RARS-T Patients Harbor SF3B1 Mutations In 90.2% and Can Be Characterized By Mutations In ASXL1 and Other Spliceosome Genes In Most Of The Remaining Cases

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2764-2764
Author(s):  
Sabine Jeromin ◽  
Christiane Eder ◽  
Sandra Weissmann ◽  
Manja Meggendorfer ◽  
Tamara Alpermann ◽  
...  
Keyword(s):  
Who Classification ◽  
Melting Curve ◽  
Gene Mutations ◽  
Curve Analysis ◽  
Lower Percentage ◽  
Refractory Anemia ◽  
Melting Curve Analysis ◽  
Employment Equity ◽  
Ring Sideroblasts ◽  
Equity Ownership

Abstract Introduction Refractory anemia with ring sideroblasts and marked thrombocytosis (RARS-T) is a rare entity with characteristics of both myelodysplastic syndromes (MDS) and myeloproliferative neoplasms and is grouped as a provisional entity in the current WHO classification. RARS-T patients have been shown to be frequently JAK2V617F and less commonly MPLW515 mutated. Recently, SF3B1 mutations (mut) were described to occur at a high frequency of up to 85% and it seems that RARS-T is genetically best characterized by SF3B1 and JAK2V617F mutations. However, a comprehensive mutational landscape analysis is still missing und genetic events in the SF3B1wild-type (wt) cases remain to be clarified. Aim Comprehensively characterize a large cohort of RARS-T patients for gene mutations. Patients and Methods We investigated 92 cases that all strictly met the criteria for RARS-T according to the WHO classification 2008. JAK2V617F and MPLW515 were analyzed by melting curve analysis. Screenings for mutations in SF3B1, SRSF2 and ASXL1 were performed by direct Sanger sequencing. ZRSR2 and TET2 were analyzed by an amplicon next generation deep-sequencing approach (NGS). U2AF1 was either analyzed by melting curve analysis or NGS. The cohort comprised 54 females (58.7%) and 38 males (41.3%). Median platelet count was 659x109/L (range: 454 – 1,500x109/L) and median percentage of ring sideroblasts (RS) was 61% (range: 18 - 97%). Cytogenetic data was available in 86 patients: 71 patients (82.6%) had normal and 15 an aberrant karyotype. Results All patients were analyzed for mutations in SF3B1, JAK2V617F and MPLW515. SF3B1 was the most frequently mutated gene (83/92, 90.2%), followed by JAK2V617F (54/92, 58.7%). MPLW515mut occurred only rarely (2/92, 2.2%) and in both cases were accompanied by SF3B1mut. SF3B1mut cases occurred concomitantly with JAK2V617F (46/83, 55.4%). However, JAK2V617F showed a tendency to be more frequent in patients with SF3B1wt (8/9, 88.9% vs. 46/83, 55.4%, p=0.076). Additionally, a subset of the cases, especially those with SF3B1wt, was analyzed for other genes. Mutations occurred with following frequencies: TET2, 14/61, 23.0%; ASXL1, 11/85, 12.9%; SRSF2, 5/86, 5.8%; U2AF1, 4/88, 4.5%; ZRSR2, 2/83, 2.4%. In 98.9% (91/92) of all patients at least one mutation in the analyzed eight genes could be found. Only one patient carried no gene mutation in any of these genes and had normal karyotype. We further analyzed this case with a pan-myeloid genes NGS panel providing data on 19 additional genes. However, no mutation could be detected. Interestingly, nearly all SF3B1wt cases carried an ASXL1mut (7/9, 77.8% vs. 4/76, 5.3%, p<0.001). Accordingly, mutations in the spliceosome genes SRSF2 (2/78, 2.6% vs. 3/8, 37.5%, p=0.005) and U2AF1 (1/79, 1.3% vs. 3/9, 33.3%, p=0.003) were rare in SF3B1mut cases, but were associated with ASXL1mut (SRSF2mut: 3/11, 27.3% vs. 1/73, 1.4%, p=0.006; U2AF1mut: 3/11, 27.3% vs. 1/74, 1.4%, p=0.006). In contrast, the only two ZRSR2 mutated cases had concomitant SF3B1mut (n.s.). TET2mut showed no association with any of the other gene mutations. Analysis of patients with mutation status of all following genes: SF3B1, JAK2V617F, MPLW515, ASXL1, SRSF2, U2AF1, ZRSR2 (n=82), revealed that only SF3B1mut occurred as a sole alteration (31/82, 37.8%). In detail, SF3B1mut cases rarely showed more than 2 gene mutations, whereas nearly all SF3B1wt cases had 3 different gene mutations (5/75, 6.7% vs. 6/7, 85.7%, p<0.001). These 6 SF3B1wt cases all carried a JAK2V617F and ASXL1mut accompanied by either an SRSF2mut (n=3) or U2AF1mut (n=3). Furthermore, SF3B1mut were associated with higher percentage of RS (mean: 61% vs. 41%, p=0.006), whereas JAK2V617F had higher platelet counts (807 vs. 599 x109/L, p<0.001). ASXL1mut had lower percentage of RS (mean: 42% vs. 61%, p=0.007), so had U2AF1mut (mean: 36% vs. 60%, p=0.028), but not SRSF2mut. Conclusions 1. RARS-T patients are characterized by high occurrence of mutations in SF3B1 (90.2%), in 37.8% detected as sole mutation. 2. Most of the SF3B1wt cases show various gene mutations, harboring a JAK2V617F and ASXL1mut together with a third mutation in either SRSF2 or U2AF1. Disclosures: Jeromin: MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Alpermann: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. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

The Recently Suggested MDS/MPN Subgroup “Refractory Anemia with Ring Sideroblasts Associated with Marked Thrombocytosis (RARS-T)” Demonstrates Unique Clinical, Cytogenetic, and Molecular Aspects: A Comprehensive Analysis of 57 Cases Strictly Defined According to WHO Criteria

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3081-3081
Author(s):  
Ulrike Bacher ◽  
Johanna Flach ◽  
Claudia Haferlach ◽  
Tamara Alpermann ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Who Classification ◽  
Normal Karyotype ◽  
Favorable Outcome ◽  
Refractory Anemia ◽  
Employment Equity ◽  
Who Criteria ◽  
Ring Sideroblasts ◽  
Equity Ownership ◽  
Wbc Count ◽  
Who 2008

Abstract Abstract 3081 Introduction: In the new WHO 2008 classification, “refractory anemia with ring sideroblasts associated with marked thrombocytosis” (RARS-T) represents a provisional entity defined by platelets ≥450 ×109/l (being lowered from 600 ×109/l; WHO 2001), proliferation of large megakaryocytes, bone marrow (BM) blasts <5%, and ring sideroblasts ≥15% of nucleated erythropoiesis. The separation of RARS-T from other myeloproliferative/myelodysplastic neoplasms is still under debate. Patients: To further characterize this subtype and to evaluate whether its separate position in the WHO classification is justified from biologic/genetic aspects, we analyzed 57 patients with a diagnosis of RARS-T (strictly defined according to WHO 2008 criteria) for peripheral blood parameters, BM morphology, cyto-/molecular genetics, and clinical profiles. The study cohort consisted of 34 females and 23 males (median age, 76 years, range, 51–92 yrs; 52 de novo; 5 therapy-associated). At the time of analysis, all pts were at diagnosis or therapy naïve. Patients with a sole del(5q) or >5% of blasts were excluded according to WHO criteria. Methods: All BM samples underwent May Giemsa Gruenwald and iron stainings. Chromosomal banding analysis (and FISH if needed) were performed in 56/57 cases. PCR was done for the following markers: JAK2V617F (investigated: n=47), MPLW515 (n=46), NRAS (n=24), TET2 mutations (TET2mut, n=14), MLL-PTD (n=13), FLT3-ITD (n=12), and CBL (n=16). Result: Median WBC count was 7.9 ×109/l (range, 3.1–60.0 ×109/l), median hemoglobin (Hb) level was 10 g/dl (range, 6–13 g/dl), and median platelet count was 572 ×109/l (range, 454-1, 737 ×109/l). The median ring sideroblast count was 60% (range, 18–92%). Karyotypes (KT) were as follows: normal KT: n=52 (52/56; 93%); +8: n=2; -Y: n=1. The most frequent mutation was the JAK2V617F (18/47; 38%); an MPLW515 mutation was detected in 3/46 (7%). From the 46 pts being analyzed both for the JAK2 as for the MPLmut, 21 (45.6%) were observed with one of both markers; there was no coincidence of the JAK2 and the MPL mutations. Furtheron, 5/14 (36%) had a TET2 mutation. Coincidences of molecular markers were observed in 3 pts who had a JAK2V617F and a TET2mut in parallel (TET2mut: 3/10; 30% in JAK2mut pts; vs. 2/4 in JAK2 wildtype pts; n.s.). No patient had a JAK2V617F and MPLW515 in parallel. There was no mutation of the NRAS, MLL-PTD, FLT3, or CBL genes in pts investigated for these markers. A positive JAK2V617F mutated status correlated significantly with higher platelets (p=0.038; T-test), whereas no significant correlations were observed for the respective medians taken as thresholds for leukocytes (≥7.9 ×109/l vs. <7.9 ×109/l vs.), Hb (≥10.0 g/dl vs. <10.0 g/dl), or ring sideroblast percentages (≥60% vs. <60%). All 3 pts with MPLW515mut had platelets ≥600×109/l. Cytogenetic aberrations were independent from the JAK2mut status (normal karyotype: 17/45 JAK2mut; 38%; vs. aberrant KT: 1/2 JAK2mut; n.s.) and the MPLmut status (normal KT: 3/44 MPLmut; 7%; vs. aberrant KT: 0/2; n.s.). Higher WBC count (≥7.9 ×109/l) was correlated to a higher Hb level (≥10 g/dl) (p=0.47) and to higher platelets (≥600 ×109/l) (p=0.011). The patients with RARS-T had a favorable outcome with 84.6% being alive at 2.5 years. Conclusion: Investigation of 57 patients strictly fulfilling the criteria of the WHO 2008 classification was able to confirm the unique profile of RARS-T in all aspects: patients with the RARS-T had a normal karyotype (>90% of all cases), had no prognostically adverse cytogenetic alterations, and frequently showed mutations of the JAK2 (V617F) or MPL (W515K/L) genes (45.6% in total). The molecular profile was even more homogeneous in RARS-T cases with ≥600 ×109/l platelets (the WHO threshold from 2001) due to significantly higher proportions of JAK2V617F positive cases when compared to cases with platelets between 450 and <600 ×109/l. However, from clinical aspects, patients with RARS-T had a favorable outcome in our study independent of the molecular state or the number of platelets. These data support to include RARS-T as definite subtype in the next edition of the WHO classification. The frequent occurrence of TET2 mutations in our cohort has to be noted for future diagnostic and classification approaches. Therefore, in cases suspicious for RARS-T but without evidence of a JAK2V617F, molecular screening should be performed including analysis for alterations of the TET2 and MPL genes. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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

Blood ◽  
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.

scholarly journals Melting Curve Analysis for Rapid Detection of Topoisomerase Gene Mutations in Haemophilus influenzae

2009 ◽  
Vol 47 (3) ◽  
pp. 781-784 ◽  
Author(s):  
S. Nakamura ◽  
K. Yanagihara ◽  
Y. Morinaga ◽  
K. Izumikawa ◽  
M. Seki ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Rapid Detection ◽  
Melting Curve ◽  
Gene Mutations ◽  
Curve Analysis ◽  
Melting Curve Analysis

scholarly journals Mutations in CALR Are Associated with Del(13q) and Are Mutually Exclusive of Monosomy 7 in BCR-ABL1-Negative MPN

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1870-1870
Author(s):  
Sabine Jeromin ◽  
Claudia Haferlach ◽  
Manja Meggendorfer ◽  
Wolfgang Kern ◽  
Torsten Haferlach ◽  
...  
Keyword(s):  
Chromosomal Aberrations ◽  
Gene Mutations ◽  
Melting Curve Analysis ◽  
Employment Equity ◽  
Monosomy 7 ◽  
Blastic Phase ◽  
Equity Ownership ◽  
Calr Mutations

Abstract Introduction: Mutations (mut) in the Calreticulin gene (CALR) were recently described in BCR-ABL1-negative myeloproliferative neoplasms (MPN). They occur frequently in essential thrombocythemia (ET; 15-30%) and primary myelofibrosis (PMF; 25-35%), but not in cases with polycythemia vera (PV). Other well-known mutations in ET and PMF are JAK2V617F (50-60%) and MPLW515 (5-10%). These three mutations are nearly mutually exclusive of each other. Furthermore, also chromosomal aberrations are frequently detected in PMF (40%), whereas they are rare in ET (3%). However, no association of CALRmut with any cytogenetic aberration has been reported yet. Aims: Investigate CALRmut and JAK2V617F in cytogenetic subgroups of BCR-ABL1-negative MPN. Patients: We studied 220 patients with cytomorphological confirmed BCR-ABL1-negative MPN excluding PV and with following chromosomal aberrations: del(20q) (n=64), trisomy 8 (+8; n=57), +1q (n=30), del(5q) (n=25), del(13q) (n=23), monosomy 7/del(7q) (-7/del(7q); n=21). Of these 220 cases, 25 and 12 patients were in accelerated and blastic phase, respectively. The cohort comprised 85 females (38.6%) and 135 males (61.4%). Median hemoglobin (Hb) level was 12.3 g/dl (range: 6.0 - 17.8 g/dl, n=167), platelet count 277,500x109/L (range: 16,000 –1,877,000x109/L; n=170) and white blood cell (WBC) count 16,000 x109/L (range: 1,600 -305,000 x109/L, n=181). Methods: Chromosome banding analysis was performed using standard G-banding. Screening for CALRmut was done by fragment analysis and subsequent Sanger sequencing of positive cases. JAK2V617F and MPLW515 were analyzed by melting curve analysis. MPLW515 was only analyzed in CALR/JAK2V617F-negative patients. Results: All 220 patients were screened for CALRmut and JAK2V617F. The frequency of CALRmut was 16.8% (37/220) and of JAK2V617F 58.2% (128/220). Mutations in these two genes were mutually exclusive (p<0.001). MPLW515 occurred in 3/55 (5.5%) of CALR/JAK2V617F-negative cases. CALR mutations presented as type 1 (p.Leu367Thrfs*46) in 56.8% (21/37) and as type 2 (p.Lys385Asnfs*47) in 27.0% (10/37) according to the nomenclature of Klampfl et al. (NEJM, 2013). The remaining 6 cases represented different mutation types all resulting in the same C-terminus of the mutated CALR protein. Analysis of gene mutations and cytogenetic aberrations showed that CALRmut associated significantly with del(13q) (with vs. without: 10/23, 43.5% vs. 27/197, 13.7%, p=0.001), whereas they were rare in +8 patients (2/57, 3.5% vs. 35/163, 21.5%, p=0.001; Figure 1). Additionally, no CALRmut was detected in patients with -7/del(7q) (0/21, 0% vs. 37/199, 18.6%, p=0.029). For JAK2V617F an association with del(20q) was detected (44/64, 68.8% vs. 84/156, 53.8%, p=0.050). Exclusion of MPN in accelerated or blastic phase from analyses resulted in the same associations between distinct cytogenetic abnormalities and CALRmut. Only the negative correlation to chromosome 7 aberrations lost its significance, probably due to low case numbers (0/9, 0% vs. 28/174, 16.1%, n.s.). For JAK2V617F the association with del(20q) was still present, even though the statistical significance was lost (37/55, 67.3% vs. 77/132, 58.3%, n.s.). Furthermore, we analyzed the distribution between type 1 and type 2 CALR mutations (n=31) in cytogenetic subgroups. Type 1 mutations were more frequent in cases with del(13q) (9/9, 100.0% vs. 12/22, 54.5%, p=0.030), whereas the frequency of type 2 mutations was higher in del(20q) (6/10, 60.0% vs. 4/21, 19.0%, p=0.040). Analysis of clinical data showed that CALRmut vs. wild-type patients had lower Hb levels (mean: 10.9 vs. 12.1 g/dl, p=0.019) and JAK2V617F cases had lower WBC counts vs. JAK2V617F-negative patients (19,308 vs. 30,786 x109/L, p=0.041). Additionally, Hb levels were higher in JAK2V617F patients compared to cases with CALRmut (12.2 vs. 10.9 g/dl, p=0.017). Conclusions: The highest CALR mutation frequency was observed in del(13q) cases (43.5%) and nearly all of them were type 1 mutations (90.0%). In contrast, CALRmut were rare in the cytogenetic subgroups with +8 and -7/del(7q). The highest JAK2V617F frequency was detected in patients with del(20q) (68.8%). Thus, in PMF and ET specific patterns are detectable based on cytogenetic and molecular data. Figure 1: Distribution of gene mutations in cytogenetic subgroups. The percentage of each mutation is depicted in the columns. Figure 1:. Distribution of gene mutations in cytogenetic subgroups. The percentage of each mutation is depicted in the columns. Disclosures Jeromin: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer: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.

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 ◽  
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.

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

Blood ◽  
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.

Prognosis of MDS Subtypes RARS, RCMD and RCMD-RS Does Not Differ by Cytomorphologic Criteria but Cytogenetics Allows to Delineate a Subgroup with Inferior Clinical Course,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3796-3796
Author(s):  
Ulrike Bacher ◽  
Wolfgang Kern ◽  
Tamara Alpermann ◽  
Susanne Schnittger ◽  
Claudia Haferlach ◽  
...  
Keyword(s):  
Multivariable Analysis ◽  
Risk Groups ◽  
Low Risk ◽  
Refractory Anemia ◽  
Employment Equity ◽  
P Values ◽  
Multilineage Dysplasia ◽  
Ring Sideroblasts ◽  
Equity Ownership ◽  
Wbc Count

Abstract Abstract 3796 Background: In 2008 the WHO classification combined the former categories RCMD (refractory cytopenia with multilineage dysplasia) and RCMD-RS (with ring sideroblasts ≥15%) thus not separating according to ring sideroblasts anymore in MDS with multilineage dysplasia, while the category refractory anemia with ring sideroblasts (RARS) was maintained separately. One aim of this study was to evaluate whether or not a separation with respect to ring sideroblasts is reasonable. Study Design: To investigate the clinical impact and genetic background of these MDS subtypes, we studied outcomes, cytogenetics, and molecular genetics in 1082 de novo MDS pts (153 RARS, 606 RCMD, 323 RCMD with ring sideroblasts ≥15% termed “RCMD-RS“): 703 m/379 f; median age, 73.1 yrs; 21.0–90.4 yrs. Cytogenetic risk groups were defined according to the International Prognostic Scoring System (IPSS; Greenberg et al., 1997). Results: Sex ratio (male preponderance in all subtypes; male/female ratio 1.9 in the whole cohort) did not differ significantly between the 3 MDS subgroups. Mean age (RARS: 71.8; RCMD: 70.1; RCMD-RS: 72.6 yrs) reached significant difference between RARS vs RCMD (p=0.020) and between RCMD vs RCMD-RS (p=0.004). Mean WBC count differed between all subgroups (RARS: 6.1; RCMD: 4.4; RCMD-RS: 5.3×10(9)/l; RARS vs RCMD p<0.001; RARS vs RCMD-RS p=0.039; RCMD vs RCMD-RS p<0.001) whereas mean platelet count (RARS: 232; RCMD: 144; RCMD-RS: 218 × 10(9)/l) and Hb level (RARS: 96; RCMD: 105; RCMD-RS: 98 g/l) differed between RARS vs RCMD and RCMD vs RCMD-RS (all p-values <0.001). IPSS categories were available in 854/1082 pts as follows: 514 pts (60.2%) low-risk, 312 (36.5%) intermediate-1, and only 28 (3.3%) intermediate-2 risk (no pt was assigned to high risk IPSS). Cytogenetics was available in all 1082 pts: The majority (918/1082; 84.4%) had good karyotypes (KTs) according to IPSS mainly due to high rates of normal KTs (821/1082, 75.9%); 112 pts had intermediate (10.4%); and only 52 (4.8%) had poor KTs. In detail, good KTs were equally distributed in RARS: 123/153; 80.4%; in RCMD: 530/606; 87.5%; and in RCMD-RS: 265/323; 82.0%. 3-yr overall survival (OS) rates did not differ significantly between the three MDS subtypes (RARS: 78.1%; RCMD: 86.7%; RCMD-RS: 80.6%). Also when entities with ≥15% ring sideroblasts (RARS + RCMD-RS) were compared to RCMD (<15% ring sideroblasts), 3-yr OS rate was similar (80.0% vs 86.7%; n.s.). Further, multilineage dysplasia per se did not impact on 3-yr OS rate (RCMD + RCMD-RS: 83.8% vs 78.1% in RARS; n.s.). In contrast, 3-year OS rate was better in good KTs compared to intermediate or poor KTs (91.4% vs 60.0% vs 29.3%; p<0.001) in the total cohort. Also in the different MDS subtypes, good karyotypes showed better 3-year OS rate than intermediate/poor karyotypes (p-values for comparison of cytogenetic risk groups: RARS: p<0.001; RCMD: p=0.032; RCMD-RS: p=0.007). In subcohorts genes were analysed and were found to be mutated only with low frequencies: RUNX1 mut: 14/213 (6.6%), NRAS mut: 1/283 (0.4%), MLL -PTD: 4/294 (1.4%), FLT3 -ITD: 0/285. This underlines the low risk profile of the cohort. In univariable analysis, good + intermediate vs poor KTs (p<0.001), aberrant vs normal KT (p<0.001), age (p=0.080), and Hb level (both continuous; p=0.007) had a significant impact on OS, while WBC count, platelets, and percentage of ring sideroblasts (all as continuous variables) were not significant. In multivariable analysis, IPSS cytogenetic risk groups (p=0.005) and Hb (p=0.008) only remained significant. Conclusions: As investigated here in 1082 pts, RARS, RCMD, and RCMD-RS all show high rates of good karyotypes as defined by IPSS and show similar clinical outcomes which clearly supports to skip the RCMD-RS category as done by the WHO in 2008. The karyotype and Hb level were the only independent prognostically relevant parameters in multivariable analysis. However, >80% of patients show a good risk cytogenetic profile making prognostication according to karyotype relevant only in a small subset of patients. To overcome this shortcoming only an increasing panel of new molecular markers in MDS can pave future investigations which presently becomes available by the advanced sequencing techniques. After diagnosed by morphology and cytogenetics especially molecular genetic information may therefore guide treatment in the near future for this low risk subgroup of MDS patients as investigated here. Disclosures: Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann: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.

DNMT3A is a Powerful Follow-up Marker in NPM1 mutated AML

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 122-122 ◽  
Author(s):  
Susanne Schnittger ◽  
Claudia Haferlach ◽  
Tamara Alpermann ◽  
Niroshan Nadarajah ◽  
Manja Meggendorfer ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Melting Curve Analysis ◽  
Employment Equity ◽  
Low Level ◽  
Mutational Status ◽  
Equity Ownership ◽  
The Stability

Abstract Background: NPM1 mutated (mut) FLT3-ITD negative acute myeloid leukemia (AML) is a distinct prognostically favorable subtype of AML. Robust data is available demonstrating that monitoring therapy response using NPM1mut-specific real time PCR is an important tool to early detect relapses and provides important information to guide therapy. Since next generation sequencing techniques have become available further gene mutations were detected that accompany NPM1mut in AML. Of these DNMT3Amut were the most frequent and stable ones (Krönke et al., Blood, 2013). Aim: 1) Analyse the stability of DNMT3Amut in paired diagnostic and relapsed samples. 2) Evaluate whether monitoring of DNMT3Amut provides additional information to monitoring of NPM1mut. Patients and Methods: Samples were selected from a cohort of 359 NPM1mut de novo AML cases with an available DNMT3Amut status.First, toevaluate the stability of DNMT3Amut paired diagnostic and relapse samples of 103 patients were analyzed (44 males, 59 females; median age 60 years, range: 26-82 years). Median time to relapse was 11 months (range: 3-68 months). NPM1mut status was assessed at diagnosis with a LightCycler melting curve analysis assay. Non type A mutations were further characterized by Sanger sequencing. Second, all diagnostic and follow-up samples (n=1,813) were quantified by real time PCR specific for the individual NPM1mut. Analysis for DNMT3Amut was performed using either the 454 technology (454 Life Sciences, Branford, CT) or the MiSeq instrument (Illumina, San Diego, CA). Deep DNMT3A sequencing of remission samples was performed using the 454 technology. Results: Out of 103 paired samples 61 (59.2%) carried a DNMT3Amut at diagnosis. 57/61 (93.4%) patients stably retained the mutation at relapse, in 4 (6.6%) the DNMT3Amut was lost. On the other hand 2 of 42 (4.8%) cases with DNMT3A wildtype at diagnosis gained the mutation at relapse. Thus, DNMT3Amut status was shown to be relatively stable (97/103; 94.1%) and thus qualifies as a promising target for follow-up controls. For comparison of DNMT3Amut and NPM1mut status during follow-up 54 patients that were NPM1/DNMT3A double mutated at diagnosis were selected according to the availability of at least one sample in first remission with an NPM1mut level <0.01%. These samples were reanalyzed by deep sequencing for the respective DNMT3A amplicons that had identified mutations at diagnosis. Two of these 54 cases (3.7%) showed morphologic relapse but NPM1mut was negative at relapse (sensitivity of 10-7). In one of these two cases at diagnosis NPM1mut and TET2mut were observed while at relapse IDH1mut and RUNX1mut were present. However, at both time points the DNMT3Amut was identified. The second case lost NPM1mut and CEBPAmut and retained the DNMT3Amut and TET2mut. Thus in these 2 cases the DNMT3Amut can be regarded as the common ancestor. 1 case retained the NPM1mut at relapse but lost the DNMT3Amut. Of note, in 32/54 (59.3%) cases the DNMT3Amut persisted in the remission samples (NPM1mut low level <0.01% or negative) with high DNMT3Amut loads (median: 20%, range: 2-59%) that was only slightly below the load at diagnosis (median: 45%, range: 38-58%). To analyze the clinical importance of these persisting DNMT3Amut survival analysis was performed. Median overall survival for patients with persisting DNMT3Amut (n=32) was 69 vs 96 months in those who lost also the DNMT3Amut in remission (n=22, p=0.053). Median event free survival was 38 vs. 96 months (p=0.031). Thus the DNMT3A mutational status in remission of NPM1mut AML is a further important parameter for prognostication. The mechanisms underlying this observation are obscure. As NPM1mut disappeared in remission and DNMT3A was retained and with the exception of 2 cases all others (n=18) relapsed with an NPM1mut (even the same type as at diagnosis) two mechanisms may be discussed: 1) Persisting DNMT3Amut cells predispose by a yet unknown mechanisms to the development of a secondary NPM1mut or 2) a residual DNMT3Amut/NPM1mut very low level survivor is able to overgrow the DNMT3Amut sole mutated clone in remission and cause relapse. Conclusions: 1) DNMT3Amut persist in remission of NPM1mut AML in the majority of cases (59.2 %). 2) DNMT3Amut analysis in remission of NPM1mut AML is an important parameter for prognostication. 3) Clones with DNMT3Amut as the sole mutation may have a normal phenotype and thus DNMT3Amut may even be regarded as premalignant mutation. Disclosures Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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