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

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

Blood ◽

10.1182/blood.v124.21.3224.3224 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3224-3224

Author(s):

Manja Meggendorfer ◽

Tamara Alpermann ◽

Claudia Haferlach ◽

Wolfgang Kern ◽

Susanne Schnittger ◽

...

Keyword(s):

Myeloproliferative Neoplasms ◽

Gene Mutations ◽

Normal Karyotype ◽

Great Majority ◽

Primary Myelofibrosis ◽

Gene Panel ◽

Prognostic Impact ◽

Prognostic Information ◽

Employment Equity ◽

Equity Ownership

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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A MAFB Associated Regulatory Network Distinguishes CMML from Other MDS/MPN Overlap Entities

Blood ◽

10.1182/blood-2018-99-115838 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3890-3890

Author(s):

Wencke Walter ◽

Claudia Haferlach ◽

Wolfgang Kern ◽

Torsten Haferlach ◽

Manja Meggendorfer

Keyword(s):

Gene Expression ◽

Regulatory Network ◽

Myeloproliferative Neoplasms ◽

Hematologic Malignancies ◽

Cell Surface Receptor ◽

Specific Gene ◽

Biological Processes ◽

Employment Equity ◽

Complex Interactions ◽

Equity Ownership

Abstract The myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are a unique group of hematologic malignancies characterized by overlapping features of myelodysplastic syndromes and myeloproliferative neoplasms. The category includes atypical chronic myeloid leukemia (aCML), chronic myelomonocytic leukemia (CMML), MDS/MPN, unclassifiable (MDS/MPN, U), and MDS/MPN with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T). The recent adaptation of next generation sequencing in genetic testing increased the knowledge of the molecular pattern of MDS/MPN. However, no specific gene signatures have been identified and underlying transcriptional mechanisms are still poorly understood. Therefore, we analyzed the transcriptome of a cohort of 49 aCML, 102 CMML, 50 MDS/MPN-U, and 72 MDS/MPN-RS-T patients, morphologically defined according to the WHO classification. Total RNA was used for 150bp paired-end RNA sequencing with a median read depth of 50 million. The edgeR package was used to perform a combined normalization and differential expression (DE) analysis of the obtained estimated gene counts by integration of trimmed mean of M-values normalization factors into the statistical model used to test for DE. Genes with an FDR < 0.05 and an absolute logFC > 1.5 were considered DE. 273 unique genes were identified differentially expressed between the entities. We found that the transcriptional profile of CMML patients was dominated by an upregulation of MAFB compared to all other entities (p < 0.001). MAFB encodes a transcription factor which is specifically expressed in myeloid cells and a master regulator of human monocytopoiesis. In order to unravel the potential effects of MAFB regulation we performed Spearman's correlation analysis to identify co-expressed genes. 51 genes showed a similar expression (correlation coefficient > 0.7) as MAFB, establishing a CMML gene expression signature (GES) (Figure 1a). The GES included the cell cycle inhibitor CDKN1A, the transcription factors IRF8, EGR2, and MYCL, and the cell surface receptor LRP1. LRP1 represents a therapeutic target and has recently been identified as a key factor in a network for multi-cancer clinical outcome prediction. MAFB was also co-expressed with KYNU, which is synthesized by indoleamine 2,3-dioxygenase, an enzyme expressed by leukemia blasts, and which has been linked with unfavorable outcome in hematologic malignancies. MAFB and KYNU were also co-expressed with multiple CD markers, which are often used as targets of immunotherapy approaches in cancer. High expressions of KYNU can block the anti-tumor effects of chimeric antigen receptor T-cell therapy, indicating complex interactions between the co-expressed genes. To better understand these underlying molecular interactions on a more global scale and to expose potential targets for intervention, a transcriptional network of the 51 MAFB co-expressed genes was reverse engineered. We used STRING, a publicly available database of protein-protein interactions, to build the scaffold of the network and subsequently pruned the network based on our gene expression data. The reconstructed, MAFB associated regulatory network (Figure 1b) identified c-FOS as an additional regulatory link. c-FOS is a known oncogene that heterodimerizes with c-JUN, ATF3, and JDP2 to form the structure of the AP-1 complex. The AP-1 complex is a key regulator of multiple biological processes such as cell proliferation and apoptosis. We observed a considerably higher expression of c-FOS compared to c-JUN, ATF3, and JDP2 in CMMLs. The MafB/cFos heterodimer is known to explicitly repress apoptosis. The co-expression network was significantly enriched for transcriptional activation of p53 responsive genes (p < 0.001). Interestingly, we also found that the p53-regulated long intergenic non-coding RNA lncPRESS1 was down-regulated in our cohort of CMML patients (p < 0.001). Conclusions: 1) MAFB represents a promising gene expression marker to distinguish CMML pts from other MDS/MPN overlap entities. 2) The identified core regulatory signature in CMML pts showed complex interactions to modulate diverse biological processes and might serve as therapeutic targets. Disclosures Walter: 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. Meggendorfer:MLL Munich Leukemia Laboratory: Employment.

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Co-Occurrence of Separate BCR-ABL1-Positve and JAK2V617F-Positive Clones in 23 Patients Reveals Biologic and Clinical Importance

Blood ◽

10.1182/blood.v124.21.3175.3175 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3175-3175

Author(s):

Pedro Martin-Cabrera ◽

Claudia Haferlach ◽

Torsten Haferlach ◽

Wolfgang Kern ◽

Susanne Schnittger

Keyword(s):

Limit Of Detection ◽

Myeloproliferative Neoplasms ◽

Melting Curve ◽

Rare Event ◽

Gene Mutations ◽

Jak2v617f Mutation ◽

Positive Clone ◽

Employment Equity ◽

Equity Ownership

Abstract Background: The simultaneous detection of a BCR-ABL1 rearrangement and a JAK2V617F mutation in the same patient is a very rare event and has previously been described in case reports or very small series of cases only. Aim: 1) To establish the incidence of cases with concurrent BCR-ABL1 rearrangement and JAK2V617F mutation. 2) Evaluate whether one clone harbours both mutations or whether there are two independent clones. 3) Establish whether these patients have additional concurrent gene mutations and how they influence the evolution of both diseases. Patients and Methods: A total of 27,907 patients with suspected myeloproliferative neoplasms (MPN) where studied in parallel for BCR-ABL1 and JAK2V617F mutation from May 2005 to June 2014 at our institution. BCR-ABL1 analysis was performed by multiplex RT-PCR and JAK2V617F mutation analysis by melting curve based LightCycler assay. A total of 23 patients (0.08%) were positive for both mutations. Eleven patients were male and 12 were female with a median age at diagnosis of 72 years (range 46-80 years). Of fifteen patients 2 or more sample time points were available for follow-up analyses (median follow-up: 4 years, range: 5 months - 9 years). Both BCR-ABL1 and JAK2V617F mutation loads were assessed by quantitative real time PCR. In addition, 22/23 cases were analyzed upon detection of co-occurrence of both clones with a pan-myeloid gene panel consisting of 25 genes: TET2, RUNX1, PHF6, ASXL1, CBL, DNMT3A, SF3B1, TP53, BCOR, BRAF, ETV6, EZH2, FLT3 (TKD), GATA1, GATA2, IDH1, IDH2, KIT, KRAS, MPL, NPM1, NRAS, SRSF2, U2AF1, and WT1. Either complete coding genes or hotspots were first amplified by a microdroplet-based assay (RainDance, Lexington, MA) and subsequently sequenced with a MiSeq instrument (Illumina, San Diego, CA). RUNX1 was sequenced on the 454 Life Sequence NGS platform (Roche 454, Branford, CT). The median coverage per amplicon was 2,215 reads (range 100-24,716). The lower limit of detection was set at a cut-off of 1.5%. Results: At the time point of detection of both mutations morphological assessment was available in 12 patients. The remaining 5 showed features typical for CML. Bone marrow blast count was <5% in all cases. Cytogenetics was available in 18/23 cases (78.3%). The classical t(9;22)(q34;q11) was identifiable in 16/18 patients. Two patients had a normal karyotype as they were in complete cytogenetic remission of their CML (due to TKI treatment) at diagnosis of the JAK2 V617F positive clone. In the majority of patients (n=16) the JAK2V617F mutation predated the BCR-ABL1 clone, in 4 patients CML was known before the detection of the JAK2V617 positive clone, in 1 patient both were diagnosed simultaneously and in another 2 patients information in this regard was lacking. BCR-ABL1 transcript types distributed as follows: b2a2 and/or b3a2 (n=18), and e1a2 (n=5). The continuous quantitative assessment of BCR-ABL1 and JAK2V617F mutational loads in 15 patients showed asynchronous patterns of courses in all cases giving proof of these aberrations representing two different clones in these cases. When treatment with TKI was initiated, the BCR-ABL1 clone decreased while the JAK2V617F clone either remained stable or increased in all 15 cases. Next generation sequencing revealed further mutations in 13/22 analyzed patients (56.5%). One mutation was detected in 8 patients, 4 patients revealed 2, and one patient even 3 different additional mutations. In detail, mutations in the following genes were detected: TET2 (n=8), ASXL1 (n=4), RUNX1 (n=2), CBL (n=1), DNMT3A (n=1), PHF6 (n=1) SF3B1 (n=1) and TP53 (n=1). These mutations were traced and quantified retrospectively. Data suggests that they were most probably present in the JAK2V617F positive clone. This again supports the theory of both clones being independent of each other. Conclusions: 1) Co-occurrence of BCR-ABL1 and JAK2V617F is a very rare event (0.08%). 2) BCR-ABL1 and JAK2V617F represent two different clones. 3) Additional gene mutations are detected in 56% of these cases and all seem to be within the JAK2V617F positive clone. 4) Clinically, the BCR-ABL1 clone is easily controlled with TKI, however, the combined management of the JAK2V617F clone is more challenging especially when a fibrotic phase of the disease takes over. The long-term effect of JAK2-inhibitors in the management of these patients is yet to be established. Disclosures Martin-Cabrera: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Does Age of Patients Influence the Composition of Gene Mutations in Myeloid Neoplasms?

Blood ◽

10.1182/blood.v126.23.3821.3821 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3821-3821

Author(s):

Manja Meggendorfer ◽

Wolfgang Kern ◽

Karolína Perglerová ◽

Susanne Schnittger ◽

Claudia Haferlach ◽

...

Keyword(s):

Healthy Aging ◽

Risk Group ◽

Gene Mutations ◽

Specific Gene ◽

Healthy Individuals ◽

Employment Equity ◽

Myeloid Neoplasms ◽

Age Dependent ◽

Equity Ownership ◽

Good Risk

Abstract Introduction: Incidences of myeloid neoplasms, i.e. AML, MDS, MDS/MPN overlap, increase with age. Cytogenetic aberrations are still the hallmark for diagnosis and prognostication especially in AML and MDS. Different patterns of relations between chromosomal aberrations and age have been described. However, in recent years, gene mutations have been depicted to further discriminate patients with respect to their diagnosis and are increasingly used for prognostication. In parallel, recent studies (Jaiswal, NEJM 2014) have demonstrated that mutations in genes occurring in hematological neoplasms are also observed in healthy individuals and increase in frequency with age. Aim: To investigate if specific molecular markers, such as DNMT3A, ASXL1, and TET2, increase in frequency with age in myeloid neoplasms as recently shown for healthy individuals. Patients and methods: We investigated 1639 patients (pts) between 20 and 93 years (yrs), 578 with de novo AML (median age: 63 yrs), 846 with MDS (median age: 73 yrs), and 215 with CMML (median age: 75 yrs). In all cases, we followed the diagnostic criteria of the WHO classification based on morphology. All patients have also been investigated by cytogenetics and for disease-oriented molecular mutations (15-36 genes/pt: 15 in AML, 36 in MDS, and 21 in CMML). Analyses were performed by melting curve analysis, gene scan, Sanger sequencing, or next generation sequencing. Results: In total we detected 3089 mutations (range: 0-9/pt) spread over all except for seven analyzed genes. Grouping the entity-specific cohorts by age of the patients into decades revealed a steady increase of the prevalence of mutations with age in MDS (at least one mut/pt, 25% in 20-29 to 93% in >80 yrs; p<0.001), less prominent in AML (77% in 20-29 to 100% in >80 yrs, p=0.007), but not in CMML (96%-100% in all decades). However, the number of mutations per patient increased according to age in all three entities, significantly in MDS (p<0.001) and AML (p<0.001). Considering AML patients separated into three cytogenetic classes (Grimwade, Blood 2010) resulted in the same findings for the intermediate risk (p=0.012) and adverse risk group (p<0.001), while the good risk group showed no change in mutation numbers over decades (median: 1 mut/pt, range 0-3). This indicates that in good risk AML (PML-RARA, CBFB-MYH11, RUNX1 -RUNX1T1) only very few additional mutations are needed for AML initiation. In contrast, an age-dependent increasing incidence of gene mutations is specific in normal karyotype and in adverse cytogenetics. We next focused on specific gene mutations according to age <60 vs ≥60 yrs within all three entities. In addition, AML patients where again subgrouped by cytogenetics. In AML good and adverse risk groups no age-dependent significant increase of specific gene mutations occurred, while in the intermediate risk group mutations in ASXL1 (3/160 vs 32/114, p<0.001), MLL-PTD (3/160 vs 17/214, p=0.01), RUNX1 (32/160 vs 20/214, p=0.001), and TET2 (4/159 vs 26/214, p=0.001) were significantly more frequent at higher age. In contrast, NRAS mutations appeared more often in younger AML patients (32/160 vs 20/214, p=0.004). In MDS, mutations in SF3B1 (27/115 vs 253/731, p=0.019), SRSF2 (10/115 vs 133/730, p=0.011), TET2 (10/115 vs 250/731, p<0.001), and TP53 (2/115 vs 50/731, p=0.035) were more frequently observed in older patients. In CMML only TET2 mutations occurred more often in older patients (5/12 vs 135/190, p=0.026). Focusing on the genes recently described to be mutated in healthy individuals showed that all of the above mentioned mutations found in myeloid neoplasms (except MLL-PTD and RUNX1) are comprised in the 10 most frequently mutated genes in the healthy aging population. However, the fact that the frequencies of these mutations are not age-dependent in some entities, e.g. ASXL1 only age-dependent in AML but not in CMML and MDS, might indicate different roles of these mutations in the pathogenesis, i.e. driver mutations independent of age, as well as their contribution to accumulation of mutations and onset of a myeloid neoplasm. Conclusions: 1) The number of mutations significantly increase with age in AML and MDS and non-significantly in CMML. 2) Several genes show age-dependent frequencies, which differ between AML, MDS, and CMML and are also related to the cytogenetic background. 3) Based on molecular mutations healthy aging and myeloid neoplasms are neighbouring scenarios. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Perglerová:MLL2 s.r.o.: Employment. Schnittger: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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Impact of Somatic Gene Mutations on Response to Lenalidomide (LEN) in IPSS Lower-Risk Myelodysplastic Syndromes (MDS) Patients (Pts) without Del(5q) and Ineligible for or Refractory to Erythropoiesis-Stimulating Agents (ESAs)

Blood ◽

10.1182/blood.v128.22.225.225 ◽

2016 ◽

Vol 128 (22) ◽

pp. 225-225 ◽

Cited By ~ 2

Author(s):

Valeria Santini ◽

Pierre Fenaux ◽

Aristoteles Giagounidis ◽

Uwe Platzbecker ◽

Alan F List ◽

...

Keyword(s):

Lower Risk ◽

Research Funding ◽

Response Rate ◽

Single Gene ◽

Gene Mutations ◽

Advisory Board ◽

Employment Equity ◽

Mutation Status ◽

Somatic Gene ◽

Equity Ownership

Abstract Background: Somatic gene mutations occur in the majority of MDS pts; specific mutations and high mutation frequency have prognostic relevance (Papaemmanuil et al. Blood. 2013;122:3616-27). Evaluation of somatic mutations may support the diagnosis of MDS and guide treatment (Tx) selection. The phase 3 randomized MDS-005 study compared LEN and placebo (PBO) Tx in red blood cell transfusion-dependent (RBC-TD) non-del(5q) lower-risk MDS pts ineligible for or refractory to ESAs. Deletions in chromosome 5q are associated with a high response rate to LEN in MDS pts; however, no mutations have been definitively associated with a predictable clinical response to LEN in non-del(5q) MDS. Aim:To investigate the relationship between somatic gene mutations detected by targeted next-generation sequencing (NGS) and response and overall survival (OS) in lower-risk non-del(5q) MDS pts treated with LEN in the MDS-005 study. Methods: Eligible pts were: RBC-TD (≥ 2 units packed RBCs/28 days 112 days immediately prior to randomization) with International Prognostic Scoring System defined Low-/Intermediate-1-risk non-del(5q) MDS; ineligible for ESA Tx (serum erythropoietin > 500 mU/mL); or unresponsive or refractory to ESAs (RBC-TD despite ESA Tx with adequate dose and duration). 239 pts were randomized 2:1 to oral LEN 10 mg once daily (5 mg for pts with creatinine clearance 40-60 mL/min) or PBO. DNA was isolated from bone marrow mononuclear cells or whole blood collected at screening from a subset of pts who gave informed consent for this exploratory biomarker analysis and had adequate tissue for analysis. Targeted NGS of 56 genes was performed at Munich Leukemia Laboratory; average sequencing coverage was 2,000-5,000-foldand the variant allele frequency detection cutoff was 3%. Target regions varied by gene, including all exons to hotspots. For association tests, mutant variants (heterozygous or homozygous) were scored as 1 (mutant) or 0 (wildtype) for gene-level analyses. A Fisher exact test was used to test association of mutation status with response. Median OS was calculated by the Kaplan-Meier method. Hazard ratios and 95% confidence intervals were determined by a non-stratified Cox proportional hazards model. A log-rank test was used to test treatment effect with OS for single gene mutation status. Results: The biomarker cohort included 198 of 239 pts (83%; LEN n = 130, PBO n = 68). At least 1 mutation was detected in 30/56 (54%) genes and 173/198 (87%) pts. The most frequently mutated genes were SF3B1 (59%), TET2 (33%), ASXL1 (23%), and DNMT3A (14%); the most frequent co-mutations were SF3B1/TET2 (23%), SF3B1/DNMT3A (10%), SF3B1/ASXL1 (10%), and TET2/ASXL1 (9%) (Figure). Of 116 pts with SF3B1 mutations, 115 (99%) had ≥ 5% ring sideroblasts. The 56-day RBC transfusion-independence (RBC-TI) response rate was significantly lower in LEN-treated ASXL1 mutant pts vs wildtype pts (10% vs 32%, respectively; P = 0.031). At 168 days, the RBC-TI response rate was still lower in LEN-treated ASXL1 mutant pts vs wildtype pts (7% vs 22%); however, the difference was not significant (P = 0.101). LEN-treated DNMT3A mutant pts had a higher 56-day RBC-TI response rate vs wildtype pts (44% vs 25%); however, this difference did not reach significance (P = 0.133) due to the small sample size. RBC-TI response rate with LEN was similar regardless of total number of mutations per pt. Higher numbers of mutations were significantly associated (P = 0.0005) with worse median OS. Mutation in any of the genes associated with a negative prognosis reported by Bejar et al. (N Engl J Med. 2011;346:2496-506) was also significantly associated (P = 0.0003) with worse median OS.However, OS was not significantly different in LEN- vs PBO-treated pts based on any single gene mutation status. Conclusions: In this group of lower-risk RBC-TD non-del(5q) MDS pts, somatic mutations in genes recurrently mutated in myeloid cancers were detected in 87% of pts. SF3B1 mutations (alone or in combination) were most frequent and not associated with response to LEN. ASXL1 mutant pts had a significantly lower LEN response rate vs wildtype pts, whereas DNMT3A mutant pts had a trend for improved LEN response. Median OS was influenced by mutations, but not significantly modified by LEN. Determining predictive clinical markers for Tx response in non-del(5q) MDS pts remains challenging; nevertheless, there is a significant need to identify pt subsets who may be responsive to LEN Tx. Figure. Figure. Disclosures Santini: Novartis: Consultancy, Honoraria; Amgen: Other: advisory board; Onconova: Other: advisory board; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Astex: Other: advisory board. Fenaux:Celgene, Janssen, Novartis, Astex, Teva: Research Funding; Celgene, Novartis, Teva: Honoraria. Giagounidis:Celgene Corporation: Consultancy. Platzbecker:Janssen-Cilag: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; TEVA Pharmaceutical Industries: Honoraria, Research Funding. Zhong:Celgene Corporation: Employment, Equity Ownership. Wu:Celgene Corporation: Employment, Equity Ownership. Mavrommatis:Discitis DX: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Employment, Equity Ownership. Beach:Celgene Corporation: Employment, Equity Ownership. Hoenekopp:Celgene Corporation: Employment, Equity Ownership. MacBeth:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties, Research Funding.

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Outcome of Refractory Anemia with Ringed Sideroblasts Associated with Marked Thrombocytosis (RARS-T) In a Large Cohort of Patients

Blood ◽

10.1182/blood.v116.21.4113.4113 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4113-4113 ◽

Cited By ~ 1

Author(s):

Francois Girodon ◽

Julien Broseus ◽

Lourdes Florensa ◽

Esther Zipperer ◽

Susanne Schnittger ◽

...

Keyword(s):

Platelet Count ◽

Myeloproliferative Neoplasms ◽

Hemoglobin Concentration ◽

Large Cohort ◽

Refractory Anemia ◽

White Blood Count ◽

Employment Equity ◽

Disease Evolution ◽

Ringed Sideroblasts ◽

Equity Ownership

Abstract Abstract 4113 Introduction: Most of the data related to RARS-T, a rare disorder, involve small cohorts of patients. We aimed to analyze more patients also considering a variety of myelodysplastic or myeloproliferative disorders. Objective: To compare a large cohort of patients with RARS-T to refractory anemia with ringed sideroblasts (RARS), refractory anemia with ringed sideroblasts and multilineage dysplasia (RARS-MD) or essential thrombocythemia (ET) at the time of diagnosis and during disease evolution, in terms of survival and complications. Materials: Data of a European multi-center study was used including 199 cases of RARS-T 173 cases of RARS, 102 cases of RARS-MD and 431 cases of ET. Results: At baseline, compared to RARS and RARS-MD patients, RARS-T patients had similar hemoglobin concentration, but a higher white blood count. The JAK2V617F mutation was observed in 43%, 12% and 5% in RARS-T, RARS and RARS-MD patients, respectively. When separated in 2 groups (450,000<platelet count <600,000 and platelet count >600,000 × 109/l), RARS-T patients were comparable for sex, age, hemoglobin level and survival. However, patients with platelet count > 600,000 × 109/l had higher WBC (11 ×109/l versus 7.5 ×109/l, p<0.001). Similarly, no difference was noted in the survival in the JAK2 positive and negative RARS-T patients. The age and sex standardised overall survival of RARS-T patients was similar to RARS and RARS-MD patients, but lower than ET patients (p<0.001). This was despite a higher risk of transformation in acute leukemia, relative to RARS-T afflicted individuals, of 2.4 and 3.5 in RARS-MD and RARS patients, respectively. Conclusion: According to our results, the outcome in RARS-T more closely mimics myelodysplastic syndromes rather than myeloproliferative neoplasms. Our results agree with the WHO 2008 classification that considers RARS-T as a separate disorder. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Gattermann:Novartis: Honoraria, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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In BCR-ABL1 Negative Myeloproliferative Neoplasms the Detection of JAK2exon12, MPLW515, CBL, KITD816V, FIP1L1-PDGFRA Mutations Are Closely Linked to Specific Entities, Whereas the JAK2V617F, TET2, or EZH2 Mutations Demonstrate a Broader Diversity: Patterns From Diagnostic Reports of 18,547 Patients Analyzed

Blood ◽

10.1182/blood.v116.21.458.458 ◽

2010 ◽

Vol 116 (21) ◽

pp. 458-458

Author(s):

Susanne Schnittger, ◽

Christiane Eder ◽

Frank Dicker ◽

Vera Grossmann ◽

Alexander Kohlmann ◽

...

Keyword(s):

Myeloproliferative Neoplasms ◽

Hematologic Malignancy ◽

Rare Event ◽

Jak2v617f Mutation ◽

Patient Specific ◽

Second Step ◽

Double Positive ◽

Employment Equity ◽

Equity Ownership ◽

Diagnostic Work

Abstract Abstract 458 The first mutation detected in BCR-ABL1 negative myeloproliferative neoplasms (MPN) was JAK2V617F that revolutionized diagnostics of MPN during the last five years. However, although this genetic marker is useful to discriminate MPN from reactive disorders, it is not specific for one entity. In addition, approximately 5% of all polycythemia vera (PV) and 50% of essential thrombocytosis (ET) and primary myelofibrosis (PMF) are not JAK2V617F mutated. In these entities other activating mutations, e.g. MPLW515 mutations or JAK2exon12 mutations, cover additional small proportions of patients without JAK2V617F mutation. To further improve the molecular genetic characterization of MPN research focuses on the identification of novel mutations and, recently, CBL, TET2, and EZH2 genes were identified to be mutated in MPN. We here report on our single centre experience in applying these markers in a daily diagnostic work flow comprizing a total cohort of 18,547 cases with suspected MPN that were investigated between 8/2005 und 8/2010 with individual patient specific combinations of these markers as soon as published. Thus, the most frequently tested marker was JAK2V617F that was applied in 17,027 pts. In 6,622/17,027 (38.9%) a definite diagnosis of MPN could be made or confirmed on the basis of the detection of JAK2V617F mutation. More detailed, the percentage of JAK2V617F positive cases varied depending on the suspected diagnoses: In patients with cytomorphologically confirmed or suspected ET 581/891 (65.2%) were JAK2V617F positive, in PMF: 168/290 (57.9%), in PV: 800/942 (84.9%), in MPN-U: 51/212 (24.0%), in CMML: 38/383 (9.9%), in “MPN” not further specified by the referring physician: 4741/11249 (42.1%), and in those with unexplained leukocytosis/thrombocytosis/splenomegaly or suspected hematologic malignancy: 139/2492 (5.6%). Many of the before mentioned cases were suspected MPN and therefore analyzed for both JAK2V617F and BCR-ABL1. Thus, in 9,924 pts BCR-ABL1 and JAK2V617F testing were performed in parallel. As such, in 541/9,924 (5.5%) analyses BCR-ABL1 positive CML was identified and 3,558 cases were JAK2V617F mutated (35.9%). Only 8 pts were BCR-ABL1/JAK2V617F double positive (0.08%), thus this is a very rare event. In cases with JAK2V617F negative PV in a second step JAK2exon12 mutation was analyzed and 27/147 (18.3%) were tested positive. JAK2V617F negative ET or PMF were analyzed in a second step for MPLW515 mutations. In ET 24/258 (9.3%) and in PMF 14/164 (8.5%) cases were tested positive. JAK2exon12 or MPLW515 were never concomitantly detected with JAK2V617 in our cohort (parallel assessments: n=3,769). PCR for detection of FIP1L1-PDGFRA was performed in 1,086 cases with suspected HES/CEL or unclear eosinophilia but only 26 (2.4%) were tested positive and a CEL could be diagnosed. However, in 36/130 (27.7%) FIP1L1-PDGFRA negative cases a KITD816V mutation was detected and thus a diagnosis of mastocytosis could be established. In addition, confirmation of mastocytosis was achieved in further 326/731 (44.6%) pts with suspected mastocytosis, three of these pts had a JAK2V617F mutation in addition. Further analyses were recently done on selected well characterized cohorts of MPN: CBL mutations were analyzed in 623 cases and tested positive in 54 (8.7%): 26/199 CMML (13.0%), 1/25 PMF, 27/293 MPN-U (9.2%), but never were detected in ET (n=61) or PV (n=45). TET2 sequencing detected mutations in 56/191 (29.3%) of pts analyzed: ET: 6/28 (21.4%), PMF: 4/12 (33.3%), PV: 10/31 (32.3%), CMML: 17/22 (77.3%) cases, MPN-U: 17/86: (19.8%), HES: 1/9 cases, Mastocytosis: 1/3 cases. Thus, TET2 mutations are widely spread in different entities and were frequently associated with other mutations: JAK2V617F: n=16, JAK2exon12: n=1, MPLW515: n=2, CBL: n=5, FIP1L1-PDGFRA: n=1, KITD816V: n=1, and EZH2: n=2. Finally, EZH2 sequence analysis detected mutations in 4/68 (5.9%) cases (1/16 PV, 2/11 PMF, 1/17 MPN-U, 0/20 ET, 0/4 CEL). In conclusion, these data show that the analysis of molecular mutations greatly improved the diagnostic work up of MPN in the last 5 years. The detection of some mutations (JAK2exon12, MPLW515, CBL) are useful to further subclassify MPNs. Others (JAK2V617F, TET2, EZH2) are widely distributed and are helpful for classification and also to discriminate MPN from reactive disorders. The individual power of each marker for prognostication in MPN remains to be defined in future studies. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Eder:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann: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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In Myelodysplastic/Myeloproliferative Neoplasms Aberrant Antigen Expression As Assessed by Multiparameter Flow Cytometry Is Related to Molecular Genetic Mutations

Blood ◽

10.1182/blood.v118.21.5152.5152 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5152-5152

Author(s):

Wolfgang Kern ◽

Susanne Schnittger ◽

Tamara Alpermann ◽

Claudia Haferlach ◽

Torsten Haferlach

Keyword(s):

Myeloproliferative Neoplasms ◽

Molecular Genetic ◽

Antigen Expression ◽

Multiparameter Flow Cytometry ◽

Employment Equity ◽

Aberrant Expression ◽

Equity Ownership ◽

Diagnostic Work ◽

Work Up ◽

Diagnostic Work Up

Abstract Abstract 5152 Background: Immunophenotyping by multiparameter flow cytometry (MFC) is increasingly used in the diagnostic work-up of patients with cytopenias and suspected myelodysplastic syndromes (MDS). Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) comprise a group of diseases with some features of MDS and is separately classified in the current WHO system. While the immunophenotype of chronic myelomonocytic leukemia has been described in detail, data is scarce on the use of MFC in myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPNu) as well as on refractory anemia with ring sideroblasts and thrombocytosis (RARS-T), which is a provisional entity in the current WHO classification. Aim: To assess patients with MDS/MPNu and RARS-T for MDS-related aberrant immunophenotypes in the context of a comprehensive diagnostic work-up including cytomorphology, cytogenetics, and molecular genetics. Patients and Methods: A total of 91 patients were analyzed in parallel by cytomorphology, cytogenetics, and MFC applying an antibody panel designed to diagnose MDS. MFC was used to detect expression of mature antigens in myeloid progenitors; abnormal CD13-CD16- and CD11b-CD16-expression patterns, aberrant expression of myeloid markers and reduced side scatter signal in granulocytes; reduced expression of myelomonocytic markers in monocytes; aberrant expression of CD71 in erythroid cells; as well as expression of lymphoid markers in all myeloid cell lines. In 77/91 patients molecular genetic markers were investigated. The median age of the patients was 75.1 years (range, 35.3–87.4). The male/female ratio was 60/31. Six patients had RARS-T and 85 had MDS/MPNu. Results: In 54/91 (59.3%) patients MFC identified an MDS-immunophenotype. This was true in 4/6 (66.7%) RARS-T and in 50/85 (58.8%) MDS/MPNu (n.s.). Cases with MDS-immunophenotype displayed aberrancies significantly more frequently than those without as follows: in myeloid progenitor cells (number of aberrantly expressed antigens, mean±SD: 0.5±0.6 vs. 0.2±0.4, p=0.002), granulocytes (2.7±1.3 vs. 1.2±1.1, p<0.001), and monocytes (1.7±1.2 vs. 0.5±0.7, p<0.001). Accordingly, there was a significant difference in the total number of aberrantly expressed antigens (4.9±2.4 vs. 2.0±1.4, p<0.001). The presence of an aberrant karyotype was not related to an MDS-immunophenotype which was observed in 11/18 (61.1%) cases with aberrant karyotype and in 43/73 (58.9%) with normal karyotype (n.s.). Mutations in RUNX1 and TET2 as well as FLT3-ITD were predominantly present in cases with an MDS-immunophenotype (10/33, 30.3%) and occurred less frequently in cases without (1/7, 9.1%, n.s.). In detail, RUNX1 mutations were present in 4/26 (10.3%) vs. 0/2, TET2 mutations were present in 4/6 (66.7%) vs. 1/2 (50%), and FLT3-ITD was present in 3/29 (10.3%) vs. 0/5. Accordingly, in cases with RUNX1 or TET2 mutations or with FLT3-ITD a significantly higher number of aberrantly expressed antigens was observed as compared to cases with none of these mutations (mean±SD, 6.4±2.0 vs. 4.4±2.5, p=0.024). In contrast, JAK2V617F mutations occurred at identical frequencies in patients with and without MDS-immunophenotype (11/38, 28.9% vs. 9/31, 29.0%). Regarding prognosis, the presence of an MDS-immunophenotype had no impact on overall survival. Conclusions: These data demonstrates that MDS-related aberrant antigen expression is present in the majority of patients with RARS-T and MDS/MPNu. While there is no association between the presence of an MDS-immunophenotype and the detection of JAK2 mutations cases with an MDS-immunophenotype tended to more frequently carry mutations in RUNX1 and TET2 as well as FLT3-ITDs. These data therefore suggests that MDS/MPNu may be subdivided based on molecular genetics and on the immunophenotype into cases with MDS-related features and those without. Further analyses are needed to validate these findings and their potential significance in RARS-T. Disclosures: Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Comprehensive Analysis Of U2AF1 Mutations In 843 Patients With Myeloid Neoplasms With Respect To Other Genetic Alterations

Blood ◽

10.1182/blood.v122.21.4973.4973 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4973-4973

Author(s):

Manja Meggendorfer ◽

Christiane Eder ◽

Sabine Jeromin ◽

Claudia Haferlach ◽

Wolfgang Kern ◽

...

Keyword(s):

Next Generation Sequencing ◽

Hot Spots ◽

Melting Curve ◽

Gene Mutations ◽

Next Generation ◽

Employment Equity ◽

Total Cohort ◽

Equity Ownership ◽

Generation Sequencing ◽

Splicing Machinery

Abstract Introduction Genes affecting the splicing machinery have been found to be frequently mutated in MDS patients. U2AF1 codes for one of these splicing components, showing two distinct mutational hot spots at amino acids Ser34 and Gln157. Mutations in U2AF1 induce global abnormalities in RNA splicing, producing intron containing unspliced RNAs. U2AF1 has been shown to be most frequently mutated in MDS cases (7-11%), but was so far investigated only in small subsets of AML and MPN and was found rarely mutated. Aim To determine the frequency of U2AF1 mutations (U2AF1mut) in different myeloid entities and to evaluate the correlation of U2AF1mut with other gene mutations, cytogenetics and clinical features. Patients and Methods The total cohort consisted of 843 patients, whereof 74 were diagnosed as AML, 201 as MDS, 243 as MPN, and 325 as MDS/MPN overlap. 331 patients were female, 512 male. Cytogenetics was available in 830 patients and these were grouped by the following karyotypes: normal karyotype (n=561), +8 (n=39), -7 (n=15), del(20q) (n=95), -Y (n=29), other aberrations (n=59), and complex karyotype (n=32). Based on the previously described association of U2AF1mut with del(20q) there was an intended selection bias to this abnormality. Mutational analyses for U2AF1 were performed by either melting curve analyses or next generation sequencing. In subcohorts we investigated mutations in ASXL1 (n=505), CBL (n=647), CEBPA (n=68), CSF3R (n=213), DNMT3A (n=260), ETV6 (n=129), EZH2 (n=355), FLT3-ITD (n=352), FLT3-TKD (n=239), IDH1/2 (n=367 and 286, respectively), JAK2 (n=681), KITD816 (n=244), KRAS (n=393), MLL-PTD (n=384), MPLW515 (n=612), NPM1 (n=477), NRAS (n=509), RUNX1 (n=516), SETBP1 (n=336), SF3B1 (n=839), SRSF2 (n=784), TET2 (n=428), and TP53 (n=239) by Sanger sequencing, next generation sequencing, gene scan, or melting curve analysis. Results In the total cohort we detected U2AF1 mutations in 55/843 (6.5%) patients, the two mutational hot spots were equally affected with 29 p.Ser34 and 26 p.Gln157 mutations, respectively. Mutation frequencies were 10.9% in MDS, 9.5% in AML, 7.1% in MDS/MPN overlap and 1.2% in MPN. U2AF1mut patients were older (median: 72.6 vs. 71.8 years; p=0.012), the mutation was more frequent in males (42/512 (8.2%) vs. 13/331 (3.9%) in females; p=0.015) and associated with lower hemoglobin levels (median: 9.5 vs. 11.0g/dL; p<0.001), and platelet counts (median: 78x109/L vs. 179x109/L; p=0.002). Regarding cytogenetics we found a high association of U2AF1mut to del(20q): in 18 of 95 cases (18.9%) with del(20q) a U2AF1 mutation was detected compared to 37 U2AF1mut in 735 cases (5.0%) with any other karyotype (p<0.001). This was true for AML (5/16 vs. 2/56; p=0.005), MDS (11/49 vs. 11/150; p=0.007) and MDS/MPN overlap cases (1/8 vs. 21/309; p=0.441). In contrast in MPN none of the 21 del(20q) patients showed a U2AF1 mutation compared to 18/74 in all other entities (p=0.01). Mutations in the two other genes of the splicing machinery, SF3B1 and SRSF2, occurred in 122/839 (14.5%) and 198/784 (25.3%) cases and were mutually exclusive with U2AF1mut. Only one case each showed an U2AF1mut and a SF3B1 (p=0.002) or SRSF2 (p<0.001) mutation. We furthermore analyzed a number of other gene mutations frequently mutated in myeloid entities and their association to U2AF1mut. There was no correlation to mutations in NPM1, FLT3-ITD and FLT3-TKD, MLL-PTD, and CEBPA in AML patients. In MDS patients there was also no correlation to mutations in ASXL1,ETV6, EZH2, TP53, RUNX1, NRAS, and KRAS. This was also true for JAK2, MPL, CBL, and TET2 mutations in MPN. However in MDS/MPN overlap patients U2AF1mut were more frequently found in cases with ASXL1mut (14/115 (12.2%) in ASXL1mut vs. 7/179 (3.9%) in ASXL1wt; p=0.01) and together with KITD816mut (3/10 (30%) in KITD816mut vs. 15/212 (7%) in KITD816wt; p=0.038). Conclusion 1) U2AF1 is most frequently mutated in MDS, followed by AML and MDS/MPN overlap and in contrast rarely mutated in MPN. 2) U2AF1mut highly correlates with del(20q) in MDS, AML and MDS/MPN overlap but not in MPN cases. 3) In MDS/MPN overlap U2AF1mut associates significantly with ASXL1mut and KITD816mut. Disclosures: Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Jeromin: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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Modulation of the Clonal Composition in Relapsed CLL: A Study Based on Targeted Deep-Sequencing of ATM, BIRC3, NOTCH1, POT1, SF3B1, SAMHD1 and TP53

Blood ◽

10.1182/blood.v126.23.1721.1721 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1721-1721

Author(s):

Sabine Jeromin ◽

Wolfgang Kern ◽

Richard Schabath ◽

Tamara Alpermann ◽

Niroshan Nadarajah ◽

...

Keyword(s):

Deep Sequencing ◽

Clonal Evolution ◽

Gene Mutations ◽

Wild Type ◽

Mutation Load ◽

Employment Equity ◽

Time Points ◽

Mutational Status ◽

Equity Ownership ◽

Time Point

Abstract Background: Relapse or refractory disease is a challenging clinical problem in the majority of chronic lymphocytic leukemia (CLL) patients. Treatment influences the clonal composition by selection and eventually induction of additional genetic abnormalities. Aim: To characterize the clonal evolution in relapsed CLL patients by deep-sequencing analysis of mutations in ATM, BIRC3, NOTCH1, POT1, SF3B1, SAMHD1 and TP53. Patients and Methods: Sequential samples of 20 relapsed CLL patients at three time-points were evaluated: A: at diagnosis (n=16) or in untreated state (n=4), B: at first relapse (n=20) and C: at second relapse (n=2). Patients were treated with diverse treatment schemes and had temporarily achieved either complete or partial remission during the course of the disease. The median time from diagnosis to first-line treatment was 13 months (1 - 169 months). All geneswere sequenced by a deep sequencing approach (Illumina, San Diego, CA). IGHV mutational status was determined by direct Sanger sequencing at time-point A. Chromosome banding analysis (CBA) and FISH data on del(17p), del(11q), trisomy 12 (+12), and del(13q) were available in 33/42 and 36/42 samples, respectively. Results: Initially, samples at first relapse were sequenced. Mutations in SF3B1 (6/20, 30%), TP53 (5/20, 25%), ATM (5/20, 25%), NOTCH1 (4/20, 20%), and SAMHD1 (3/20, 15%) were detected at high frequencies. No mutations were detected in BIRC3 and POT1. In total, 75% of cases presented with at least one mutation (Figure 1): 8 (40%) cases had one, 6 (30%) cases had two and one patient had three genes concomitantly mutated (mut). Patients were also analyzed for IGHV mutational status at diagnosis and presented with unmutated status at a frequency of 85% (17/20). Subsequently, samples from cases with mutations were analyzed at time-point A. In 12/15 (80%) cases the mutations at relapse were already detectable at time-point A with a similar load indicating presence of the main clone before and after chemotherapy. However, in 7/15 (47%) patients new gene mutations were acquired either additionally to existing mutations (n=4) or in previously wild-type cases (n=3). In 5/7 (71%) cases mutations were located in TP53. TP53 mut were the only mutations that were not detected in samples before treatment (sensitivity of 3%). Thus, TP53 mutations might have been initiated by chemotherapy or exist in a minor subclone subsequently selected by chemotherapy. Furthermore, only 4 cases had low-level mutations (3-6% mutation load) at diagnosis in either SAMHD1 or SF3B1 that eventually increased in their burden during disease course. Of note, in two patients a multibranching clonal evolution could be identified (#2, #9). For patient #2 three time-points were analyzed. At diagnosis 2 ATM mutations were detected with mutation loads of about 20%, each. In the course of the disease these mutations were lost, whereas SF3B1 mut showed a stable mutation load in all three time-points of about 40%. In contrast, mutation load of SAMHD1 increased over time from 4% to 87%. CBA was performed at diagnosis and detected independent clones with del(11q) and del(13q). Accordingly, del(11q) detected by FISH at diagnosis was lost and the percentage of cells with del(13q) increased from diagnosis to time-point C. Therefore, patient #2 shows different genetic subclones in parallel that were eradicated or selected by chemotherapy. In patient #9 two SF3B1 mutations were initially detected with the same mutation load of 10%. After treatment one mutation was lost, whereas the load of the second mutation increased indicating at least two different subclones with only one of them being sensitive to chemotherapy. This might be due to different additional aberrations. Indeed, CBA identified two clones: one with +12 alone and one in combination with del(13q). FISH revealed unchanged percentage of +12 at time-point B, whereas del(13q) positive cells were diminished. Conclusions: In 75% of relapsed CLL cases mutations in SF3B1, TP53, ATM, NOTCH1, and SAMHD1 are present at high frequencies. 80% of these mutations are already detectable before treatment initiation representing the main clone. Remarkably, TP53 mutations were the only mutations that were not detected before but only after chemotherapy. Figure 1. Distribution of gene mutations in 15 CLL cases with mutations at diagnosis or before treatment (D) and at relapse (R). Red = mutated, grey = wild-type, white = not analyzed. Figure 1. Distribution of gene mutations in 15 CLL cases with mutations at diagnosis or before treatment (D) and at relapse (R). Red = mutated, grey = wild-type, white = not analyzed. Disclosures Jeromin: MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schabath:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Schnittger: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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