Clinical and Biological Implications of CUX1 Mutations in Myeloid Neoplasms

Abstract Recurrent somatic mutations of CUX1 are described in myeloid neoplasms. CUX1 is located at chromosome 7q22.1; -7/del(7q) involving CUX1 locus are common abnormalities in myelodysplastic syndromes (MDS). Mutations and loss of heterozygosity involving CUX1 have been also described in breast, lung and uterine cancers. Preliminary functional studies, lack of a mutational hotspot and coincidental deletions suggest loss of function/hypomorphic consequences of these molecular defects. CUX1 (p200), contains 4 evolutionarily conserved DNA-binding domains, including 3 CUT repeats and a CUT homeodomain. Functionally, CUX1 regulates many genes involved in DNA replication and chromosome segregation. Cell-based assays have established a role for CUX1 in the control of cell-cycle progression, cell motility, and invasion .The objective of this study is to assess the molecular context and clinical significance of CUX1 mutations and deletions in myeloid neoplasms. We analyzed a subset of 1478 patients [24% lower-risk MDS, 17% higher-risk MDS, 22% primary (p)AML, 14% secondary AML, 14% MDS/myeloproliferative neoplasms (MPN) and 9% MPN] for the presence of CUX1 mutations and deletions. No CUX1 mutations were found in core binding factor AML. We correlated the presence of these lesions with clinical parameters, cytogenetic abnormalities, and molecular features including clonal architecture and associated somatic mutations. Copy number variation and their boundaries were analyzed by Single Nucleotide Polymorphism (SNP) arrays and mutations by multiamplicon deep sequencing utilizing a panel targeting 60 most commonly mutated genes in myeloid neoplasms. In total cohort 4 % of patients had CUX1 mutations and 6% had locus deletions (affecting ch 7q commonly deleted region: 7q22.1) including 90% of del (7q) cases. Expression of CUX1 is significantly lower in AML with -7/del(7q) compared to AML with normal cytogenetics (p<.00001) and also in MDS with -7/del(7q) compared tohealthy controls (p=.004). Additionally, decreased expression of CUX1 was found in 15% of MDS and 8% of AML patients without -7/del(7q) or related mutations. Cases with lower expression had worse OS compared to patients with higher expression (p=.002). In terms of configuration, most mutations were heterozygous, 5% of mutations were hemizygous and 4% were homozygous (due to UPD). Among 75 somatic CUX1mutations; 72% were missense, 20% where frame shift and 8% where non sense. CUX1 mutations were associated with either lower-risk MDS (p=.0001) and pAML (p=.04) while deletions involving the CUX1 locus were significantly related to higher-risk MDS (p=.05). Heterozygous CUX1 mutations were more commonly associated with normal cytogenetics (p=.01). Patients with -7/del(7q) frequently represented del(5q) (p=.04) and thrombocytopenia (p=.001). The OS of patients with CUX1 mutations was shorter (p=.04) as was that of patients with CUX1/deletions (p=.02) when compared to wild type. We subsequently studied the molecular background of CUX1 alterations. CUX1 mutations (vs. wild type) were associated with TET2 (31% vs. 14%, p=.006), ASXL1 (29% vs. 9%, p=.0005), BCOR (28% vs. 8%, p=.0004), and cohesion mutations (26%, vs. 5%, p=.0005), while NPM1 mutations showed the reverse relationship (1% vs. 7%, p=.03). RAS and CUX1 mutations were mutually exclusive (0% vs. 6%, p=.03). When we analyzed clonal hierarchy in the context of CUX1 mutations; dominant CUX1 mutations (24%; mean VAF=49%); were accomplished by ASXL1 (21%) and SRSF2 (14%) mutations which were the most common secondary events in this context. Phenotypically, dominant CUX1 mutations were associated with MDS/MPN (42%) and MDS (33%). 14% of CUX1 mutant cases did not harbor any other alterations and were not associated with a discernable phenotype. Secondary CUX1 lesions (62%; mean VAF=22%) were found in the context of dominant TET2 mutations (16%). The pathomorphologic context of secondary CUX1 mutation did not differ from that of primary lesions. AML seemed to be underrepresented (p=.006) and MPN overrepresented (p=.019) among dominant CUX1 mutant cases. In conclusion, CUX1 lesions including locus deletions with haploinsuffciency, mutations and a fraction of cases with decreased CUX1 expression can be encountered in MDS and related neoplasms, chiefly AML. CUX1 dysfunction is associated with poor survival likely due to its distinct molecular background. Disclosures Makishima: The Yasuda Medical Foundation: Research Funding. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Response to Treatment Among SF3B1 Mutated Myelodysplastic Syndromes (MDS): A Case-Control Study from the MDS Clinical Research Consortium (MDS CRC)

Blood ◽

10.1182/blood.v126.23.1697.1697 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1697-1697 ◽

Cited By ~ 2

Author(s):

Rami S. Komrokji ◽

Amy E. DeZern ◽

Katrina Zell ◽

Najla H. Al Ali ◽

Eric Padron ◽

...

Keyword(s):

Board Of Directors ◽

Lower Risk ◽

Research Funding ◽

Somatic Mutations ◽

Response To Treatment ◽

Wild Type ◽

Advisory Committees ◽

Baseline Characteristics ◽

Matching Criteria

Abstract Introduction Somatic mutations in SF3B1 ,a gene encoding a core component of RNA splicing machinery, have been identified in patients (pts) with myelodysplastic syndrome (MDS). The SF3B1 mutation (MT) is more commonly detected in pts with ring sideroblasts (RS) morphology and is associated with favorable outcome. The pattern of response among SF3B1 mutated MDS pts to available treatment options, including erythropoiesis stimulating agents (ESA), hypomethylating agents (HMA) and lenalidomide is not known. The distinct underlying disease biology among such pts may alter response to treatment. Methods Pts treated at MDS CRC institutions with MT vs wild-type SF3B1 (WT) controls were matched 1:2. Matching criteria were age at diagnosis, year of diagnosis and International Prognostic Scoring System (IPSS) category at diagnosis. IPSS category was split into two groups (Low or Int-1 vs. Int-2 or High). Matching was performed using the R package by calculating a propensity score, which was then used to determine the two most similar WT SF3B1 patients for each SF3B1-mutated pt, without replacement. Additionally, to be included in the population, pts also had to have been treated with one of the following: ESAs, HMA, or lenalidomide. Response to treatment was evaluated by international Working Group criteria (IWG 2006) and classified as response if hematological improvement or better was achieved (HI+). Survival was calculated from date of treatment until date of death or last known follow-up, unless otherwise noted. Results: We identified 48 Pts with MT and 96 matched controls. Table 1 summarizes baseline characteristics comparing MT vs WT SF3B1 cohorts. SF3B1 MT was detected more often in association with RS, as expected. The majority of pts had lower-risk disease by IPSS and revised IPSS (IPSS-R). Pts with MT had higher platelets than controls. The most common concomitant somatic mutations observed were TET2 (30%), DNMT3A (21%), and ASXL1 (7%). Median follow-up time from diagnosis was 35 months (mo). Median overall survival (OS) from diagnosis was significantly longer for patients with SF3B1 MT (108.5 mo (68.8, NA)) than wild-type controls (28.3 mo (22.3, 36.4); p < 0.001). Patients with an SF3B1 MT had a decreased hazard of death (hazard ratio [HR]: 0.49 (95% confidence limits [95% CL]: 0.29, 0.84); p = 0.009) ESA was the first line therapy for 43 pts (88%) with MT and 55 WT Pts (56%). For ESA treated pts, 14 out 40 MT Pts responded (35%) compared to 9/56 among WT Pts (16%), p 0.032. Among those treated with HMA therapy, 5 out 21 (24%) MT pts responded compared to 11/46 (24%) WT Pts (p 0.99). Finally, for Pts treated with lenalidomide 4/16 (25%) and 4/21 (19%) responded among SF3B1 MT and WT Pts respectively, p 0.7. Conclusions SF3B1 somatic mutation in MDS is commonly associated with RS, lower risk disease, and better OS. Pts with SF3B1 mutation had higher response to ESA compared WT SF3B1. No difference in response to HMA or lenalidomide was observed compared to WT patients. Response rates to lenalidomide and HMA were low in both MT patients and controls. Biologically rational therapies are needed that target this molecular disease subset. Table 1. Baseline characteristics SF3B1 MT (n=48) SF3B1 WT (n=96) P value Age median 65 67 0.6 Gender male 29 (60%) 64(67%) 0.5 Race White 44/45 (98%) 83/90 (92%) 0.34 WHO classification RA RARS RCMD RARS-T Del5 q RAEB-I RAEB-II MDS-U MDS/MPN CMML 3 24 8 4 1 3 3 2 0 0 6 9 17 2 6 10 9 3 11 9 IPSS Low Int-1 Int-2 High 29 (60%) 16 (33%) 3 (6%) 0 21 (22%) 69 (72%) 4 (4%) 2 (2%) < 0.001 IPSS-R Very low Low Intermediate High Very High 15 (31%) 26 (54%) 5 (10%) 2 (4%) 0 11 (11%) 37 (39%) 26 (27%) 18 (19%) 4 (4%) <0.001 Lab values (mean) Hgb Platelets ANC myeloblasts 9.7 274 2.63 1 9.6 108 1.92 2 0.46 <0.001 0.04 0.05 Disclosures Komrokji: Novartis: Research Funding, Speakers Bureau; Celgene: Consultancy, Research Funding; Incyte: Consultancy; Pharmacylics: Speakers Bureau. Padron:Novartis: Speakers Bureau; Incyte: Research Funding. List:Celgene Corporation: Honoraria, Research Funding. Steensma:Incyte: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Onconova: Consultancy. Sekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Haploinsufficiency and Deletions of G3BP1 on Chromosome 5q Result in Induction of TP53

Blood ◽

10.1182/blood.v124.21.784.784 ◽

2014 ◽

Vol 124 (21) ◽

pp. 784-784

Author(s):

Naoko Hosono ◽

Mahfouz Reda ◽

Bartlomiej P Przychodzen ◽

Chantana Polprasert ◽

Latifa Zekri ◽

...

Keyword(s):

Growth Inhibition ◽

Board Of Directors ◽

Cell Lines ◽

Lower Risk ◽

Chromosomal Instability ◽

Somatic Mutations ◽

Clonal Evolution ◽

Loss Of Function ◽

Advisory Committees ◽

Low Expression

Abstract Interstitial deletion of the long arm of chromosome 5 (del(5q)) is the most common chromosomal abnormality in MDS. The extent of individual defects vary, which may account for observed clinical diversity. Del(5q) pathogenesis has been related to haploinsufficiency of genes contained in the common deleted regions (CDR), including RPS14, miR-145/146a and SPARC. Driver mutations or pathogenic microdeletions were not identified for these genes, suggesting that multiple genes must function in combination to promote clonal evolution and phenotypic heterogeneity. Hence, we performed a comprehensive analysis of somatic mutations in genes located on chromosome 5 (chr5), both in patients with diploid 5q and in those with del(5q), to clarify the role of germline and somatic mutations in disease pathogenesis. In parallel, expression analysis was performed to correlate haploinsufficiency with the frequency of mutational events, in particular for diploid 5q cases. Applying SNP-array karyotyping to samples from 146 patients with del(5q), the lesion was identified in 5q31.1q33.1. Two retained regions (CRRs) were also observed in q11.1q14.2 (CRR1) and q34qter (CRR2). Lower-risk MDS is frequently affected by CDR, while in higher-risk MDS and secondary AML CRR1/2 are commonly co-involved. Using whole exome sequencing, we identified 11 hemizygous mutations located within the deleted area in del(5q) (N=59), while in cases diploid for 5q (N=330), 243 heterozygous mutations were found. One of the mutations discovered on chr5q afflicted a gene G3BP1 (5q33.1), located within the CDR and present in 2 patients. Both were missense mutations (one heterozygous and the other homo/hemizygous). A mutant case showed good responses to lenalidomide even though diploid 5. In addition, other somatic mutations of driver genes including TET2, CUX1 and EZH2 were concomitantly observed. Whole transcriptome sequencing demonstrated hemizygous loss of G3BP1 resulting in haploinsufficiency. G3BP1 was haploinsufficient in 48% of RAEB as well as low-risk MDS cases with del(5q). Overall, defective G3BP1 is associated with shorter overall survival (P<.001) in AML, consistent with the reports that del(5q) is a worse prognostic factor in myeloid neoplasms with aggressive phenotype. G3BP1 is a nuclear RNA-binding protein and is ubiquitously expressed in bone marrow, CD34+ progenitors and leukemic cell lines. Furthermore, G3BP1 binds to TP53 and its expression leads to the redistribution of TP53 from the nucleus to the cytoplasm. Similar to RPS14, haploinsufficient of G3BP1 resulted in TP53 up-modulation. Moreover, low expression of G3BP1 in diploid 5q cases was indeed associated with higher TP53 expression. Next, we generated haploinsufficient G3BP1 cell lines using shRNA transduction. Decreased expression of G3BP1 led to growth inhibition and impaired colony formation by transduced cells lines and hematopoietic progenitor cells, respectively. Knockdown of G3BP1 in K562 cell line increased TP53 in the nucleus, and when treated with CPT11, DNA-damaged induced G1-arrest was more prominent in knockdown cells. Furthermore, after knockdown of G3BP1 in TP53-null HL60 cells, we observed increased aneuploidy, suggesting that the loss of function of G3BP1 and TP53 may result in chromosomal instability. Most significantly, G3bp1-/+ mice showed lower blood counts and defective, dysplastic hematopoiesis, similar to lower-risk MDS. As previously described, TP53 defects are associated with advanced disease but recently it became apparent that TP53 may be one of the most common somatic lesions found in the context of del(5q). We stipulate that loss of TP53 function might overcome TP53 tumor suppressor effects and induce leukemic evolution in the defective G3BP1 status. In our cohort, TP53 mutations were more frequently present in high-risk phenotype with G3BP1 haploinsufficient expression. In conclusion, novel somatic mutations of G3BP1 suggest that it could be a candidate gene associated with the clonal evolution of del(5q). Loss of function or low expression of G3BP1 has been shown to up-modulate TP53 and result in dysplasia and growth inhibition, hallmarks of early stages of MDS. Additional events constitute loss of function of TP53, resulting in chromosomal instability, which is associated with leukemogenesis. Disclosures Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen Corp: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim Corp: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Molecular Predictors of Response in Patients with Myeloid Neoplasms Treated with Lenalidomide

Blood ◽

10.1182/blood.v124.21.4665.4665 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4665-4665 ◽

Cited By ~ 1

Author(s):

Chantana Polprasert ◽

Tomas Radivoyevitch ◽

Naoko Hosono ◽

Hideki Makishima ◽

Bartlomiej P Przychodzen ◽

...

Keyword(s):

High Risk ◽

Board Of Directors ◽

Odds Ratio ◽

Lower Risk ◽

Somatic Mutations ◽

Low Risk ◽

Similar Response ◽

Advisory Committees ◽

Myeloid Neoplasms ◽

Significant Difference

Abstract While patients with del(5q) MDS treated with Lenalidomide (LEN) have a response rate as high as 70%, the efficacy of this drug is lower in non-del(5q) cases. Aside from the presence of del(5q), up front identification of potentially responsive patients is difficult, particularly as the mechanistic underpinnings of LEN response have not been elucidated. Although expression signatures of responders were described in 2008, they have not yet been translated into an actionable diagnostic test. Analyses of molecular lesions including somatic mutations and chromosomal defects may predict response to LEN in MDS. We performed deep targeted DNA sequencing on 62 genes in 111 cases of myeloid neoplasms (MDS, MDS/MPN, and MPN) treated with Len for at least 3 months for whom fully annotated clinical outcomes were available. Clinical parameters, FISH, SNP array-based karyotyping and metaphase cytogenetics were also included in our analysis. We assessed response according to IWG 2006 criteria and performed analyses for responses at 3 or 6 months of therapy. Of 111 LEN-treated patients, 77% had lower-risk MDS (IPSS Low /Int-1) and 23% higher-risk disease (IPSS Int-2/High/sAML). Regimens included either LEN alone (52%), or in various combinations (29%) LEN+azacytidine, TLK+LEN (1.8%) or high-dose chemotherapy (7+3)+LEN (0.9%). Any hematologic improvement, cytogenetic response, and complete response (BM) were achieved in 58%, 19% and 18% at 3 months and 84%, 44% and 30% at 6 months, respectively. Responders had better survival, with HR=0.55 (0.32, 0.94; P=.03). The mean age did not differ between responders and non-responders. Using IPSS scoring criteria, there was no difference in proportion of patients with lower-risk disease among responders and non-responders (73% vs. 81%). When IPSS-revised (-R) score was applied, there also was no significant difference between responder and non-responders with very low risk (4% vs. 7%), low risk (30% vs. 41%), intermediate risk (22% vs. 15%), high risk (29% vs. 22%), and very high risk (14% vs.12%). Refractory patients showed significantly lower platelet counts compared to responders (117 vs. 215 K/uL; P=.01). Responders tended to have higher reticulocyte counts prior to therapy compared to non-responders (0.5 vs. 0.3 M/uL; P=.07) and had significantly higher MCVs compared to refractory cases (99 vs. 91fL; P<.01). Focusing on karyotype, there was no difference between responders and non-responders in the proportion of patients with +8, -7/del(7q), and those with normal cytogenetics. Del(20q) was marginally associated with treatment failure (6/8 failed; P=.07). In this highly selected cohort, among all del(5q) patients (N=38) 63% responded, compared to 53% in non-del(5q) (N=73), (P=.4). Among lower-risk del(5q) MDS (blast<5%) 75% (12/16) had a response vs. 50% (16/32) in lower-risk non-del(5q) MDS (P=.06). In del(5q) patients both interstitial and long del(5q) (including q11.1-q14.2 and/or q34-qter) showed similar response rates. TP53 mutations were found coinciding with del(5q) and marginally correlated with failure to respond to LEN (P=.07), but not precluded response. Using multiplex amplicon panels of 62 genes commonly mutated in MDS, we confirmed 143 somatic mutations in responders vs. 137 mutations in non-responders. Mutations in RUNX1 correlated with LEN responses (OR=3.62 [0.63-20.87]). Mutations in DDX41 correlated with LEN response (8/8 responded; P=.009, odds ratio OR=Infinity), while mutations in U2AF1 correlated with failure to respond to LEN (1/9 responded; P=.01, OR = .08 [0, 0.66]) as did mutations in IDH1/2 (1/8 responded; P=.02, OR = .1 [0, 0.81]). DDX41 pooled with DDX54 had an odds ratio of OR=5.66 [0.58, 54.85] and DNMT3A, TET2 and IDH2pooled yielded OR=.12 [0.04-0.38]. Bayesian Model Averaging (BMA) was then applied to these and other covariates. BMA fits all submodels of a full model and then forms a weighted average of them wherein the weight of each model is the probability that it is correct relative to all other models in the model space. This yielded the linear predictor S=0.76 - 1.91•DNMT3A.TET2.IDH2 - 2.15•U2AF1 + 0.77•DDX41.54 + 0.06•del5q - 0.61•del20q-0.14•TP53.cmplx + 0.39•RUNX1 + 0.05•KDM6A that awaits validation using an independent set of patient data. In conclusion, in addition to the presence of del(5q), the various molecular lesions including specific somatic mutations may help to better predict responses to LEN. Disclosures Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen Corp: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim Corp: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Distinct Clinical and Biological Implications of Various DNTMT3A Mutations in Myeloid Neoplasms

Blood ◽

10.1182/blood.v128.22.2872.2872 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2872-2872

Author(s):

Suresh Kumar Balasubramanian ◽

Mai Ali ◽

Taha Bat ◽

Bhumika Patel ◽

Bartlomiej P Przychodzen ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Clinical Features ◽

Bone Marrow Failure ◽

Missense Mutations ◽

Negative Consequences ◽

Wild Type ◽

Advisory Committees ◽

Myeloid Neoplasms ◽

Significant Difference

Abstract DNMT3A, a member of the DNA methyltransferases family along with DNMT1 and DNMT3B, is located on chromosome 2p23. Recurrent somatic mutations in DNMT3A are typically heterozygous and found mostly in non-CBF AML, less frequently in MDS and MPN. DNMT3A mutations are reported with other common myeloid mutations including NPM1, FLT3 and IDH1/2. The most canonical DNMT3A mutations are missense alteration in the R882 codon, accounting for >60% of all DNMT3A mutations and they imply dominant negative consequences. Overall, DNMT3A mutations carry a poor prognosis compared to the AML or MDS with wild type (WT) DNMT3A, although data within different subgroups (e.g., incorporating cytogenetic profiles) are conflicting. We hypothesized that molecular consequence of R882 mutations will differ from those of other somatic alterations of DNMT3A and may also result in distinct clinical features and outcomes. To test this theory, we analyzed a cohort of 1174 patients with myeloid neoplasias including 32% AML, 33% MDS, 13% MDS/MPN, 6% MPN and 16% other bone marrow failure disorders. These cases were subjected to multiamplicon targeted deep NGS including all ORFs of DNMT3A and other recurrently mutated genes. After application of various bioanalytic algorithms, confirmatory sequencing and thus stringent exclusion of all artifacts and germline alterations, we identified 140 somatic mutant cases (12% of the cohort), including 89 missense mutations (53 at R882, 19 at R693 and 17 other non-canonical missense alterations) and 51 truncations/frame shifts (all heterozygous). There was an age-related increase in the incidence of DNMT3A mutations, with the peak occurrence at 35-40 yrs. of age. Mutations in DNMT3A were most common in AML (54% in primary (p) AML, 8% in secondary (s) AML) followed by MDS (28%), MDS/MPN (4%), MPN (3%) and other bone marrow failure disorders (3%). Mutation in the R693 codon and truncating mutations were most commonly associated with MDS (p=.013) and sAML (p=.0013) whereas mutation occurring in codon R882 and other non-canonical missense mutations were frequently associated with pAML (p=.00001). For the whole cohort, DNMT3A mutations were most frequently associated with NPM1 (21% vs 8%, p=.014), FLT3 (24% vs. 2%, p=.0001), and IDH1/2 (26% vs. 8%, p=.001), compared to wild type DNMT3A. However, PRC2 complex mutations were less likely to occur in the context of DNMT3A mutations (6% vs. 24%, p=.0006). Canonical R882 mutation was commonly associated with FLT3 (p=.03) mutations, while truncating mutations were not (p=.03). Analyses of clonal hierarchy by ranking of VAF values demonstrated that 53% of DNMT3A mutations were dominant (mean VAF 39%, range 5-93%) (n=74/140). When DNMT3A mutations were dominant, IDH 1/2 (14%), TET2 (9%), ASXL (5%), PRC2 complex (3%) and BCOR (3%) mutations were common secondary events. In subgroup analyses, 55% of mutations in the R693 codon were dominant compared to 45% in R882 and 47% in truncating mutations. TET2 mutations were the most common associated secondary hits in dominant R693 mutations (n=10) compared to truncating (n=24) and R882 mutations (n=23) (40% vs. 8% vs. none, p=.0001). When DNMT3A mutations are secondary (mean VAF 34%, range 1-60%), as in 47% of our cases (n=66/140), then the common first hits were TET2 (10%), U2AF1 (8%) and cohesin complex (RAD21, SMC3, STAG2) mutations (6%). Dominant DNMT3A mutations correlated with MDS/MPN (60%, p=.007), while secondary DNMT3A mutations correlated with sAML (73%, p=.001). DNMT3A mutant myeloid neoplasms showed worse survival (p<.0001) compared to WT cases. Among different subgroups, there was significant difference in OS between R882, R693, truncating and other non-canonical missense mutations (p=.013). The R882 mutations had worse survival compared to other DNMT3A mutations (p=.003). Non-canonical mutations (truncating and other missense) vs. canonical mutations (R882 and R693) had better survival (p<.04). Survival for mutant R882 DNMT3A was worse compared to truncating mutations (p=.005) while there was no difference between R693 and truncating mutations. Among AML cases, R882 mutations vs. other mutations had worse survival (p=.01) while in MDS and MDS/MPN there was no significant difference in OS. DNTMT3A mutations often occur as founder lesion in AML. Our study shows that different types of mutations other than canonical R882 alterations may have a differential impact on OS and distinct clinical features. Disclosures Carraway: Celgene Corporation: Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees; Baxalta: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Clinico-Molecular Features and Treatment Outcomes in Primary Myelofibrosis Phenotypes with Protracted Disease Dynamics

Blood ◽

10.1182/blood-2021-152026 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2571-2571

Author(s):

Luis E. Aguirre ◽

Akriti G Jain ◽

Somedeb Ball ◽

Najla Al Ali ◽

Sara Marie Tinsley-Vance ◽

...

Keyword(s):

Board Of Directors ◽

Active Surveillance ◽

Research Funding ◽

Myeloproliferative Neoplasms ◽

Primary Myelofibrosis ◽

Lower Percentage ◽

Cancer Center ◽

Blast Phase ◽

Advisory Committees ◽

Molecular Features

Abstract Background Primary myelofibrosis (PMF) is the most aggressive subtype among classical BCR-ABL1 negative myeloproliferative neoplasms (MPN). Driven by constitutive activation of the JAK/STAT pathway, its prognosis is defined by cardinal clinical, cytogenetic and molecular features. While most patients require therapy for symptomatic splenomegaly, disease-related symptoms, or cytopenias, asymptomatic lower-risk patients may be appropriately monitored with active surveillance. The aim of this study was to explore disease characteristics and outcomes among pts who remained on prolonged active surveillance compared to those who received early treatment. Methods We identified patients with confirmed MF (inclusive of primary MF and MF occurring after essential thrombocythemia or polycythemia vera) treated at Moffitt Cancer Center between 2003-2021. Patients were stratified into two cohorts: those remaining on active surveillance for ≥ 36 months following diagnosis and those who received within 36 months of diagnosis. Results Between August 2000 and March 2021, we identified 626 patients with a diagnosis of MF. Among these, 48 (8%) did not receive treatment for at least 3 years. Table 1 summarizes the baseline characteristics comparing those pts who remained on active surveillance for ≥ 36 months (LTO-MF) to those who received treatment within 36 months of diagnosis (ET-MF). The LTO cohort presented at a younger age (median age 63 vs 68; p = 0.001), but otherwise demographic variables were balanced between the two cohorts. LTO patients were more likely to have primary MF (85.4% vs 60.9%, p=0.003). LTO patients were less likely to have leukocytosis (28.2% vs 49.9%, p=0.01), and constitutional symptoms (29.8% vs 44.6%, p=0.05), while having a higher reticulocyte percentage (81.4% vs 64.1%, p=0.02). LTO patients also had lower platelet counts (mean: 274k vs 359k, p=0.006), lower percentage of circulating blasts (0.4% vs 1.2%, p<0.001), and lower percentage of marrow myeloblasts (1.3% vs 1.9%, p<0.001) at baseline. Cohorts had comparable rates of anemia, thrombocytopenia, transfusion dependence, LDH levels and splenomegaly at baseline. Interestingly, the cohorts were well-balanced in terms of risk score based across all major prognostic scoring systems: IPSS (p=0.356), DIPSS (p=0.764), DIPSS+ (p=0.148), GIPSS (p=0.125), MIPSS70 (p=0.924) and MIPSS70+ (p=0.407). There was no association between GPSS karyotype risk and need to start treatment earlier (p=0.481) (Table 1). LTO patients were less likely to harbor JAK2 mutations (58.3% vs 72.4%, p=0.04). No significant differences were seen regarding CALR (p=0.144), MPL (p= 0.271), or triple-negative disease (p=0.521) (Table 2). The median OS (mOS) for the entire population was 82.5 months (95%CI 69.4-95.5). LTO patients had longer OS (mOS 170.3 mo vs 63.9 mo; (p<0.001). Rates of transformation to blast phase were comparable (6.2% vs 9.7%;p=0.441), but median time to blast phase transformation was longer for LTO MF: 66.3 mo vs 29 mo, p=0.011). Expectedly, time to first treatment longer for LTO patients (62.1 mo vs 0.9 mo; (p<0.001). No differences were noted between cohorts in terms of response to ruxolitinib, duration of response to ruxolitinib or response to lenalidomide/thalidomide (p = 0.91, 0.90, 0.83, respectively) Conclusion In this single-center study of patients seen at a tertiary referral center, the vast majority of MF patient required treatment within 36 months of diagnosis. Those monitored with active surveillance were younger, had less proliferative signs/symptoms, were less likely to have JAK2 mutations, and more favorable outcomes. Figure 1 Figure 1. Disclosures Tinsley-Vance: Fresenius Kabi: Consultancy; Novartis: Consultancy; Incyte: Consultancy, Speakers Bureau; Abbvie: Honoraria; Jazz: Consultancy, Speakers Bureau; Taiho: Consultancy; Celgene/BMS: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Sallman: Magenta: Consultancy; Takeda: Consultancy; Syndax: Membership on an entity's Board of Directors or advisory committees; Incyte: Speakers Bureau; Agios: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Aprea: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees; Intellia: Membership on an entity's Board of Directors or advisory committees. Sweet: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; AROG: Membership on an entity's Board of Directors or advisory committees. Lancet: AbbVie: Consultancy; BerGenBio: Consultancy; ElevateBio Management: Consultancy; Celgene/BMS: Consultancy; Daiichi Sankyo: Consultancy; Astellas: Consultancy; Agios: Consultancy; Millenium Pharma/Takeda: Consultancy; Jazz: Consultancy. Padron: Incyte: Research Funding; BMS: Research Funding; Taiho: Honoraria; Kura: Research Funding; Blueprint: Honoraria; Stemline: Honoraria. Kuykendall: Novartis: Honoraria, Speakers Bureau; Incyte: Consultancy; BluePrint Medicines: Honoraria, Speakers Bureau; Protagonist: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Prelude: Research Funding; PharmaEssentia: Honoraria; Abbvie: Honoraria; Celgene/BMS: Honoraria, Speakers Bureau; CTI Biopharma: Honoraria. Komrokji: AbbVie: Consultancy; Geron: Consultancy; Acceleron: Consultancy; BMSCelgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Taiho Oncology: Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Germline Variants of RUNX-1 in Myeloid Malignancy

Blood ◽

10.1182/blood.v128.22.3926.3926 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3926-3926

Author(s):

Alek d Nielsen ◽

Sayer Alharbi ◽

Cassandra M. Hirsch ◽

Bartlomiej P Przychodzen ◽

Mikkael A. Sekeres ◽

...

Keyword(s):

Amino Acid ◽

Board Of Directors ◽

Dominant Negative ◽

Missense Mutations ◽

Wild Type ◽

Runt Domain ◽

Advisory Committees ◽

Myeloid Malignancy ◽

Myeloid Neoplasms ◽

Platelet Disorder

Abstract Mutation of the master transcription factor RUNX1 (NM_001754) has a well characterized role in the pathogenesis of myeloid neoplasms. RUNX1 is located at 21q22 and is composed of a c-terminal transactivation domain (TAD) (269-480) and an n-terminal RUNT domain (amino acid 76-209) that binds DNA and mediates heterodimerization with CBF-β. If the RUNT domain is disrupted a RUNX1 mutant will behave in a dominant negative fashion. Mutations leaving the RUNT domain intact result in haploinsufficiency compounded by CBF-β binding site competition with the wild-type allele. Germline (GL) RUNX1 mutations cause the autosomal dominant predisposition syndrome familial platelet disorder with propensity to myeloid malignancy (FPDMM). In FPDMM, RUNX1 mutations, including RUNT domain missense mutations or nonsense mutations throughout the gene, confer mild chronic platelet disorders and a lifetime risk of MDS/AML. As with other GL predisposition syndromes a secondary event is considered necessary for progression, typically an additional mutation in the wild type RUNX1 allele. Onset of MDS/AML occurs at a median age of 33 years but the latency can be variable and ease of detection in younger cases likely distorts summary statistics. Inherited mutations in adults may be difficult to distinguish and have not been systematically explored. We hypothesize that a fraction of patients with otherwise typical RUNX1-positive MDS or related disorders are in fact carriers of a RUNX1 GL mutation. DNA obtained from a cohort of 1451 patients with myeloid neoplasia was analyzed using a multiamplicon deep next-generation sequencing (NGS) panel including all ORFs of RUNX1. A total of 124 patients (8.5%) were found to carry 117 unique RUNX1 mutations. Of these, 59 hits were missense and 58 nonsense; and in 94 (76%) of cases the RUNT domain was disrupted. Eighteen (15.3% RUNX1 positive patients) were previously described in FPDMM. We applied various bio analytic criteria to designate somatic status in 57 patients. The remaining 64 RUNX1 mutations were further investigated, when possible, by paired capillary sequencing of CD3- mononuclear DNA and in-vitro expanded CD3+ T-Cell DNA. We found 5/31 (16%) of these cases carried mutation in both myeloid and lymphoid lineages and were designated GL. This included 2 post-RUNT truncations (p.Y281*, p.S410*) and two RUNT domain missense mutations (p.R80C, p.S141L) all of which are expected to produce a dominant negative phenotype. A 5th GL mutation of uncertain significance in the TAD (p.M310I) was also discovered. Excluding this TAD missense mutation, each of these have been described as somatic factors in myeloid disease. The truncation at amino acid 410 is further downstream than all reported FPDMM variants to date. Chart review revealed an anamnestic presence of thrombocytopenia in 80% of proposed FPD patients. Suggestive family history was found in both truncated cases, with hematologic malignancy presenting in a 1st degree relative before 30 years of age. Median age at diagnosis of MDS/AML in GL cases was 46 years (range: 18-68 years) compared to 65 years in the cases designated somatic (range: 37-87 years). Two cases (p.S410* and p.M310I) had cytogenetic abnormalities on the 21st chromosome (trisomy 21 and t(8;21) respectively) and had no other molecular abnormalities detected by our NGS panel. A mutation in GPR98 of unknown significance was found with p.Y281*. Lastly the p.S141L mutant had cooperating mutations in chromatin modifiers ASXL1 and BCOR, and in RNA splicing gene LUC7L2. This study of an exemplary GL leukemia gene suggests that systematic searches for known and potential GL predisposition genes in otherwise typical adult cohorts may reveal a GL role in the evolution of myeloid neoplasms. Future work to determine genetic predisposition to leukemia in adults is essential. Disclosures Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees. Carraway:Celgene: Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Overall Survival in Patients with JAK2 and ASXL1 Positive Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2019-128309 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5383-5383

Author(s):

Murtadha Al-Khabori ◽

Shoaib Al-Zadjali ◽

Iman Al Noumani ◽

Khalil Al Farsi ◽

Salam Al-Kindi ◽

...

Keyword(s):

Overall Survival ◽

Board Of Directors ◽

Myeloproliferative Neoplasms ◽

Prognostic Significance ◽

Jak2 V617f ◽

World Health ◽

Prognostic Impact ◽

Advisory Committees ◽

Myeloid Neoplasms ◽

The Impact

Objectives: Mutations in additional sex combs-like transcriptional regulator 1 (ASXL1) have been previously described in myeloid neoplasms (21% in non-Myeloproliferative [MPN; Tefferi A, Leukemia, 2010) and have been associated with a more aggressive disease [Rocquain J et al, BMC Cacer, 2010]. They can also be found in patients with JAK2 positive MPN [Abdel-Wahab O et al, Cancer Research, 2010). Disruption of ASXL1 gene leads to MPN phenotype in zebrafish model (Gjini E, Dis Model Mech, 2019). The co-expression and the prognostic significance of ASXL1 in patients with JAK2 positive MPN are not yet fully defined. We therefore planned to define the prognostic impact of ASXL1 mutations on the Overall Survival (OS) of patients with JAK2 positive MPN. Methods: We included patients with JAK2 V617F positive MPN diagnosed according to the World Health Organization 2016 criteria and treated at the three largest hematology centers in Oman. The entire coding region of ASXL1 gene was sequenced using Next Generation Sequencing (NGS; Ion PGM Sequencer; Thermo Fisher Scientific®). The library was constructed and the templates were prepared using the PGM tool and the variants were annotated using the ClinVar database and the prediction from the Scale-Invariant Feature Transform (SIFT) and or Polymorphism Phenotyping (Polyphen) algorithms. The NGS analysis was done on the frozen diagnostic bone marrow samples. The survival probability was estimated using Kaplan-Meier estimator and Cox regression was used to assess the impact of predictors on the OS outcome. An alpha threshold of 0.05 was used. The R program (version 3.1.2) was used for all statistical analyses. Results: A total of 58 patients with JAK2 V617F positive MPN were included. All of these patients were found to have mutated ASXL1 using the NGS (ASXL1 p.Leu815Pro was found in all patients). The median age of this cohort was 62 years (InterQuartile Range [IQR]: 44 - 70) and female to male ratio was 25:33. The median hemoglobin, hematocrit, white blood cell count and platelet count was 14.7 g/dL, 58%, 11.5 x109/L and 518 x109/L respectively. Out of the 58 patients included, 28 had polycythemia vera, 20 had essential thrombocythemia, 8 had myelofibrosis and 2 had MPN-Unclassified. The median time from diagnosis to last follow up or death was 13 months (IQR: 3-39). During this period, 5 patients died. The probability of OS at 3 years was 88%. The median OS was not reached. In the univariable analysis, age was a statistically significant predictor of OS (p = 0.0355) but not gender (p = 0.434) and MPN subtype (p = 0.7). In the multivariable analysis model of the previous three factors, age remained statistically significant (Hazard ratio = 1.13, p = 0.041). Conclusions: ASXL1 is mutated in high proportion of patients with JAK2 positive MPN. Despite the negative impact of ASXL1 in patients with non-MPN myeloid neoplasms, the patients with combined positivity of JAK2 and ASXL1 in this study had a very good OS probability. Age was a predictor of OS in the univariable and multivariable models. We recommend the development and the assessment of ASXL1 inhibitors as therapeutic strategies in patients with MPN. Disclosures Al-Khabori: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; NovoNardisk: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Shire (Takeda): Membership on an entity's Board of Directors or advisory committees; SOBI: Honoraria; AstraZeneca: Honoraria; Abbvie: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Targeted Sequencing Reveals a Relationship Between Mutational Burden and Clinical Phenotype in MPNs

Blood ◽

10.1182/blood.v126.23.4061.4061 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4061-4061

Author(s):

Bartlomiej Getta ◽

Franck Rapaport ◽

Sean Devlin ◽

Chen Zhao ◽

Kristina Marie Knapp ◽

...

Keyword(s):

Board Of Directors ◽

Somatic Mutations ◽

Clinical Phenotype ◽

Disease Phenotype ◽

Advisory Committees ◽

Mutational Burden ◽

Normal Cytogenetics ◽

Serial Samples ◽

Over Time ◽

Stat Pathway

Abstract The Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) Essential Thrombocytosis (ET), Polycythemia Vera (PV), and Myelofibrosis (MF) are characterized by mutations, which drive JAK-STAT pathway activation. Several studies have demonstrated the presence of recurrent somatic mutations outside of the JAK-STAT pathway, which accumulate over time, and may impact disease phenotype and outcome. We sought to determine the influence of somatic mutations on clinical phenotype and prognosis. We sequenced a total of 30 genes recurrently mutated in myeloid malignancies in a cohort of 162 MPN patients (pts) using a next generation sequencing platform. The cohort included 49 pts with ET, 26 PV, 38 Primary Myelofibrosis (MF), 11 Post ET MF, 14 Post PV MF, 12 with leukemic transformations of MPN (LT), 7 with MPN-unclassified (MPN-U) and 5 others. Median age was 59 years and 79 were men. A Total of 288 gene mutations were identified with the most commonly mutated genes being JAK2 (n=121, 74%), TET2 (n=31, 19%), DNMT3A (n=18, 11%), ASXL1 (n=16, 10%), IDH2 (n=10, 6%), RAS (n=12, 7%), TYK2 (n=8, 5%) and TP53 (n=7, 4%). We did not find any mutations in NPM1, CBL, SRSF2 and no FLT3 -ITD. CALR was not assessed in 20 pts and these were excluded from mutation number analysis. Importantly, we identified a relationship between the absolute number of mutations found per pt, disease phenotype, and age (table 1). Pts with/without prior chemotherapy or radiotherapy exposure did not have a difference in mutation number (1.5 vs. 1.9). Cases of ET or PV with fibrotic transformation had more mutations in ASXL1, RAS, EZH2, PHF6 and MPL than pre fibrotic ET or PV suggesting these may be relevant in disease progression and development of fibrosis. Mutations in TET2, RAS and PHF6 were more frequent in cases with LT compared to those with chronic phase MPN. Pts over 40 were more likely to have mutations in TET2 (p=0.026) and JAK2 (p=0.019) and ASXL1 mutations were more common in pts with abnormal cytogenetics than in those with normal cytogenetics (p=0.003). Thrombotic events, which are an important cause of morbidity in MPN patients, negatively correlated with mutations in ASXL1 (p=0.044). Prognosis as measured by DIPPS and DIPSS-Plus scores appeared to correlate with the average number of mutations found in MF patients (table 2). We examined several cases for which serial samples were available, and noted the acquisition of new mutational events despite ongoing therapy. We noted that the most commonly acquired mutations occurred in epigenetic modifying (DNMT3A, TET, IDH, ASXL1) and in growth signaling pathway (RAS, CBL) genes. These occurred despite active therapy and often without an overt change in clinical phenotype. Further details of these serial samples will be presented. We conclude that the number and spectrum of somatic mutations correlate with disease phenotype of MPN. Younger pts have fewer mutations, as do pts with normal cytogenetics. JAK2 and TET2 mutations were more common in older pts. We show that a subset of pts acquire mutations in epigenetic modifiers and in genes involved in growth signaling pathways during disease course, and that mutations in TET2, RAS and PHF6 were enriched at the time of leukemic transformation. Taken together, these results indicate that mutations outside the JAK-STAT pathway influence disease phenotype, and that the acquisition of mutations over time may predict for disease progression. Serial evaluation of mutational burden over time therefore warrants exploration in the clinical setting. Table 1. Average number of mutations appeared to correlate with disease phenotype, age and abnormal cytogenetics. Average Number of Mutations N Mean (SD) P-value Age < 40 years 13 1.4 (0.9) 0.026 Age > 40 years 12 2 (1) No Thrombosis 113 2 (1) 0.712 Thrombosis 28 1.9 (1) Normal Cytogenetics 64 1.8 (0.9) 0.016 Abnormal Cytogenetics 40 2.3 (1.2) ET/PV/PMF 99 1.8 (0.8) 0.029 LT 10 3 (1.5) ET/PV 66 1.6 (0.7) 0.01 Post ET/PV MF 22 2.3 (1.1) ET 44 1.6 (0.7) < 0.001 PV 22 1.5 (0.9) PMF 33 2.2 (0.9) Post ET/PV MF 22 2.3 (1.1) LT 10 3 (1.5) Table 2. Disease prognostic scores in MF appear to correlate with the average number of mutations found per patient. Risk category N Average number mutations DIPSS Low 8 1.5 Intermediate-1 19 2.4 Intermediate-2 9 1.8 High 1 5 DIPSS-Plus Low 6 1.5 Intermediate-1 12 2 Intermediate-2 14 2.2 High 1 5 Figure 1. Comutation map of genomic alterations. Each hash mark on x-axis represents an individual patient. Figure 1. Comutation map of genomic alterations. Each hash mark on x-axis represents an individual patient. Disclosures Levine: CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.

Download Full-text

Impact and Function of Somatic PHF6 Mutations in Myeloid Neoplasms

Blood ◽

10.1182/blood.v124.21.3581.3581 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3581-3581

Author(s):

Brittney Dienes ◽

Bartlomiej P Przychodzen ◽

Michael Clemente ◽

Wenyi Shen ◽

Chantana Polprasert ◽

...

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Board Of Directors ◽

Somatic Mutations ◽

Suppressor Gene ◽

Advisory Committees ◽

Nonsense Mutations ◽

Dysmorphic Features ◽

Normal Bone ◽

Myeloid Neoplasms

Abstract Borjeson−Forssman−Lehmann syndrome (BFLS), a hereditary X-linked disorder characterized by mental retardation, truncal obesity, gynecomastia, hypogonadism and other dysmorphic features, is known to be caused by germline (GL) mutations of plant homeo domain finger protein 6 (PHF6). PHF6 is a highly conserved 41kDa protein showing ubiquitous expression in a variety of tissues, including bone marrow, CD34+ cells and blood leukocytes. Human PHF6 is located on chrXq26.2. Recently, rare somatic nonsense mutations and deletions have been detected in patients with T-ALL and AML and found in some T-ALL cell lines. Patients with BFLS with PHF6 mutations have been reported to develop leukemia, suggesting PHF6 mutations may predispose cancer. Although the actual function and molecular pathogenesis is unknown, PHF6 has been suggested to be a tumor suppressor gene involved in the control of myeloid development. In an index case of a young adult female patient with proliferative CMML with dysmorphic features, we have identified remarkable GL mosaicism for PHF6 mutation (p.K44fs), confirmed by deep sequencing of marrow, CD3+ cells and skin tissue. Subsequently, we screened patients with myeloid neoplasms by targeted multi-amplicon sequencing to determine the prevalence and distribution of PHF6 gene alterations. Sequencing results from 1072 cases were analyzed (728 by targeted deep sequencing and 344 by whole exome sequencing). In total, we identified 21 cases with PHF6 mutations, 13 of which were frameshift or nonsense mutations. Previously, PHF6 have been included in screening panels by Haferlach et al., (Leukemia 2014) and Papaemmanuil et al., (Blood 2013) and somatic mutations were found in 24/944 and 21/738 cases of MDS, respectively. These results along with ours suggest that PHF6 mutations are common driver events. The somatic nature of these defects was confirmed by analysis of non-clonal CD3+ lymphocytes, thus, PHF6 mutations occur at a frequency of 2.0% and are most frequently observed among patients with secondary AML (33%, P=.0021). Gender distribution showed a strong male predominance (76%), likely due to the location of PHF6 on chrX and indicating that retention of a single copy of PHF6 may be protective. SNP-array karyotyping showed that deletions of Xq, involving the PHF6 locus (Xq26), were present in about 1.2% of myeloid neoplasms and affect only female patients. As a family, plant homeo domain (PHD) finger genes are affected by mutations associated with various cancers. JARID1A, PHF23, NSD1 and NSD3 were described to serve as fusion partners with the NUP98 in a subset of AML cases. The most frequent chromosomal aberration observed in conjunction with PHF6 mutations was trisomy-8 (P=.08). The most commonly associated somatic mutations were in RUNX1 (N=7; P=.001), U2AF1 (N=5), ASXL1 (N=5), IDH1 (N=4), and DNMT3A (N=4). Interestingly, 6/7 cases with concomitant PHF6 and RUNX1 mutations showed a poor prognosis AML. Subsequent analysis of clonal architecture using variant allelic frequency calculations and serial samples for these cases suggested that PHF6 may function as a founder driver gene while RUNX1 mutations are acquired as secondary events. Recent studies proposed that PHF6 deficiency leads to impaired cell proliferation, cell cycle arrest at G2/M phase and an increase of DNA damage. To examine DNA damage and quantify double stranded breaks (DSBs) in primary cells from PHF6-mutants, those with wild-type (WT) PHF6 and normal bone marrow we used a flow cytometric anti-γH2AX assay, following induction of DNA damage with Camptothecin. As judged by greater percentages of anti-γH2AX labeled cells, DSBs were more common in mutant cases consistent with more DNA damage present in PHF6 mutant compared to WT MDS and normal bone marrow cells. In conclusion, our results indicate that PHF6 mutations are generally present in more aggressive types of myeloid neoplasms, frequently associated with RUNX1 mutations. Our functional in vitro studies along with recently published reports suggest an association of PHF6 deficiency with genomic instability and thereby provide a basis for a mutator phenotype conveyed by ancestral lesions, consistent with its role as a tumor suppressor gene. Disclosures Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen Corp: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim Corp: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Mutation Profiling of Therapy-Related Myeloid Neoplasms Using Next-Generation Sequencing Demonstrates Distinct Profiles from De Novo Disease

Blood ◽

10.1182/blood.v124.21.4611.4611 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4611-4611

Author(s):

Alan H. Shih ◽

Franck Rapaport ◽

Stephen S. Chung ◽

Emily K Dolezal ◽

Sean Hobson ◽

...

Keyword(s):

Next Generation Sequencing ◽

Board Of Directors ◽

Tp53 Mutation ◽

Somatic Mutations ◽

De Novo ◽

Next Generation ◽

Advisory Committees ◽

Myeloid Neoplasms ◽

Mutation Profile ◽

Generation Sequencing

Abstract Therapy-related Myeloid Neoplasms (tMN) comprise a poor risk subset of myelodysplastic syndromes and acute myelogenous leukemia, are increasing in incidence, and represent a serious complication following treatment for primary malignancies. In our previous study of 11 genes in 38 tMN patient samples, the data suggested that the mutational spectrum of tMN was distinct from de novo myeloid malignancies. To confirm this finding and to refine the tMN mutation profile, we investigated the mutation profile in samples from 88 patients and 28 genes using Sanger and next-generation sequencing approaches. We performed amplification using RainDance microfluidic PCR, followed by HiSeq next-generation sequencing. Mutations were identified using a modified pipeline for SNP calling employing variant detection software programs. Our study cohort included 88 patients, 71 of whom had complete clinical data for analyses. Patients had a history of epithelial and hematologic malignancies (³2 malignancies n=11; breast n=9; colorectal n=5; head and neck n=4; genital-urinary n=6; lung n=1; lymphoma n=25; melanoma n=2; ovarian n=1; sarcoma n=2; other, n=5). Treatment of primary cancers included chemotherapy alone (n=27), radiation alone (n=8), autologous stem cell transplant (n=11), or chemotherapy plus radiation (n=25). The median latency time between primary malignancy treatment and tMN diagnosis was 5.7yrs (range, 0.7 - 30.9 yrs). Median age at tMN diagnosis was 64yrs (range, 26 - 85 yrs). International Prognostic Scoring System (IPSS) risk group for MDS at tMN diagnosis were Low risk (n=8), Int-1 (n=11), Int-2 (n=30), High risk (n=9). We identified somatic mutations in 56 of 88 (64%) patients (83 patients were evaluated by next-generation sequencing and 5 by Sanger sequencing only). Mutations in TP53 were most common and were detected in 27/88 patients (30.7%), followed by mutations in TET2 in 12/88 (13.6%), DNMT3A in 9/88 (10.2%), NRAS in 8/83 (9.6%), KRAS in 5/83 (6.0%), and KIT in 5/83 (6.0%). Gene mutations detected at lower frequencies included those in ASXL1 in 5/88 (5.7%), RUNX1 in 2/83 (2.4%), EZH2 in 1/88 (1.1%), and SF3B1 in 1/88 (1.1%). Of the 58 patients with complete sequencing and FISH data, 4 patients exhibited biallelic somatic TP53 mutations and 3 patients had TP53 mutation combined with del 17p TP53 loss, demonstrating that 7 of 58 evaluable patients (12.1%) experienced biallelic loss of TP53. We also identified biallelic mutations in TET2 and DNMT3A in 2 separate patients. 25 patients had 2 or more concurrent somatic mutations. The highest number of co-occurring mutations in one patient was 5 mutations; 12 patients had 2 somatic mutations. The most common co-occurrence was TP53 and TET2, which was observed in 5 patients. All 5 ASXL1 mutations co-occurred with additional mutations. By analyzing variant allele frequencies (VAFs) in patients with multiple mutations, we observed that some tMN patients harbored multiple clones with distinct VAFs. This observation was also supported by the co-occurrence of typical class I driver mutations in the same patient, (e.g. KRAS 6% and NRAS 21% VAF; NRAS 9% and KIT 34%; NRAS 26% and KIT 9% in individual patients). The allele frequency data also suggested that ASXL1 is likely an early occurring mutation as the VAF was higher than for other co-occurring mutations (mean VAF ASXL1 50%, other co-occurring genes 23.5%, p<.05 t-test). Because of previous reports on the prognostic significance of point mutations in myeloid malignancies (e.g. TP53 in MDS and TET2 in AML), we tested the impact of individual mutations on prognosis. TP53 mutation or loss was associated with worse prognosis in tMN (OS 17.6 vs 25.2 mos, n=72, p<.11 log-rank test) (Fig A). TET2 mutation and KRAS or NRAS mutations did not predict for a difference in prognosis, although analysis was limited by cohort size. TP53 mutation was also associated with del 5q / monosomy 5 (p<.0001, Chi-square test, n=51). Our data reveal that tMNs display distinct mutation profile compared to de novo disease (Fig B). TP53 mutations and loss are the most common abnormalities and predict for adverse outcome. Epigenetic modifier mutations also occur in tMNs and can serve as disease-initiating mutations. Collectively our results demonstrate that characterizing these mutation profiles can enhance our understanding of disease mechanisms in tMNs and may guide the development of future therapies for these difficult to treat disorders. Figure 1 Figure 1. Disclosures Sekeres: Celgene Corp.: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees.

Download Full-text