a median of 5.1 years (p<0.001). Stereotypy rate was too low for reliable statistics. At univariate analysis, TTT was only influenced by: IGHV-U (relative risk (RR): 3.9, p<0.001), TP53 mutation (RR: 3.7, p=0.03), % CLL cells (RR: 1.2 per 10% increase, p=0.013), and number of mutations (RR: 1.8 per each mutation, p=0.031). Multivariate Cox regression analysis showed an independent role for IGHV-U status (RR: 3.3, p=0.002) and % CLL cells (RR: 1.2 per 10% increase, p=0.038) Only age showed an impact on OS (RR: 1.2 per decade, p<0.001). Conclusions: 1. The CLL subset without any genomic event by CBA/FISH/genomic array is characterized by very low frequency of IGHV-U status; 2. IGHV-U subgroup showed higher gene mutation rate compared to IGHV-M subgroup, in particular higher NOTCH1, XPO1 and POT1 mutation rate; 3. BCR stereotypy is less frequent than in CLL in general. 4. IGHV-U, as well as the higher disease burden (i.e. % CLL cells), has an independent negative impact on TTT. 5. Requirement for treatment is low and prognosis very favorable in CLL without any genomic event by CBA/FISH/genomic array and a mutated IGHV status. Disclosures Vetro: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Baer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Molecular Analysis of Myelodysplastic Syndrome with Isolated Del(5q) Reveals a Broad Spectrum of Clinically Relevant Mutations: A Study on 119 Patients and 26 Genes

Blood ◽

10.1182/blood.v124.21.4608.4608 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4608-4608

Author(s):

Manja Meggendorfer ◽

Tamara Alpermann ◽

Claudia Haferlach ◽

Susanne Schnittger ◽

Wolfgang Kern ◽

...

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndrome ◽

Specific Treatment ◽

Gene Mutations ◽

Gene Panel ◽

Prognostic Impact ◽

Treatment Needs ◽

Employment Equity ◽

Ring Sideroblasts ◽

Equity Ownership

Abstract Introduction: The WHO category of “Myelodysplastic syndrome with isolated del(5q)” shows a good prognosis. It also demonstrates sensitivity for specific treatment such as lenalidomide. However, in some patients it evolves to secondary AML. Underlying pathobiological mechanisms are still under debate. Aim: To determine the frequency of mutations in a 26 gene panel and to investigate a mutation pattern combined with clinical data and prognostic information. Patients and Methods: We investigated 119 patients (85 female, 34 male) having MDS with isolated del(5q), strictly classified according to WHO classification 2008 with respect to cytomorphology and cytogenetics (blasts below 5% in the bone marrow and 5q deletion sole). All patients underwent molecular analyses by a myeloid gene panel containing ASXL1, BCOR, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3-TKD, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, NRAS, KRAS, MPL, NPM1, PHF6, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1, and WT1. The library was generated with the ThunderStorm (RainDance Technologies, Billerica, MA) and sequenced on a MiSeq instrument (Illumina, San Diego, CA). Results: Most patients harbored 1 mutation (60/119, 50%), while 2, 3, and 4 mutations per patient occurred less frequently (18/119, 15%; 2/119, 2%; and 1/119, 1%, respectively). In 38/119 patients (32%) no gene mutation was identified in addition to the del(5q). In the total cohort the most frequently mutated genes were DNMT3A and TP53 (21/119, 18% each), followed by SF3B1 (20/119, 17%), TET2 (14/119, 12%), ASXL1 (9/119, 8%), and JAK2 (7/119, 6%). Although these 6 gene mutations overlapped rarely and occurred frequently as sole mutations, they were not completely mutually exclusive. The mutation frequencies of all other analyzed genes were below 5%. Dividing the patients in groups defined by a bone marrow blast count of <2% and 2-5%, as described in IPSS-R, we could not detect any correlation to the mutation number per patient. However, patients that had no mutation were younger compared to patients with at least 1 mutation (70 vs. 76 years, p=0.009). But there was no difference between these 2 patient groups in white blood cell count, hemoglobin level, or platelet count. Taking single genes into account revealed that TP53 and SF3B1 mutations (mut) correlated with higher age (78 vs. 73 years, p=0.047; 78 vs. 73 years, p=0.050, respectively). Addressing the correlations of ring sideroblasts (RS) >15% and SF3B1mut showed that also in MDS with isolated del(5q) these two parameters significantly correlate with a mean of 19% RS (range: 0-80%) in SF3B1mut and only 1% RS in SF3B1 wildtype patients (wt; range: 0-12%, p<0.001). Looking at prognostic relevance of gene mutations surprisingly showed that SF3B1mut patients had a significantly worse outcome than SF3B1wt patients (median overall survival (OS) 31 vs. 91 months, p=0.008). Comparing the mutation frequency of TP53 in MDS with isolated del(5q) with all other MDS (Haferlach et al, Leukemia 2014) resulted in a significant higher mutation rate in MDS with isolated del(5q) (21/119 (18%) vs. 49/781 (6%), p<0.001). However, we did not find a prognostic impact of TP53mut in our cohort. Our patients were unselected and median OS was 91 months. Thus, our cohort may include a larger proportion of patients earlier in their clinical course compared to cohorts enrolled in treatment studies. Therefore the negative impact of TP53mut may become obvious later or even not before treatment needs to be started. Of note, 50% (60/119) of our patients were only under observation or received red blood cells or erythropoietin only. In contrast, an increasing number of gene mutations per patient showed a very strong trend towards a worse outcome with a median OS of 90 months in patients with no or 1 additional mutation in comparison to patients with more than 1 mutation (median OS: 36 months, p=0.061). Conclusion: 1) In myelodysplastic syndrome with isolated del(5q) the 5 most frequently mutated genes are comparable to all other MDS (Haferlach et al, Leukemia 2014). 2) In contrast, TP53 is more frequently mutated in MDS with isolated del(5q). 3) Ring sideroblasts >15% correlate with SF3B1mut. 4) SF3B1mut lead to significantly worse OS. 5) Increasing numbers of mutations show negative prognostic impact. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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SF3B1 Mutations in AML, MDS and MDS/MPN-RS-T Are Accompanied By Different Other Gene Mutations: Impact for Targeted Treatment Studies

Blood ◽

10.1182/blood.v128.22.1976.1976 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1976-1976

Author(s):

Manja Meggendorfer ◽

Sabine Jeromin ◽

Karolína Perglerová ◽

Claudia Haferlach ◽

Wolfgang Kern ◽

...

Keyword(s):

Myeloproliferative Neoplasms ◽

Gene Mutations ◽

Specific Gene ◽

Prognostic Information ◽

Employment Equity ◽

Additional Mutation ◽

Potential Biomarker ◽

Leukemia Treatment ◽

Equity Ownership ◽

Relevant Gene

Abstract Introduction: Precision medicine aims at the molecular profiling of patients to specifically target gene mutations. Targeted therapies now enter leukemia treatment, e.g. by targeting FLT3-ITD or mutations in DNMT3A, TET2, IDH1/2 or JAK2. Recently, luspatercept, a fusion protein (ACE-536), was shown to inhibit different signaling cascades, resulting in differentiation and maturation of erythropoietic progenitors in anemic patients (Platzbecker et al., Haematologica 2015). Interestingly, only patients with myelodysplastic syndrome (MDS) and ring sideroblasts (RS) responded to luspatercept, suggesting SF3B1 to be a potential biomarker. Besides MDS, SF3B1 mutations occur also in acute myeloid leukemia (AML) and MDS/myeloproliferative neoplasms with RS and thrombocytosis (MDS/MPN-RS-T). However, concomitant gene mutations bearing prognostic information and/or also being therapeutic targets as well as the cytogenetic background may need to be addressed in addition before further investigation. Aim: To investigate the mutation pattern and cytogenetic background of patients with AML, MDS and MDS/MPN-RS-T carrying SF3B1 mutations. Patients and Methods: In a cohort of 365 patients - all showing SF3B1 mutations and the diagnosis of AML (n=51), MDS (n=263) or MDS/MPN-RS-T (n=51) - cytomorphology, cytogenetics and mutation status were available. The cohort comprised 145 females and 220 males, the median age was 75 yrs (range: 42-93 yrs). In all patients ASXL1, RUNX1, TP53 as important prognostic markers as well as DNMT3A, FLT3-TKD, IDH1/2, JAK2, K/NRAS and TET2 as optional targets were analysed for mutations. Furthermore, additional entity specific gene mutations were investigated in respective subcohorts (AML: CEBPA, FLT3-ITD, MLL-PTD, NPM1; MDS: ETV6, EZH2, SRSF2, U2AF1, ZRSR2; MDS/MPN-RS-T: MDS genes, CBL, MPL). Results: 73% of all patients (268/365) showed normal karyotypes. Addressing molecular genetics resulted in 370 mutations beside SF3B1 in 238 patients, leaving only 23% of patients (84/365) showing no other aberration than in SF3B1. The variant allele frequencies (VAF) of SF3B1 mutations were high in nearly all cases with only few (9/353) subclonal cases (VAF <10%). In AML the median VAF of SF3B1 was 45% (range: 5-70%) with 3 cases showing subclonal mutations, likewise in MDS with a VAF of 39% (range: 3-50%) and 6 subclonal cases, while in MDS/MPN-RS-T the VAF was also 39% (range: 15-50%) without any subclonal case. In detail, 63% of AML cases showed normal karyotypes. Looking at gene mutations revealed that 49/51 patients (96%) had additional gene mutations (median: 2, range: 0-4), while 28/51 cases (55%) showed mutations in at least one of the therapeutically relevant genes. Of note, 37/51 patients (73%) had a mutation known to be associated with adverse prognosis. Therefore, in AML just one patient had a sole SF3B1 mutation and only 3/51 cases (6%) showed only other targetable mutations beside SF3B1. In MDS 73% of patients showed normal karyotypes. MDS patients showed in median 1 additional mutation (range: 0-4), leaving 115/263 (44%) patients without additional mutations. Furthermore, 124/263 (47%) patients carried mutations in a therapeutically relevant gene, while only 32/263 cases (12%) had mutations worsening prognosis. This results in 74/263 MDS patients (28%) without any additional aberration and 85/263 patients (32%) showing only other targetable mutations beside SF3B1. Furthermore, 84% of MDS/MPN-RS-T showed normal karyotypes. In median 1 additional mutation (range: 0-7) was identified in MDS/MPN-RS-T patients, while 10/51 cases (20%) showed no additional mutation. Looking at therapeutically relevant gene mutations revealed in 39/51 patients a respective mutation, while 7/51 patients carried prognostically adverse mutations. Therefore, MDS/MPN-RS-T patients show also a high proportion of cases without additional aberration (9/51, 18%) and even 47% (24/51) of patients having only targetable gene mutations. Conclusion: 1) SF3B1 mutation is supposed to be in the main clone. 2) AML, MDS and MSD/MPN-RS-T differ in their respective patterns of molecular aberrations beside SF3B1 mutations. 3) MDS patients show most frequently SF3B1 mutations as sole abnormality and might therefore benefit best from SF3B1 targeting treatment. 4) Treatment decisions should in all cases consider additional targetable mutations but also those worsening prognosis. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Other: Part Owner MLL Munich Leukemia Laboratory.

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Comprehensive Analyses of Mantle Cell Lymphoma with a 17 Gene Panel Reveal an Accumulation of TP53 Mutations in SOX11 Negative Patients

Blood ◽

10.1182/blood.v124.21.3012.3012 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3012-3012

Author(s):

Manja Meggendorfer ◽

Wolfgang Kern ◽

Claudia Haferlach ◽

Torsten Haferlach ◽

Susanne Schnittger

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

Gene Mutations ◽

Gene Panel ◽

Prognostic Impact ◽

Employment Equity ◽

Mutational Status ◽

Mantle Cell ◽

Sox11 Expression ◽

Equity Ownership

Abstract Introduction: Mantle cell lymphoma (MCL) belongs to the mature B-cell neoplasms and is characterized by t(11;14)(q13;q32)/IGH-CCND1 rearrangement resulting in overexpression of CyclinD1 that is encoded by CCND1. CCND1 is a weak oncogene, requiring additional cooperating oncogenic events. MCL show mainly an aggressive course of disease, although a subset of patients have been identified with an indolent clinical course. This has been associated with a mutated IGHV status and the lack of SOX11 expression. However, some additional gene mutations have been identified in recent years, but the underlying biological heterogeneity is still under debate. Also the prognostic impact of SOX11 remains controversially discussed. Aim: To analyze 1) recently identified molecular mutations in CCND1, UBR5, WHSC1 for their specificity for MCL, 2) in more detail SOX11 negative MCL to get more insights in this group of MCL by sequencing a 17 gene panel. Patients and Methods: In total 184 patients with mature B-cell neoplasms were investigated. All cases were diagnosed according to WHO classification by cytomorphology, immunophenotyping and cytogenetics. 81 cases were diagnosed as MCL, 78 as chronic lymphocytic leukemia/prolymphocytic (CLL/PL), and 25 as CLL. The cohort comprised 67 females and 117 males. CyclinD1 and SOX11 expression levels were quantified by real time PCR. The 17 gene panel included ATM, BIRC3, BRAF, CCND1, FBWX7, IGHV, KLHL6, KRAS, MYD88, NOTCH1, NRAS, POT1, SF3B1, TP53, UBR5, WHSC1, and XPO1. Next generation sequencing was performed on MiSeq instruments (Illumina, San Diego, CA), except for CCND1, UBR5, WHSC1, and IGHV mutational status, which were analyzed by Sanger sequencing. The latter 4 genes and gene expression levels were analyzed in the total cohort, while the 17 gene panel was applied to 26 MCL patients only (13 SOX11 negative and 13 SOX11 positive cases matched for CCND1 mutations and IGHV mutation status). Results: 23/184 (13%) patients had CCND1 mutations, while only 3/184 patients (2%) carried a UBR5 mutation, and 4/184 patients (2%) a WHSC1 mutation. Of note, CCND1 and UBR5 mutations occurred exclusively in MCL patients, WHSC1 mutations were found in MCL and CLL/PL. Therefore CCND1 mutation was, as expected, specific for MCL in comparison to the other 2 mature B-cell neoplasms (p<0.001). Of 81 MCL patients 68 (84%) showed SOX11 overexpression, 23 (28%) had CCND1 mutations and 3 (4%) each had UBR5 and WHSC1 mutations, respectively. The IGHV mutational status was evaluable in 73/81 cases, revealing 32/73 (44%) patients with a mutated IGHV status. A negative correlation of CCND1 mutations and SOX11 overexpression was found: 8/13 (62%) SOX11 negative patients were CCND1 mutated as compared to 15/68 (22%) SOX11 positive patients (p=0.007). Furthermore, CCND1 mutations were more frequent in patients with a mutated IGHV status than in those with unmutated status (15/32 (47%) vs. 8/41 (20%), p=0.021). Accordingly, SOX11 overexpression occurred more often in patients with an unmutated than with a mutated IGHV status (88% vs. 75%; n.s.). Regarding clinical data, more males were SOX11 positive (51/54, 94% vs. 17/27, 63%; p=0.001), and correspondingly more females were CCND1 mutated (12/27, 63% vs. 11/54, 20%; p=0.036). To get more insight in the SOX11 negative patients (n=13) we addressed all these cases and a SOX11 positive control group, matched for IGHV mutational status and CCND1 mutations (n=13), by comprehensive mutational analyses. Overall, beside CCND1 the most frequently mutated gene was TP53 (8/26, 31%), followed by ATM (6/26, 23%), BIRC3 (2/21, 10%), and KRAS (2/26, 8%). No mutations were detected in any of the other genes analyzed. Addressing differences in gene mutations between SOX11 negative and SOX11 positive cases revealed that TP53 mutations were found more frequently in SOX11 negative cases (6/13, 46% vs. 2/13, 15%; n.s.), while ATM mutations were more frequent in SOX11 positive cases (5/13, 39% vs. 1/13, 8%; n.s.). Conclusions: 1) CCND1 mutations are specifically found in MCL, correlate with SOX11 negativity and IGHV mutated status, and are more frequent in females. 2) TP53 is frequently mutated in SOX11 negative patients and its prognostic impact has to be further evaluated. 3) Thus, the differences in clinical course between SOX11 positive and negative MCL patients might correlate with a different spectrum of additional molecular alterations. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Comprehensive Genetic Analyses Of 213 PMF Patients Revealed 13% Of SRSF2 Mutations Being Strongly Correlated To ASXL1 Mutations

Blood ◽

10.1182/blood.v122.21.2828.2828 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2828-2828

Author(s):

Manja Meggendorfer ◽

Tamara Alpermann ◽

Christiane Eder ◽

Claudia Haferlach ◽

Wolfgang Kern ◽

...

Keyword(s):

Molecular Marker ◽

Scoring System ◽

Sanger Sequencing ◽

Gene Mutations ◽

Normal Karyotype ◽

Prognostic Impact ◽

Wild Type ◽

Employment Equity ◽

Cytogenetic Aberrations ◽

Equity Ownership

Abstract Introduction Primary myelofibrosis (PMF) is a myeloproliferative neoplasm, characterized amongst others by stem-cell derived clonal myeloproliferation, bone marrow fibrosis, anemia, splenomegaly, constitutional symptoms and leukemic progression. Diagnosis is based in most cases on cytomorphology/histology demonstrating fibrosis as well as on mutations in JAK2 or MPL. The Dynamic Prognostic Scoring System (DIPSS)-plus is the current base for prognostication using different clinical parameters including karyotype. Furthermore, molecular genetic alterations are currently addressed to provide additional prognostic information. Recently, besides JAK2 and MPL further gene mutations have been described in a limited number of patients, including ASXL1 and SRSF2. Aim To analyze in a large cohort the frequency of SRSF2 and ASXL1 mutations in PMF patients, and to identify their prognostic impact in the context of other previously described gene mutations. Patients and Methods Diagnosis was done according to WHO classification. The cohort comprised 82 female and 131 male patients. In all cases a BCR-ABL rearrangement was excluded by RT-PCR or fluorescence in situ hybridization. JAK2V617F mutation was analyzed in all cases by melting curve analysis, MPLW515 mutation was subsequently analyzed in JAK2V617 wild type (wt) patients. In addition, we analyzed all patients for SRSF2 mutations by Sanger sequencing of the mutational hot spot region coding for amino acid Pro95. Cytogenetics was available in 139 patients. Patients were grouped in favorable (n=121) and unfavorable (n=18) karyotypes based on the DIPSS-plus-scoring system. Based on the previously described correlation of SRSF2mut with ASXL1mut and SETBP1mut in other myeloid entities, SRSF2 mutated cases were also analyzed for mutations in ASXL1 and SETBP1by Sanger sequencing. Follow-up data was available for 136 patients. Results 56% (120/213) of the patients showed JAK2V617F mutations and 18.0% (16/89) of JAK2wt patients carried a mutation in MPLW515 summing up to 65.1% of patients with an already established molecular marker. Of note, SRSF2 was mutated in 12.7% (27/213) of all PMF patients. Patients with SRSF2 mutation had higher white blood cell counts in comparison to SRSF2wt patients (20.00x109/L vs. 7.35x109/L; p=0.005), but there was no correlation to gender, age, hemoglobin level, platelet count or % of myeloblasts in the peripheral blood. In 17 SRSF2mut cases the karyotype was available, 12 were normal karyotype, while two cases showed an unfavorable karyotype according to DIPSS-plus with +8 and i(17)(q10), respectively. The remaining three aberrations belong to the favorable aberration group (del(20q), del(13q), and der(14)). There was no correlation of SRSF2 mutations to the cytogenetic subgroups normal karyotype (n=91) or DIPSS categories favorable and unfavorable aberrations. SRSF2 mutations were also equally distributed between both JAK2V617 or MPLW515 mutated and wild type cases. 18/27 SRSF2mut cases carried also either a JAK2 or MPL mutation, while 9 cases showed no additional JAK2 or MPL mutation. Therefore 30.6% patients remain that carry no mutation in at least one of the three genes investigated first. Interestingly, ASXL1 was frequently mutated in SRSF2 mutated patients (16/23 analyzed SRSF2mut patients) while none of the 24 analyzed SRSF2 mutated cases showed a mutation in SETBP1. To evaluate a potential influence of gene mutations on clinical outcome the overall survival (OS) was calculated. We could confirm that JAK2V617F had no prognostic impact. The same was true for MPLW515 mutations. In contrast to other studies we could not find any impact of SRSF2mutations on OS. Only cytogenetics, i.e. the normal karyotype showed a trend to a prognostic relevance: the median 3 year OS was 70.8% in patients with normal karyotype (n=56) but 58.8% in patients with cytogenetic aberrations (n=29; p=0.153). Conclusion 1) SRSF2 is mutated in 13% of PMF patients. 2) SRSF2 mutated patients show frequently an additional ASXL1 mutation but no coincidence with SETBP1. 3) The prognostic relevance of cytogenetic aberrations was confirmed, while the molecular marker SRSF2 shows no impact on prognosis. Disclosures: Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Diagnostic and Prognostic Utility Of a 26-Gene Panel For Deep-Sequencing Mutation Analysis In Myeloid Malignancies

Blood ◽

10.1182/blood.v122.21.1547.1547 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1547-1547 ◽

Cited By ~ 1

Author(s):

Alexander Kohlmann ◽

Sabrina Kuznia ◽

Niroshan Nadarajah ◽

Tamara Alpermann ◽

Sandra Weissmann ◽

...

Keyword(s):

Overall Survival ◽

Deep Sequencing ◽

Limit Of Detection ◽

Gene Panel ◽

Target Sequence ◽

Prognostic Impact ◽

Employment Equity ◽

Poor Overall Survival ◽

Myeloid Malignancies ◽

Equity Ownership

Abstract Introduction Molecular mutation analyses are performed in myeloid malignancies either in a stepwise procedure, i.e. one target gene after each other or are not performed at all, e.g. in low-risk MDS. A comprehensive pan-myeloid panel to simultaneously target mutations in 26 genes allows a comprehensive analysis with the perspective to detect disease defining mutations in the majority of patients. Aims To test the utility of a pan-myeloid panel in routine diagnostics. Methods We developed sensitive next-generation deep-sequencing (NGS) assays comprising in total 26 genes: ASXL1, BCOR, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3 (TKD), GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PHF6, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1, and WT1. With the exception of RUNX1, which was sequenced on the 454 Life Sciences NGS platform (Branford, CT), all remainder genes were studied using a combination of a microdroplet-based assay (RainDance, Lexington, MA) and the MiSeq sequencing instrument (Illumina, San Diego, CA). The assay's turn-around time was less than 6 days, loading up to eight patients per sequencing run. In summary, 389 amplicons were designed with a median length of 206 bp (range 150-240 bp), representing a total target sequence of 78.15 kb. The sequencing library was constructed starting off 2.2 μg genomic DNA per patient, purified from isolated mononuclear cells. Using the 500 cycles sequencing-by-synthesis chemistry in median 7.644 millions of paired-end reads were generated per run. This resulted in a median coverage per gene of 7,626 reads (range 174-12,256). The lower limit of detection was set at a cut-off of 3%. Results Thus far, 191 prospectively collected cases have been analyzed during routine operations. In all cases the assay was successfully performed. Mutations (range 0-7) have been found in 119/191 (62.3%) cases. The major disease categories were as follows: MDS (n=76), suspected MDS (n=28), MDS/MPN (n=10), reactive bone marrow conditions (n=46), AML (n=8), CML (n=3), other conditions (n=20). We first were interested to address the utility of the panel in MDS when the analysis was restricted to the five prognostically relevant predictors of poor overall survival according to Bejar et al. (N Engl J Med. 2011;364:2496-506), i.e. ASXL1, ETV6, EZH2, RUNX1, and TP53. In detail, 69 cases with MDS were studied and in 42.0% (29/69) of cases mutations had been detected in these five genes while 40 patients showed no mutation. Interestingly, upon extending the analysis to the remainder 21 genes, at least one more mutation was discovered in 72.5% (29/40) of these cases, thereby extending the number of cases with at least one mutation to 84.1% (58/69) of patients. Of note, in 65.5% (19/29) of these latter cases, spliceosome mutations occurred in a mutually exclusive manner (SRSF2, SF3B1, U2AF1), thus also detecting mutations conferring a favorable clinical outcome, i.e. SF3B1 alterations. We next studied in more detail 28 cases with suspected MDS according to cytomorphology, i.e. cases with dysplastic features not sufficient to diagnose MDS. When again in a first step the five predictors of poor overall survival according to Bejar et al. were analyzed, mutations in ASXL1, ETV6, EZH2, RUNX1, and TP53 were observed in 25.0% of cases (7/28). In the group of 75.0% (21/28) of samples with no mutations according to Bejar et al., 28.6% (6/21) of cases harbored a mutation in the group of the 21 remainder genes analyzed simultaneously in the gene panel assay. Thus, in total the number of cases with at least one mutation increased to 46.4% (13/28) of patients. Of note, 6 of the 13 suspected MDS cases with mutations had a normal karyotype. In summary, with respect to correlations between these two cohorts, we observed that morphologically confirmed MDS cases (n=69) showed a higher number of mutated genes compared to “suspected MDS” cases (n=28) (1.88 vs 0.71; p<0.001). Conclusion A pan-myeloid screening assay using NGS allows to address 26 relevant gene mutations in myeloid malignancies with diagnostic or prognostic impact. This approach is scalable and adoptable to accommodate the inclusion of novel gene targets according to the latest evidence from the literature. Importantly, given the broad spectrum of mutations in myeloid diseases covered by such a panel, mutations can be identified in the majority of patients and enable to support a more comprehensive classification in these complex diseases. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Kuznia:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Albuquerque:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Clinical relevance of mutations in patients with myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms with normal karyotype.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.7053 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. 7053-7053 ◽

Cited By ~ 1

Author(s):

Guillermo Montalban-Bravo ◽

Ana Alfonso Pierola ◽

Koichi Takahashi ◽

Marina Konopleva ◽

Elias Jabbour ◽

...

Keyword(s):

Myelodysplastic Syndromes ◽

Lower Risk ◽

Myeloproliferative Neoplasms ◽

Univariate Analysis ◽

Normal Karyotype ◽

Median Number ◽

Gene Panel ◽

Prognostic Impact ◽

Risk Patients

7053 Background: Clinical outcomes of patients with myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are heterogeneous. Specific mutations and mutation patterns are known to define prognostic groups in normal karyotype acute myeloid leukemia. Whether this is the case in MDS and MDS/MPN remains unknown. Methods: We evaluated 325 previously untreated patients with MDS or MDS/MPN with normal karyotype evaluated from 2012 to 2016. Next generation sequencing (NGS) on whole bone marrow DNA analyzing a panel of 28 or 53 genes was performed at the time of diagnosis. Results: A total of 225 (69%) patients had MDS and 100 (31%) had MDS/MPN including 77 (24%) patients with chronic myelomonocytic leukemia (CMML). Median age was 69 years (31-92). Among patients with MDS, 189 (84%) had lower-risk and 36 (16%) had higher-risk based on IPSS. NGS data was obtained by 53-gene panel in 93 (29%) patients and by 28-gene panel in 232 (71%). A total of 202 (62%) patients had detectable mutations. Median number of mutations was 1 (range 0-6). Detected mutations are detailed in Table 1. A total of 111 (34%) patients, 70 with MDS and 41 with MDS/MPN, received therapy with hypomethylating agents. Median follow up was 12 months (0-167). By univariate analysis, NRAS (HR 3.28, CI 1.25-8.62, p=0.016) and TP53 (HR 4.9, CI 1.44-16.67, p=0.011) predicted for shorter overall survival (OS) among MDS patients. After multivariate analysis including IPSS-R, only TP53retained its impact in OS (HR 5.25, CI 1.44-19.13, p=0.012). Among MDS/MPN patients, no mutation was found to significantly impact OS. Conclusions: With the exception of TP53mutations, no other identified mutation seemed to independently define prognosis of patients with MDS or MDS/MPN with normal karyotype. In view of the high proportion of lower-risk patients, longer follow up is required to better define prognostic impact of mutations in this population. [Table: see text]

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Mutations in Triple-Negative Patients with Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2019-128764 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5395-5395

Author(s):

Maria Carolina Costa Melo Svidnicki ◽

Paula De Melo Campos ◽

Moisés Alves Ferreira Filho ◽

Caio Augusto Leme Fujiura ◽

Tetsuichi Yoshizato ◽

...

Keyword(s):

Essential Thrombocythemia ◽

Triple Negative ◽

Myeloproliferative Neoplasms ◽

Biological Significance ◽

Primary Myelofibrosis ◽

Prognostic Impact ◽

Hematopoietic Stem ◽

Missense Variants ◽

Myeloid Neoplasms ◽

Equity Ownership

Background Myeloproliferative neoplasms (MPNs) are chronic hematopoietic stem cell disorders, including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (MF). JAK2, MPL, and CALR mutations are considered as "driver mutations" and are directly implicated in the disease pathogenesis by activation of JAK/STAT signaling. However, some patients do not harbor any of these mutations. Since such triple-negative MPNs are very rare, no specific molecular markers were established to use for a precise differential diagnosis yet. So far, the introduction of next generation sequencing (NGS) technologies in research of myeloid neoplasms has provided valuable contributions on the identification of new molecular biomarkers, establishing more accurate risk rating and selection of more specific therapeutic interventions. This study aimed to identify, through targeted deep sequencing, specific genetic variants in patients with triple-negative MPNs. Methods We performed NGS targeted sequencing in 18 Brazilian triple-negative patients (11 MF and 7 ET). The median age at diagnosis was 64 years for primary myelofibrosis (range 42-78), and 52 years for essential thrombocythemia (range 19-79). In 14 cases, we used the Illumina TruSight Myeloid Panel covering 54 genes and in 4 cases we used a custom Sure Select Agilent panel containing more than 300 genes previously reported to be related to myeloid neoplasm. The inclusion criteria for variant filtering was quality score>30, read count>50, minor allele frequency<0.05, frameshift, nonsense, splice site and 5`UTR variants, and missense variants described as deleterious for at least three prediction softwares. Results Possible pathogenic mutations were identified in 33 genes by Illumina and/or Agilent panels. Frameshift/nonsense or missense variants previously described as pathogenic correspond to 11 variants (Table 1). Out of these, mutations in TET2 were the most frequently identified (in 9/18 (50%) of the cases). In three MF patients with TET2 mutations no other considered pathogenic mutation was identified, indicating a possible role of TET2 as a driver gene. According to previous reports, the frequency of TET2 mutations in triple-negative MPNs patients were only 7%. Phenotypically, in our triple-negative MPNs, 6/11 (54.5%) MF and 3/7 (42.9%) ET patients harbored TET2 mutations. Clinically, the adverse prognostic impact of TET2 mutations in MPN had not been consistently shown by previous studies. In addition, mutations in SF3B1, CEBPA, and KMT2A genes were the second most frequent ones detected in 2/18 each (11%) of the patients, some of which were concomitant with TET2 mutations, suggesting additional clonal advantage due to these genetic events. Other potentially pathogenic variants were also detected is genes that have been reported to be related to other myeloid neoplasms (KMT2A, CDKN2A, TERT, DIS3, ZFPM1, PCDHA8, SAMD9, SAMD9L, DCLRE1C,ERBB3, SDHA, PCDHA6, SVEP1, MAP2K1 and EP300). Conclusions We have characterized the genomic alterations in 18 Brazilian patients with MPN triple-negative for either JAK2, CALR or MPL main mutations. Using a sensitive NGS platform, we identified significantly more frequent mutations in TET2 gene (in as many as a half of the cases) compared to JAK2, MPL, CALR mutation-positive MPN cases. We also uncovered mutations in genes not previously related with in MPN. Our novel findings call for further studies validating the frequencies, biological significance, and prognostic impacts of somatic mutations in triple-negative MPNs. Disclosures Ogawa: Qiagen Corporation: Patents & Royalties; RegCell Corporation: Equity Ownership; Kan Research Laboratory, Inc.: Consultancy; Asahi Genomics: Equity Ownership; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding.

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WT1 Mutations Are Secondary Events In AML and Show Varying Frequencies Within Genetic Subgroups and Different Impact On Prognosis

Blood ◽

10.1182/blood.v122.21.2542.2542 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2542-2542

Author(s):

Susanne Schnittger ◽

Christiane Eder ◽

Tamara Alpermann ◽

Frank Dicker ◽

Madlen Ulke ◽

...

Keyword(s):

Multivariate Analysis ◽

Median Time ◽

Univariate Analysis ◽

Normal Karyotype ◽

Adverse Impact ◽

Prognostic Impact ◽

Employment Equity ◽

Genetic Aberrations ◽

Equity Ownership ◽

The Impact

Abstract Background Mutations (mut) in the WT1 gene belong to the first genetic aberrations described in AML. In contrast to recurrent fusion genes or NPM1mut WT1mut do not seem to be disease defining. Also in contrast to other mutations in AML, for most of which a certain prognostic value has been established, the impact of WT1mut still is discussed controversially. Aim Analyze the frequency and prognostic impact of WT1 mutations in comparison to other genetic aberrations. Patients and Methods 3,157 unselected AML patients (pts) were analyzed (de novo: n=2,699, s-AML: n=234, t-AML: n=224). 1,708 pts were male and 1,449 female. Median age was 67.1 years (y) (range: 17.8-100.4 y) with 1,108 pts <60 y and 2,049 ≥60 y. The mutational hot spot regions of WT1 (exons 7 and 9) were analyzed by direct Sanger sequencing with a sensitivity of ∼10%. Karyotype and WT1 mutation status was available in all cases. Other mutations were assessed in subsets: ASXL1 (n=1,951), CEBPA (n=2,670), DNMT3A (n=1,293), FLT3-ITD (n=3,149), FLT3-TKD (n=3,004), IDH1R132 (n=2,431), IDH2R140 (n=2,380), IDH2R172 (n=2,412), KRAS (n=1,409), NRAS (n=1,780), NPM1 (n=3,003), MLL-PTD (n=2,961), RUNX1 (n=2,390), TET2 (n=1,016) and TP53 (n=1,215). Results A total of 189 WT1 mutations were detected (exon 7: n=151, exon 9: n=38). The total frequency of WT1mut pts was 175/3,157 (5.5%). 11 pts were double to quadruple mutated. The frequency was heterogeneous with respect to AML subtypes. Compared to all others, significantly higher frequencies were detected in biallelic CEBPAmut (15/110; 13.6%; p=0.001), followed by t(15;17)/PML-RARA (18/164; 11.0%, p=0.004), and FLT3-ITD (58/682; 8.5%, p<0.001). Lower frequencies were observed in DNMT3Amut (18/412; 4.3%, p=0.014, ASXL1mut (6/355; 1.7%, p<0.001), IDH2R140 (5/286; 1.7%, p=0.001), and IDH1R132 (2/222; 0.9%, p<0.001). WT1mut were never detected in pts with complex karyotypes (0/175; p=0.047) or those with IDH2R172 (0/68; p=0.020). Further, WT1mut were more frequent in females (95/1,449, 6.6%) than in males (80/1,708, 4.7%) (p=0.014) and in younger pts (<60 y: 102/1,108, 9.2% vs ≥ 60 y: 73/2,049, 3.6%; p<0.001). Median age of pts with WT1mut was 55.5 y compared to 63.6 in WT1wt (p<0.001). Further, WT1mut were associated with lower platelet count (58.4 vs 84.7 x109/L; p<0.001) and lower hemoglobin level (8.8 vs 9.3 g/dL, p=0.001). There was no association to the history of the disease or white blood cell count. Stability of WT1mut was analyzed in 35 paired diagnostic and relapse samples (median time of relapse after diagnoses: 11.1 months (m); range: 2.6-60.6 m). In 23 cases (65.7%) the WT1mut was retained at relapse and in 12 cases (34.3%) it was lost. In 5 cases a sample at 2nd relapse was available (median time from 1st relapse: 8.5 m, range: 6.0-18.0 m). 3 of these cases retained and 2 lost the WT1mut. Analysis of prognostic impact was restricted to intensively treated pts (n=1,936, WT1mut: n=132, 6.8%). In the total cohort, there was no impact of WT1mut on prognosis. In pts ≥60 y there was a trend to shorter event free survival (EFS) for WT1mut (9.3 vs 12.3 m, p=0.052). In the two prognostically favorable groups with high WT1mut incidences (biallelic CEBPAmut and PML-RARA) no effect on outcome was seen. When restricting the analysis to normal karyotype AML (WT1mut: n=85, WT1wt: n=1,093) WT1mut pts had shorter EFS (10.8 vs 17.9 m, p=0.008). This was true for the younger (12.2 vs 29.0 m, p=0.007) as well as for the older pts (9.3 vs 13.9 m, p=0.016). In a multivariate analysis all parameters with significant impact on EFS in univariate analysis were included: age (p<0.001, HR: 1.24), ASXL1mut (p<0.001, HR: 1.36), FLT3-ITD (p<0.001, HR: 1.55), NPM1mut/FLT3-ITD wild-type (p<0.001, HR:1.55), RUNX1 (p=0.019, HR: 1.23, and WT1mut (p=0.009, HR: 1.64). In multivariate analysis WT1mut was found to have independent adverse impact on EFS (p=0.002, HR: 1.64) besides FLT3-ITD status (p<0.001, HR: 1.71) and age (p<0.001, HR: 1.28). Conclusions WT1 mutations are 1) more frequent in females and younger AML, 2) more frequent in t(15;17)/PML-RARA, biallelic CEBPAmut, FLT3-ITD mutated AML, and nearly mutually exclusive of ASXL1, IDH1, IDH2 and complex karyotype. 3) The distribution pattern in different genetic subtypes and the instability during follow-up as shown by paired sample analyses clearly emphasize a secondary character of this mutation. 4) For AML with normal karyotype an independent adverse impact of WT1mut on EFS was shown. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Eder:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Ulke:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kuznia:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Characterization of MDS Harboring TET2 Mutations and/or TET2 Deletions

Blood ◽

10.1182/blood.v128.22.4288.4288 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4288-4288 ◽

Cited By ~ 1

Author(s):

Claudia Haferlach ◽

Anna Stengel ◽

Manja Meggendorfer ◽

Wolfgang Kern ◽

Torsten Haferlach

Keyword(s):

Overall Survival ◽

Relative Risk ◽

Molecular Genetic ◽

Normal Karyotype ◽

Prognostic Impact ◽

Complex Karyotype ◽

Wild Type ◽

Employment Equity ◽

Total Cohort ◽

Equity Ownership

Background: TET2 mutations and deletions have been reported in MDS. A detailed analysis of the prognostic impact of TET2 deletions and their association to TET2 mutations is lacking. Aim: To characterize MDS with TET2 mutations (mut) and/or TET2 deletions (del) with respect to accompanying cytogenetic and molecular genetic abnormalities and their impact on prognosis. Patients and Methods: First 788 unselected MDS cases (cohort A) were evaluated. As in this cohort only 8 cases with TET2 deletion were detected, further MDS were screened for TET2 deletions. In total 77 MDS harboring a TET2 deletion were identified and included in cohort B. Both cohorts were analyzed by chromosome banding analysis, FISH, genomic arrays and mutation analysis of TET2. Cases from cohort A were also analyzed for mutations in ASXL1, ATM, BCOR, BRCC3, CBL, CTCF, DNMT3A, ETV6, EZH2, FBXW7, IDH1, IDH2, JAK2, KRAS, LAMB4, MPL, NCOR1, NCR2, NF1, NRAS, PHF6, PRPF8, PTPN11, RAD21, RUNX1, SETBP1, SF3B1, SMC3, SRSF2, STAG2, TET2, TP53, U2AF1 and ZRSR2. Results: In cohort A 248 cases (31%) with TET2mut were identified. TET2del and a normal karyotype were more frequent in MDS with TET2mut as compared to those with TET2 wild-type (wt) (3% vs 1%, p=0.006; 89% vs 78%, p<0.001). SF3B1 and ASXL1 were frequently mutated in both TET2mut and TET2wt MDS (32% and 34%, 22% and 18%, respectively). In MDS with TET2mut compared to MDS with TET2wt the following genes were less frequently mutated: ATM (0.5% vs 3%, p=0.05), DNMT3A (9% vs 15%, p=0.02), ETV6 (0.5% vs 3%, p=0.03), IDH1 (0.5% vs 3%, p=0.02), IDH2 (1% vs 5%, p=0.002), TP53 (2% vs 7%, p=0.004), U2AF1 (4% vs 9%, p=0.04), while the following genes were more frequently mutated: CBL (6% vs 2%, p=0.01), EZH2 (8% vs 2%, p<0.001), SRSF2 (27% vs 12%, p<0.001), and ZRSR2 (15% vs 3%, p<0.001). Overall spliceosome genes were more frequently mutated in TET2mut than in TET2wt MDS (77% vs 56%, p<0.001). In the total cohort A neither TET2mut nor TET2del had an impact on overall survival (OS). In TET2mut MDS and TET2wt MDS SF3B1mut were associated with favorable outcome, while TP53mut were associated with shorter OS in both subsets (table 1). However in TET2mut MDS mutations in RUNX1 (p<0.0001), CBL (p=0.001), and U2AF1 (p=0.03) were independently associated with shorter OS, while in TET2wt MDS mutations in KRAS (p=0.03), EZH2 (p=0.02), NRAS (p=0.02), SRSF2 (p=0.007), IDH2 (p=0.05), and ASXL1 (p=0.01) were independently associated with shorter OS. In cohort B 40/77 (52%) MDS with TET2del also harbored a TET2mut. The 4q deletion encompassing the TET2 gene was < 10 MB in size and thus cytogenetically cryptic in 77% of cases with TET2mut, while the TET2 deletion was cryptic in only 24% of cases without TET2mut. A normal karyotype was present in 37 cases (48%), a complex karyotype in 29 (38%) and other abnormalities in 11 cases (14%). TET2mut were frequent in cases with a normal karyotype (68% vs aberrant karyotype: 32%, p<0.001) and were rare in cases with a complex karyotype (13%). Relating the mutation load of TET2mut to the proportion of cells with TET2del as determined by FISH revealed in 60% of cases that both TET2 alterations were present in the main clone, while in 23% of cases the TET2mut was present in a subclone only and in 17% the TET2del was observed in a subclone only. In the subset of patients with TET2del in a subclone only, 83% showed a normal karyotype and none a complex karyotype, while in the subset of cases with TET2mut in a subclone only, 43% showed a normal and 29% a complex karyotype. In the total cohort B the presence of a TET2mut in addition to the TET2del had no prognostic impact, while the presence of a complex karyotype was associated with shorter OS (RR: 8.0, p=0.004). Conclusions: 1) TET2 deletions are rare in TET2 mutated MDS (3%). 2) TET2 mutations are frequent in MDS with TET2 deletion (52%). 3) TET2 mutations are highly correlated to a normal karyotype and are rare in complex karyotype. 3) Neither TET2 mutations nor TET2 deletions have a prognostic impact in MDS. 4) In TET2 mutated MDS mutations in RUNX1, TP53, CBL, and U2AF1 have the strongest negative independent impact on OS, which in TET2 wild-type MDS is the case for mutations in TP53, KRAS, EZH2, NRAS, SRSF2, IDH2 and ASXL1. Table The relative risk of parameters significantly (p<0.05) associated with overall survival are depicted in TET2 mutated and TET2 wild-type MDS Table. The relative risk of parameters significantly (p<0.05) associated with overall survival are depicted in TET2 mutated and TET2 wild-type MDS Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Stengel:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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