Somatic PHF6 Mutations In Myeloid Malignancies

Abstract X chromosome genomics is an important area of hematologic malignancy research because of frequent acquired X-abnormalities, location of important genes on this chromosome, and issues surrounding Lyonization (X-inactivation). For example, we previously described somatic mutations of UTX (KDM6A), a H3K27 demethylase located on chromosome Xp11.3, in aggressive myeloid neoplasms. In a companion abstract to the the results here, we also report loss of function somatic mutations of BRCC3 (Xq28), encoding a subunit of the BRCA1-BRCA2-containing complex. In an index young female case of a proliferative CMML with dysmorphic features, we have identified PHF6 mutation mosaisism (p.K44fs), confirmed by deep sequencing of bone marrow, skin and spleen tissues. Subsequently, we screened our MDS exome project data set, involving 206 patients with MDS and related neoplasms, and have detected and confirmed additional somatic PHF6mutations. Plant homeodomain finger protein 6 (PHF6) is a ubiquitously expressed 41 kDa protein that is conserved and vertebrate-specific. Human PHF6 is located on chrXq26.3. Germline mutations of PHF6 cause Borjeson−Forssman−Lehmann syndrome (BFLS), an X-linked mental retardation disorder characterized by truncal obesity, gynaecomastia, hypogonadism and other dysmorphic features. BFLS patients have been reported to develop leukemias. More recently, rare somatic PHF6mutations were detected in patients with T-ALL, but rarely also in AML. To assess the clinical associations and significance of PHF6 mutations, we analyzed NGS results in a total of 809 patients with MDS, MDS/MPN, MPN and AML. In addition we also investigated for the presence of PHF6 mutation in the TCGA AML data sets (n=199). All mutations in our patients were confirmed by Sanger sequencing and targeted deep NGS. In total, we identified 19/809 cases with PHF6 mutations; they were located throughout the gene including 15 SNVs and 4 indels. In addition TCGA pAML NGS results revealed PHF6 mutations in 6/199 cases, including 4 SNVs and 2 indels. Thus, PHF6 mutation occurs at a frequency of 2.5% in myeloid neoplasm and are most frequently observed in pAML (36%) together with sAML (32%) phenotypes. Gender distribution showed male predominance (84%), likely related to PHF6 locus on chrXq26.3. SNP-array karyotyping showed that deletions of Xq, involving PHF6locus (Xq26) were present in about 2% of myeloid neoplasms. Chromosome 7 abnormalities, including del(7q), were the most frequent lesions seen in conjunction with PHF6 mutations. Most commonly coinciding mutations were in RUNX1 (n=8), TET2 (n=4), ASXL1 (n=3) and U2AF1 (n=3) and unbiased statistical analysis confirmed the significant association between PHF6 and RUNX1 mutations (P=.002). Interestingly, all of 8 cases with concomitant RUNX1 and PHF6 mutations were diagnosed as high-risk diseases; 1 RAEB-2 and 7 AMLs. Deep sequencing analysis of 5 cases with coexisting PHF6 and RUNX1 mutations showed that PHF6 mutated clones were always significantly larger than RUNX1 mutated clones. Such a serial clonal acquisition pattern of ancestral PHF6 and secondary RUNX1 mutations was also observed clearly in an illustrative case with evolution from aplastic anemia (AA) to sAML, in which small clone of PHF6 was detected in AA sample and expanded during MDS stage, followed by secondary driver RUNX1 mutations at the stage. These findings suggest that RUNX1 mutations were acquired as a subclone of the main population with primary driver PHF6mutations. In conclusion, our results indicate that PHF6 mutations, as a recurrent genetic abnormality, were frequently mutated in more aggressive types of myeloid malignancies. Newly identified ancestral nature of PHF6 mutations specifically favor being followed by secondary driver RUNX1 mutations during leukemic evolution. Disclosures: Polprasert: MDS foundation: Research Funding. Maciejewski:Aplastic anemia&MDS International Foundation: Research Funding; NIH: Research Funding. Makishima:Scott Hamilton CARES grant: Research Funding; AA & MDS international foundation: Research Funding.

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Whole Exome Sequencing Detecting Kinesin Family Gene Defects In Myeloid Neoplasm

Blood ◽

10.1182/blood.v122.21.2762.2762 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2762-2762

Author(s):

Chantana Polprasert ◽

Hideki Makishima ◽

Bartlomiej P Przychodzen ◽

Naoko Hosono ◽

Wenyi Shen ◽

...

Keyword(s):

Research Funding ◽

Somatic Mutations ◽

Snp Array ◽

Chromosome 17 ◽

Therapeutic Window ◽

Myeloid Malignancies ◽

Myeloid Neoplasm ◽

Myeloid Neoplasms ◽

Whole Exome ◽

Kinesin Family

Abstract Clinical and pathomorphologic diversity in MDS is a reflection of heterogeneity of molecular lesions. Somatic mutations and chromosomal deletions/amplifications affect various pathways in a convergent and divergent fashion, generate phenocopy and can occur in a variety of combinations. Recent technological advances, including high density arrays and the new generation sequencing (NGS) led to the discovery of novel pathway mutations or gene families affected by somatic defects, e.g., cohesin or spliceosomal mutations. We have performed whole exome NGS of paired (tumor/germ line) samples in 222 patients with myeloid neoplasms from the Cleveland Clinic and University of Tokyo. Clinical parameters were studied including age, gender, overall survival (OS), bone marrow blast count, and metaphase cytogenetics. Additionally, we also used in our analysis data sets from 197 AML included in the Cancer Genome Atlas (TCGA). We found 1.4% (6/419) of non-canonical somatic mutations of KIF2Bwhich is a member of kinesin13 family located on the long arm of chromosome 17; 3 cases from our cohort (p.V32M (c.G94A), p.T113M (c.C338T), p.R163C (c.C487T)) and 3 cases from TCGA database (p.T47M (c.C140T), p.T310M (c.C929T), p.H551N (c.C1651A)). By analyzing clonal architecture and intra-tumor heterogeneity in 2 cases (RCMD and RAEB) by targeted deep sequencing, allelic frequencies of KIF2B mutations were more than 45% and larger than for any other concomitant mutations, suggesting that KIF2B mutations might consequently constitute ancestral events followed by subclonal acquisitions of the other mutations. Of note is that 6 non-sense mutations were also reported in lung cancer. Based on SNP-array mapping of chromosomal abnormalities, deletions of 17q involving the KIF2B locus (17q22) was present about 3% (6/215) of myeloid neoplasm. KIF2B defects were frequently detected in higher-risk MDS and AML phenotypes (9%). KIF2B performed an important role in regulation of kinetochore-microtubule attachment. Previous studies showed that the velocity of chromosomes’ movement in KIF2B-deficient cells is reduced 80% comparing to control and fail to perform cytokinesis. In our series, 56% of myeloid neoplasms with KI2B defects had complex cytogenetics and 67% cases of them were also UPD, suggesting that KIF2B defects might lead to inducing abnormal chromosomal movements and segregations. We then, expanded our study to the whole kinesin gene family: 17 somatic mutations and 57 deletions were identified in KIF1A (n=6), KIF23 (n=1), KIF26A (n=1), KIF27 (n=7), KIF1C (n=9), KIF21B (n=2), KIF13A (n=10), KIF14 (n=2), KIF17 (n=15), KIF25 (n=1), KIF3C (n=8), KIF6 (n=2) and CENPE (n=10). All mutations were heterozygous and mutually exclusive. By survival analysis of such mutated cases, a tendency towards worse prognosis was observed (HR; 1.72, 95%CI 0.86-3.37). Analysis of concomitant mutations associated with whole kinesin family mutations or deletions showed that most frequently affected genes are TET2 (n=14), DNMT3A (n=8), IDH1/2 (n=8) and MLL (n=5), all involved in epigenetic regulation. In conclusion, somatic mutations in kinesin family genes are found in myeloid malignancies and might be responsible for another pathogenesis of the disease. KIF2B is most frequently found in myeloid malignancies and associated with aggressive type of MDS. Since knockout mice of multiple kinesin family genes (KIF5A, KIF16B and EG5) were lethal in embryo and all the mutations occur in a heterozygous configuration, it is likely synthetic lethal approach might create therapeutic window between defective malignant cells and healthy controls. Kinesin family of motor proteins may be an emerging novel therapeutic target. In fact some kinesins have been already successfully targeted in solid tumors. Disclosures: Polprasert: MDS foundation: Research Funding. Makishima:AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

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Profiling the Genomic Landscape at the Early Stages of CLL: Low Genomic Complexity and Paucity of Driver Mutations in MBL and Indolent CLL

Blood ◽

10.1182/blood.v128.22.3214.3214 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3214-3214 ◽

Cited By ~ 1

Author(s):

Andreas Agathangelidis ◽

Viktor Ljungström ◽

Lydia Scarfò ◽

Claudia Fazi ◽

Maria Gounari ◽

...

Keyword(s):

Deep Sequencing ◽

Research Funding ◽

Somatic Mutations ◽

Driver Mutations ◽

Whole Genome ◽

Sequencing Analysis ◽

Driver Genes ◽

Hematopoietic Stem ◽

Genomic Complexity ◽

Targeted Deep Sequencing

Abstract Chronic lymphocytic leukemia (CLL) is preceded by monoclonal B cell lymphocytosis (MBL), characterized by the presence of monoclonal CLL-like B cells in the peripheral blood, yet at lower numbers than those required for the diagnosis of CLL. MBL is distinguished into low-count (LC-MBL) and high-count (HC-MBL), based on the number of circulating CLL-like cells. While the former does not virtually progress into a clinically relevant disease, the latter may evolve into CLL at a rate of 1% per year. In CLL, genomic studies have led to the discovery of recurrent gene mutations that drive disease progression. These driver mutations may be detected in HC-MBL and even in multipotent hematopoietic progenitor cells from CLL patients, suggesting that they may be essential for CLL onset. Using whole-genome sequencing (WGS) we profiled LC-MBL and HC-MBL cases but also CLL patients with stable lymphocytosis (range: 39.8-81.8*109 CLL cells/l) for >10 years (hereafter termed indolent CLL). This would refine our understanding of the type of genetic aberrations that may be involved in the initial transformation rather than linked to clinical progression as is the case for most, if not all, CLL driver mutations. To this end, we whole-genome sequenced CD19+CD5+CD20dim cells from 6 LC-MBL, 5 HC-MBL and 5 indolent CLL cases; buccal control DNA and polymorphonuclear (PMN) cells were analysed in all cases. We also performed targeted deep-sequencing on 11 known driver genes (ATM, BIRC3, MYD88, NOTCH1, SF3B1, TP53, EGR2, POT1, NFKBIE, XPO1, FBXW7) in 8 LC-MBL, 13 HC-MBL and 7 indolent CLL cases and paired PMN samples. Overall similar mutation signatures/frequencies were observed for LC/HC-MBL and CLL concerning i) the entire genome; with an average of 2040 somatic mutations observed for LC-MBL, 2558 for HC-MBL and 2400 for CLL (186 for PMN samples), as well as ii) in the exome; with an average of non-synonymous mutations of 8.9 for LC-MBL, 14.6 for HC-MBL, 11.6 for indolent CLL (0.9 for PMN samples). Regarding putative CLL driver genes, WGS analysis revealed only 2 somatic mutations within NOTCH1, and FBXW7 in one HC-MBL case each. After stringent filtering, 106 non-coding variants (NCVs) of potential relevance to CLL were identified in all MBL/CLL samples and 4 NCVs in 2/24 PMN samples. Seventy-two of 110 NCVs (65.5%) caused a potential breaking event in transcription factor binding motifs (TFBM). Of these, 29 concerned cancer-associated genes, including BTG2, BCL6 and BIRC3 (4, 2 and 2 samples, respectively), while 16 concerned genes implicated in pathways critical for CLL e.g. the NF-κB and spliceosome pathways. Shared mutations between MBL/CLL and their paired PMN samples were identified in all cases: 2 mutations were located within exons, whereas an average of 15.8 mutations/case for LC-MBL, 8.2 for HC-MBL and 9 for CLL, respectively, concerned the non-coding part. Finally, 16 sCNAs were identified in 9 MBL/CLL samples; of the Döhner model aberrations, only del(13q) was detected in 7/9 cases bearing sCNAs (2 LC-MBL, 3 HC-MBL, 2 indolent CLL). Targeted deep-sequencing analysis (coverage 3000x) confirmed the 2 variants detected by WGS, i.e. in NOTCH1 (n=1) and FBXW7 (n=1), while 4 subclonal likely damaging variants were detected with a VAF <10% in POT1 (n=2), TP53 (n=1), and SF3B1 (n=1) in 4 HC-MBL samples. In conclusion, LC-MBL and CLL with stable lymphocytosis for >10 years display similar low genomic complexity and absence of exonic driver mutations, assessed both with WGS and deep-sequencing, underscoring their common low propensity to progress. On the other hand, HC-MBL comprising cases that may ultimately evolve into clinically relevant CLL can acquire exonic driver mutations associated with more dismal prognosis, as exemplified by subclonal driver mutations detected by deep-sequenicng. The existence of NCVs in TFBMs targeting pathways critical for CLL prompts further investigation into their actual relevance to the clinical behavior. Shared mutations between CLL and PMN cells indicate that some somatic mutations may occur before CLL onset, likely at the hematopoietic stem-cell level. Their potential oncogenic role likely depends on the cellular context and/or microenvironmental stimuli to which the affected cells are exposed. Disclosures Stamatopoulos: Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses. Ghia:Adaptive: Consultancy; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Honoraria, Research Funding.

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Novel Recurrent Mutations in the Ras-Like GTP-Binding Gene Rit1 in Myeloid Malignancies

Blood ◽

10.1182/blood.v120.21.558.558 ◽

2012 ◽

Vol 120 (21) ◽

pp. 558-558

Author(s):

Inées Góomez-Seguíi ◽

Hideki Makishima ◽

Andres Jerez ◽

Kenichi Yoshida ◽

Bartlomiej P Przychodzen ◽

...

Keyword(s):

Research Funding ◽

Somatic Mutations ◽

Small Gtpases ◽

Refractory Anemia ◽

Myeloid Malignancies ◽

Gtp Binding ◽

Myeloid Neoplasms ◽

Relative Ratio ◽

Recurrent Mutations ◽

Ras Family

Abstract Abstract 558 In addition to chromosomal and epigenetic abnormalities, somatic mutations constitute key pathogenic lesions in myeloid neoplasms. Individual somatic mutations or various combinations may be both valuable prognostic markers and targets for new rational therapies. Among them, RAS family genes are ubiquitous oncogenes associated with various cancers. Recurrent canonical mutations in the nucleotide binding domains in NRAS and KRAS result in constitutively activated proteins. In myeloid neoplasms, RAS mutations convey a poor prognosis and are often found in acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and, rarely, myeloproliferative neoplasms (MPN). We applied whole exome sequencing to paired germline vs. leukemia samples in 65 cases of MDS, 36 MDS/MPN and 32 sAML. We focused our study on the RAS protein superfamily of small GTPases and identified mutations in 3% and 6% of KRAS and NRAS, respectively. Most significantly, we identified somatic recurrent mutations in the F82 residue of Ras-like without CAAX1 (RIT1) gene in 2 patients with chronic myelomonocytic leukemia (CMML) and secondary AML (sAML), respectively. We confirmed the somatic nature of both mutations in sorted CD3+ cells from each patient (pt). RIT1 gene encodes a member of Ras-related GTPases, involved in the p38 MAPK and AKT signaling pathway that mediates cellular survival in response to stress. RIT1 gene amplification has been found in 26% of hepatocellular carcinoma. However, neither amplification nor mutations of this gene have been reported in myeloid malignancies. We thus focused this line of experimentation on this somatic mutation. To establish clinical associations we further studied a cohort of 322 patients with various myeloid malignancies by Sanger sequencing and detected somatic RIT1 mutations in an additional 6 (2%) cases. All mutations were located in exon 5, in the 81 and 82 residues, which encode the switch II domain of this protein, an effector region very close to the GTP-binding site G3, and which is highly conserved among species. Among the 8 mutant cases, 5 (63%) pts had CMML, resulting in a higher frequency of mutations in this subcohort of pts (5 out of 57 CMML, 9%). The other 3 mutations were found in one primary (p)AML (M5b subtype) (1 out of 58 pAML, 2%) and two high-grade MDS, one refractory anemia with excess blasts (RAEB)-2 and one sAML(RAEB-T in the FAB-classification) (2 out of 80, 2.5%). RIT1 mutations were heterozygous in all cases except for one case with trisomy 1 and duplication of the mutant allele. In the cases of WES, we estimated an allelic frequency of ∼35%, consistent with the presence of a heterozygous mutation in ∼70% of sample cells. Because of the large size of the clone and serial samples showing RIT1 mutation since the time of initial diagnosis, it is likely that RIT1 may be of ancestral origin. As RAS-family gene amplifications have been described in cancer, we also studied the presence of amplifications of the RIT1 locus (1q22) by SNP-A. We found 10 cases characterized by a gain involving the RIT1 region (1q21.1-q44): 4 (40%) cases had a diagnosis of CMML, 4 (40%) had myelofibrosis, whereas the remaining patients had MDS (one RAEB-1 and a RA). Quantitative RT-PCR showed RIT1 overexpression in mutants and in patients with 1q amplification (median normalized relative ratio 0,51 and 0,40, respectively) compared to patients with wild type RIT1 and no amplification in 1q (median normalized relative ratio 0,15; P=.039). We theorized that activating RIT1 mutations may constitute a suitable therapeutic target. Because AKT inhibitors can block AKT phosphorylation and therefore reverse the antiapoptotic effect of mutant RIT1, we tested whether AKT inhibitor V (Triciribine) can selectively abrogate the growth of primary cells with RIT1 mutation. In in vitro suspension cultures, a 65% of reduction proliferation was observed with significant effects even at 0.1μM concentrations. In sum, somatic recurrent RIT1 mutations are novel lesions involved in the molecular pathogenesis of myeloid cancers, presumably early in the development of the disease. Moreover, amplifications of RIT1 also lead to overexpression of this Ras-like GTP-ase. Specifically, these abnormalities appear to be more frequent in patients with CMML, but also can be found in other types of MDS. Disclosures: Makishima: Scott Hamilton CARES Initiative: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

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Molecular Defects In BRCC3 Complex, a Novel Pathogenic Pathway In MDS

Blood ◽

10.1182/blood.v122.21.264.264 ◽

2013 ◽

Vol 122 (21) ◽

pp. 264-264 ◽

Cited By ~ 1

Author(s):

Dayong Huang ◽

Hideki Makishima ◽

Yang Du ◽

Naoko Hosono ◽

Wenyi Shen ◽

...

Keyword(s):

Deep Sequencing ◽

Research Funding ◽

Chromosomal Abnormalities ◽

Snp Array ◽

K562 Cells ◽

Strand Break ◽

Clinical Analysis ◽

Single Strand ◽

Myeloid Neoplasms

Abstract In addition to the known cytogenetic heterogeneity of MDS, systematic application of new generation sequencing technologies (NGS) and SNP-arrays have further unraveled the complexity of MDS, revealing previously unknown somatic mutational patterns and chromosomal abnormalities. While many of the mutational events are secondary and acquired during disease progression, some may be ancestral in nature. Discovery of novel somatic defects may contribute to the understanding of molecular pathogenesis of MDS and lead to the introduction of new prognostic biomarkers or new therapeutic targets. When we performed analysis of whole exome NGS in patients with MDS and other myeloid neoplasms, including 205 from our own MDS cohort as well as 201 primary AML cases from TCGA, we noted that the BRCC3 complex gene was recurrently mutated in 9 patients (2%). These somatic mutations were confirmed by both Sanger and targeted deep sequencing. UIMC1, FAM175A, BABAM1 and FAM175B gene mutations were each found in separate single patients. The most commonly affected gene was BRCC3, found in 5 patients (1%): 1 with primary AML, 2 with CMML and 2 with MDS. There were 3 canonical mutations at exon 4 (p.R81X) and 2 in exon 1 (p.Q7X). SNP-array analysis (N=682) showed deletion of the BRCC3 complex gene in 45 patients (7%). Deletion involving the UIMC1 locus (5q35.2) and the BRCC3 locus (Xq28) were found in 31 (4.5%) and 8 (1%) patients, respectively. Evaluation of deep sequencing results demonstrates that the variant allelic frequencies of BRCC3 mutations were more than 48% in the early MDS stage, suggesting that BRCC3 mutations are initial events. The BRCC3 complex is located in the nucleus and participates in DNA double-strand break (DSB) repair. BRCC3 is a member of the JAMM/MPN+ family of zinc metalloproteases and specifically cleaves Lys-63 linked polyubiquitin chains. BRCC3 is a component of two complexes, the BRCA1-A complex and the BRISC complex. The BRCA1-A complex consists of UIMC1, FAM175A, BABAM1, BRE, BARD1, BRCC3 and BRCA1. DSB defects may be deleterious to genomic stability and instigate tumorigenesis. Clinical analysis revealed that mutations and deletions of BRCC3 complex genes were more common in MDS than in pAML (P<.01). When 684 patients genotyped for these defects were analyzed, the presence of BRCC3 complex defects was associated with shorter survival (HR=2.44 95%CI; 1.73-3.34; P<.001). The presence of BRCC3 complex gene defects affected survival in all subgroups of MDS, MDS/MPN, sAML, and pAML when analyzed separately. Overall, analysis of the additional mutational spectrum of patients with BRCC3 complex defects showed that the most frequently mutated genes were KRAS/NRAS, TET2 and U2AF1, raising the possibility of synergistic leukemogenic effects of multiple mutations. However, multivariate analysis identifies a BRCC3 complex gene defect as an independent adverse prognostic factor (HR=2.3 95%CI; 1.63-3.14; P<.001). For functional studies, we first generated a BRCC3 shRNA lentivirus vector and used it in in vitro immortalization assays utilizing a serial replating principle. BRCC3 knockdown resulted in decreased colony formation and lacked any immortalization properties. To further elucidate the functional consequences of BRCC3 lesions in the pathogenesis of MDS we performed silencing studies targeting BRCC3 in K562 cells and normal human CD34+ cells. While depletion of BRCC3 alone was not lethal, it led to enhanced etoposide-induced apoptosis. Consistent with these results, overall cell viability was substantially lower in shRNA-BRCC3–treated cells following etoposide when compared with control cells (46% vs. 55%). RPA2 expression, a single-strand DNA-binding protein used as a marker, was higher in BRCC3 knockdown cells than control cells and was further enhanced by etoposide treatment, thus indicating an increased end resection of single strand breaks. We concluded that defects in BRCC3 decrease non-homologous end joining and increased homologous recombination. In sum, our study demonstrates for the first time detection of BRCC3 complex defects, leading to impairment of DSBs repair in patients with myeloid neoplasms. Furthermore, BRCC3 defects are associated with an aggressive phenotype and shorter survival. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Polprasert:MDS foundation: Research Funding. Maciejewski:NIH: Research Funding; AA/MDS foundation: Research Funding.

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

Blood ◽

10.1182/blood.v126.23.2853.2853 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2853-2853 ◽

Cited By ~ 1

Author(s):

Eiju Negoro ◽

Chantana Polprasert ◽

Tomas Radivoyevitch ◽

Vera Adema ◽

Naoko Hosono ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Somatic Mutations ◽

Snp Array ◽

Low Risk ◽

Clinical Parameters ◽

Complex Karyotype ◽

Advisory Committees ◽

Myeloid Neoplasms ◽

Response Expression

Abstract Up to 70% of patients with del(5q) MDS may respond to Lenalidomide (LEN). However, the success rates in non-del(5q) cases, while substantial, are much lower (ranging from 20-40% depending on selection criteria). Aside from the presence of del(5q), up front identification of potentially responsive patients is difficult, particularly as the mechanistic underpinnings of LEN response are still under investigation. Initial attempts to prospectively predict LEN sensitivity resulted in a description of response expression signatures, but they have not been robust enough to serve as an actionable diagnostic test. In the outset of this study, we stipulated that apart of clinical selection (low risk MDS, transfusion-dependence, normal/low risk cytogenetics, etc.), analyses of molecular lesions including somatic mutations and chromosomal defects may help to predict LEN responsiveness. To that end, we performed deep targeted NGS (using multiamplicon panel of the top most commonly mutated genes in MDS). In total we analyzed 143 cases of myeloid neoplasms (MDS, MDS/MPN, or MPN) treated with LEN (median duration 6 months) for whom annotated clinical outcomes were available (83 responders vs. 60 refractory cases). Clinical parameters including IPSS-R, cytogenetics (FISH, SNP-array or metaphase cytogenetics) were used to characterize patients, whose responses were assessed by 2006 IWG criteria. Initially, in a combined analysis, we included both del(5q) (N =37) and non-del(5q) patients (N =106). Very low/low, intermediate, high/very high IPSS-R scores were found in 47%, 23%, 34% cases, respectively. Of 143 LEN-treated patients, regimens included LEN (80%), or LEN+5-Aza (20%). Any hematologic improvement (HI), partial response (PI), and complete response (CR) were achieved in 44%, 14% and 42%, respectively. Responses were associated with a better survival (median survival time 6.2 yrs. vs. 3.7 yrs. in refractory cases; P =.003). Non-responders showed significantly lower platelet levels compared to responders (median 169 vs. 89 K/uL; P =.007) but intricate analysis of clinical parameters (age, other blood counts, blasts and IPSS-R score) failed to identify other factors that would help to select potential responders. As expected, when sub-analysis of patients with del(5q) was performed, combined response was achieved in 78% (OR 13.14 [4.34-39.75]; PR 16%, CR 35%) of patients, respectively, while in non-del(5q) the responses were as predicted lower at 51% (P =.004). Of note is that both del(5q) involving and excluding commonly retained regions (CRR; q11.1-q14.2 and/or q34-qter) also was associated with sensitivity (CRR affected; OR=9.9 [1.4-102], vs. CRR not affected; OR=6.3 [1.3-37.6]). When we analyzed impact of karyotype on LEN sensitivity, -7/del(7q), -20/del(20q), complex karyotype and normal cytogenetics did not correlate with response, but in addition to del(5q); the presence of +8 (7/10 responded; OR 12.25 [1.33-113.06]) was significantly associated with responsiveness. Using targeted deep NGS, we confirmed 168 somatic mutations in responders vs. 142 mutations in non-responders (OR .85; .67-1.07). The number of mutational events per patient did not correlate with responses (P =.38). Among genes sequenced mutations in DDX41 (100% vs. 0%; OR infinity [6.7-infinity]) and RUNX1 mutation+deletion (75% vs. 25%; OR=8.1 [1.1-84.6]) were overrepresented in responders vs. refractory cases while in U2AF1 mutationswere more common among non-responders (20% vs. 80%; OR=.075 [.004-.76]). When reverse analysis was performed DDX41 mutations correlated with LEN response (10% mutant cases among responders vs. 0% in refractory; P =.009), while mutations in U2AF1 correlated with LEN failure (2.4% vs. 13.3% of mutant cases in responders and refractory, respectively; P =.02). The presence of all combined or any of the other spliceosomal mutations (SRSF2, SF3B1, ZRSR2, LUC7L2, and PRPF8) did not influence the results of the therapy. All TP53 mutations were found with complex karyotype with del(5q) and 5/7 (71%) TP53 mutant cases were treatment failures (OR .19 [.01-2.41]). In conclusion, in addition to the presence of del(5q), low platelet count and the presence of various molecular lesions (+8 and RUNX1, DDX41 mutations or wild type status of U2AF1) may help to predict responses to LEN. Disclosures 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. Santini:celgene, Janssen, Novartis, Onconova: Honoraria, Research Funding. List:Celgene Corporation: Honoraria, Research Funding.

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Somatic Mutations in Schinzel-Giedion Syndrome Gene SETBP1 Determine Progression in Myeloid Malignancies

Blood ◽

10.1182/blood.v120.21.2.2 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2-2 ◽

Cited By ~ 1

Author(s):

Hideki Makishima ◽

Kenichi Yoshida ◽

Nhu Nguyen ◽

Masashi Sanada ◽

Yusuke Okuno ◽

...

Keyword(s):

Research Funding ◽

Somatic Mutations ◽

Bone Marrow Cells ◽

Germ Line ◽

Cancer Susceptibility ◽

Gain Of Function ◽

Myeloid Malignancies ◽

Runx1 Expression ◽

Myeloid Progenitors

Abstract Abstract 2 MDS and other chronic myeloid malignancies such as MDS/MPN are characterized by a frequent progression to secondary AML (sAML), a likely multistep process of acquisition of genetic abnormalities. Genes involved in congenital genetic cancer susceptibility syndromes are often targets of somatic mutations in various tumors. For instance, germ-line mutations of SETBP1 are associated with Schinzel-Giedion syndrome (SGS), which is characterized by skeletal malformations, mental retardation and frequent neuroepithelial tumors. While SETBP1 overexpression in myeloid malignancies links to poor prognosis, somatic mutations of SETBP1 were not previously identified in leukemias. When we performed whole exome sequencing of 20 cases with myeloid malignancies, in addition to detecting previously described lesions, such as TET2, CBL and ASXL1, we identified a somatic SETBP1 mutation (D868N) in 2 cases with RAEB. Analysis of DNA from CD3+ cells from these patients confirmed its somatic nature. Sanger sequencing was applied to all coding exons in an additional 48 cases, leading to detection of 2 additional somatic mutations (G870S and I871T) in 2 patients with CMML and sAML, respectively. These findings prompted us to further expand our screening cohort: targeted SETBP1 sequencing was performed in a total of 734 patients (283 with MDS, 106 with sAML, 167 with MDS/MPN, 138 MPN and 146 with primary AML): 52 mutations were detected in 52 patients (7.1%); D868N, G870S and I871T alterations were more frequently observed (N=27, N=16 and N=5, respectively), while D868Y, S869N, D880E and D880N were less prevalent. These mutations, of which 92% (48 out of 52) were identical to those in the SGS germ line, were detected in 15% with CMML (24/156), 15% with sAML (16/106) and 7% CML blast phase (2/28). Clinically, mutant cases were associated with higher age (p=.014), deletion of chromosome 7q (p=.0005) and shorter median survival (28 vs. 13 months, p<.0001). As shown in the analysis of 11 paired samples of progressing MDS patients, all SETBP1 mutations were acquired during leukemic evolution. In addition to mutations, SETBP1 overexpression can be found in 12% and 26% of cases of MDS and sAML, respectively, a finding linking higher activity of SETBP1 to leukemic progression. To directly test whether SETBP1 mutations represent gain-of-function, we performed retroviral transduction of murine Setbp1 engineered with two of the somatic mutations, D868N and I871T, and evaluated the ability of the mutants to immortalize normal murine myeloid progenitors. With a low viral titer of 1 x105 cfu, both Setbp1 mutants caused efficient immortalization of myeloid progenitors, similar to overexpressed WT Setbp1. In addition, cells immortalized with mutant Setbp1 proliferated faster than cells with WT Setbp1. These data suggest that mutations of SETBP1 in our study represent gain-of-function in leukemias. The in vitro immortalization effect of overexpressed WT Setbp1 was associated with and dependent on Hoxa9 and Hoxa10 overexpression. We performed quantitative RT-PCR and western blot experiments to evaluate expression of these genes in our mutant cases. Relative HOXA9 and HOXA10 mRNA expression values were higher in all mutant cases (N=7) than median of those in WT cases (N=4). Also, both HOXA9 and HOXA10 proteins were detected in all cases with SETBP1 mutations, suggesting that HOXA9 and HOXA10 induction is consistently associated with SETBP1 mutations similar to observations in forced expression of WT Setbp1. Moreover, in agreement with findings in primary cells showing that SETBP1 mutations or high SETBP1 expression share a common genetic association with RUNX1 mutations, Runx1 expression was reduced after in vitro immortalization of normal bone marrow cells by forced Setbp1 overexpression and two Runx1 promoter sequences were amplified after ChIP performed with antibody specific for exogenous Setbp1 protein. Moreover, Setbp1 shRNA knockdown resulted in enhanced Runx1 transcription consistent with the negative regulation of this gene by Setbp1. These results indicate that SETBP1 is associated with decreased activity of RUNX1 due to hypomorphic mutations or by direct down-modulation WT RUNX1 expression bypassing the need for mutations. In sum, somatic recurrent SETBP1 mutations are lead to gain of function and are associated with molecular pathogenesis of myeloid leukemic transformation of various primary myeloid subentities. Disclosures: Makishima: Scott Hamilton CARES Initiative: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

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Spectrum Of Genetic Alterations In Acquired Aplastic Anemia

Blood ◽

10.1182/blood.v122.21.2464.2464 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2464-2464

Author(s):

Tetsuichi Yoshizato ◽

Bogdan Dumitriu ◽

Kohei Hosokawa ◽

Hideki Makishima ◽

Kenichi Yoshida ◽

...

Keyword(s):

Aplastic Anemia ◽

Exome Sequencing ◽

Research Funding ◽

Somatic Mutations ◽

Older Age ◽

Myeloid Malignancies ◽

Japanese Cohort ◽

Immune Mediated ◽

The Mean ◽

Allelic Burden

Abstract Acquired aplastic anemia (AA) is a prototype of idiopathic bone marrow failure, which is caused by immune-mediated destruction of hematopoietic progenitors. However, its natural course could be more complicated than expected for a simple immune-mediated disorder, as in the development of apparently acquired (somatic) clonal disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML), during its course. Although these evidences suggest a pathogeneic link between these disorders, the clonal architecture in AA has not been fully explored. In order to genetically define the origin of clonal hematopoiesis in patients with acquired AA, we sought gene mutations, by targeted deep sequencing of peripheral blood DNA from 192 Japanese patients with AA for mutations, using a panel of 51 genes including common mutational targets in myeloid malignancies using a SureSelect custom kit. An extended cohort of 293 American AA patients was further analyzed for targeted sequencing of granulocyte-derived DNA; for these cases, multiple sequential specimens with germline controls and complete clinical follow-up data were available. Exome sequencing was also performed for selected cases. In the Japanese cohort, about 40% were severe or very severe diseases with an excellent response to immunosuppressive therapies (IST). In total, 43 somatic mutations were detected in 18% of the cases with the mean allelic burden of 18%. Mutations were most frequent in DNMT3A (3.6%), followed by ZRSR2 (3.1%), ASXL1 (2.6%), BCOR (2.0%) and more biased to nonsense (25.6%), frameshift (14.0%), splice site changes (7.0%) and non-frameshift indel (11.6%), indicating driver roles of these mutations in many cases. Mutations were associated with older age (p=0.014) and a better response to IST (p=0.040). We next examined an extended cohort of 293 AA cases from the United States, for which samples were collected at 6 months after treatment in the patients of severe or very severe disease. Except for 12 cases, CD3(+) cells were available and used to confirm the somatic origin of mutations by comparison with CD3 cells representing germline sequence. All patients had received IST, with overall response about 65%. As of the date of submission, data analysis was completed for 86 of the 293 cases, in whom we confirmed somatic mutations detected in BM samples from 45 cases (53%) 6 months after IST, with the mean number of mutations and the mean allelic burden were 1.08 and 15.3%, respectively. Similar to the finding in the Japanese cohort, BCOR (13.8%), DNMT3A (11.5%), and ASXL1 (10.3%) were most frequently mutated. PIGA (6.9%) and CSMD1 (4.6%) were also mutated in 6.9% and 4.6%, respectively. Again, mutations were associated with older age. Although there was no significant difference in response to IST (p=0.133) between mutation (+) and (-) cases, responders showed significantly higher numbers of mutations compared with non-responders (p=0.033). Evolution to MDS/AML occurred in 12 out of the 45 cases with mutations, while 13 out of the 62 cases without mutations developed MDS/AML. Therefore, candidate genes associated with some but not most evolution events. We further performed whole exome sequencing in 6 cases, for whom sequential samples were available: in 5 of 6 cases, somatic mutations were detected and the mean number of mutations was 9. There was evidence over time of clonal selection with or without progression to MDS or AML. Small clones of cells containing RUNX1 and U2AF1-mutated clones present in the initial specimen showed expansion in size with progression to MDS (0.003 to 0.46 and 0.013 to 0.097, respectively). In conclusion, mutations in common target genes in myeloid malignancies can drive clonal evolution during the course of AA. However, overall there was no correlation between the presence of mutations and clinical evolution to MDS/AML, as many patients with evidence of clones containing mutations remained stable. Clonal expansion and the appearance and disappearance of clones occurred in some cases without clinical changes. The marrow failure environment may favor selection of mutant clones. In addition, other genetic/epigenetic alterations, including chromosomal instability induced by telomere shortening (accompanying abstract by Dumitriu and Feng) provide a mechanism of oncogenesis. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:Aplastic anemia&MDS International Foundation: Research Funding; NIH: Research Funding.

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Impact of Eltrombopag on Expansion of Clones with Somatic Mutations in Refractory Aplastic Anemia

Blood ◽

10.1182/blood.v126.23.300.300 ◽

2015 ◽

Vol 126 (23) ◽

pp. 300-300 ◽

Cited By ~ 1

Author(s):

Bhumika J. Patel ◽

Bartlomiej Przychodzen ◽

Michael J. Clemente ◽

Cassandra M. Hirsch ◽

Tomas Radivoyevitch ◽

...

Keyword(s):

Aplastic Anemia ◽

Board Of Directors ◽

Deep Sequencing ◽

Somatic Mutations ◽

Clonal Evolution ◽

Clonal Expansion ◽

Quantitative Detection ◽

Sequencing Analysis ◽

Advisory Committees ◽

Before And After

Abstract Despite documented success of immunosuppressive therapy (IST) in the treatment of aplastic anemia (AA), a significant minority of patients remain refractory, most responses are incomplete, and allogeneic stem cell transplantation is not available for older patients or those with significant comorbidities. Until introduction of the cMpl agonist eltrombopag, anabolic steroids were the most commonly used salvage drugs. At least theoretically, engaging growth factor receptors with eltrombopag has the potential to promote the evolution or expansion of mutant clones and thereby increase the rate of progression to secondary MDS, a feared complication of AA occurring in 10-20% of patients. Recently we and others reported detection of clonogenic somatic mutations typical of MDS in patients with AA and PNH. Subsequent study demonstrated that mutations characteristic of sMDS can be found in some patients at presentation of AA and may constitute risk for subsequent progression to MDS. As the risk of MDS evolution was a prominent concern when filgrastim was more widely used in management of AA, now similar questions have been raised regarding use of eltrombopag, be it as salvage therapy or to complement IST. Recently, one of our primary refractory patients receiving eltrombopag progressed to AML. This clinical observation led to investigation of the impact of eltrombopag on evolution and clonal expansion using deep sequencing of a cohort of patients with AA. DNA from bone marrow cells was sequenced before and after initiation of eltrombopag to evaluate clonal expansion or evolution using a targeted multi-amplicon deep sequencing panel of the top 60 most commonly mutated genes in MDS. Among 208 AA patients treated at Cleveland Clinic, we identified 13 patients (median age 68 yrs.) who were treated with eltrombopag for IST-refractory AA; median duration of treatment was 85 wks. The overall response rate, defined as sustained improvement in blood counts and transfusion independence after 12 weeks of therapy, was 46% (6/13), while 38% (5/13) of patients showed stable disease with intermittent transfusions (one of whom underwent HSCT). Among the two non-responders, one patient developed a PNH clone and another progressed to AML (see below). Expansion of PNH granulocytes after eltrombopag treatment was observed in 2 patients. Two patients had chromosomal abnormalities at initial diagnosis, one with t (10; 18) in 2 metaphases, and one with an extranumeral Y chromosome. Use of next generation sequencing (NGS) allows for the quantitative detection of clonal events. We hypothesized that serial analysis by NGS before and after eltrombopag therapy may provide clues as to potential effects of this drug on clonal evolution. Sequencing analysis before eltrombopag treatment revealed the presence of a sole clonal mutational event in 3/13 cases, including CEBPA, EZH2, and BCOR. In the patient with a CEBPA mutation, the mutation persisted during treatment with minimal clonal expansion evidenced by a change in VAF from 53% to 65%. In the second patient, NGS results revealed the initial presence of an EZH2 mutation. A post eltrombopag sample clearly identified acquisition of additional clonal events in genes highly associated with advanced disease and clonal evolution (RUNX1 and U2AF1), as well as slight expansion of a persistent EZH2 clone from 2 to 8%. The third patient harbored a BCOR mutation which expanded markedly, increasing from 8% to 21%, and was accompanied by a hematological response. Sequencing results after eltrombopag treatment revealed the acquisition of new somatic mutations in 5/13 (38%) cases: 2 new CEBPA mutations, 1 new BCOR mutation, and, as discussed, one case with an initial EZH2 mutation in which RUNX1 and U2AF1 mutations were later discovered. In the 5th patient, evolution to AML was observed and accompanied by a large DNMT3A and U2AF1 clone that was absent on initial evaluation. In conclusion, we did observe occasional expansion of clones with potentially leukemogenic mutations during treatment with eltrombopag. At our institution a case control study of patients with refractory aplastic anemia without treatment with eltrombopag is ongoing; ideally a prospective trial would be needed to confirm results. Our results suggest that the initial detection of certain somatic mutations (CBL, SETBP1 and RUNX1) associated with post-AA MDS may contraindicate use of eltrombopag in AA. Disclosures Sekeres: Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees.

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Recurrent Somatic Genomic Alterations in Follicular NHL (FL) Revealed By Exome and Custom-Capture Next Generation Sequencing

Blood ◽

10.1182/blood.v126.23.574.574 ◽

2015 ◽

Vol 126 (23) ◽

pp. 574-574 ◽

Cited By ~ 1

Author(s):

Todd A Fehniger ◽

Kilannin Krysiak ◽

Brian S White ◽

Matthew Matlock ◽

Chris Miller ◽

...

Keyword(s):

B Cell ◽

Clinical Outcomes ◽

Research Funding ◽

Somatic Mutations ◽

Prognostic Score ◽

Notch Pathway ◽

Negative Regulator ◽

Discovery Cohort ◽

Data Set ◽

Recurrent Mutations

Abstract Background: Follicular lymphoma (FL) is the most common indolent NHL (iNHL), exhibits a variable clinical course, and remains largely incurable. The pathogenesis of FL is complex and involves over expression of Bcl2 via t(14;18) translocation, as well as copy number alterations, recurrent somatic mutations, and changes in the tumor microenvironment. In line with recent publications, we hypothesized that recurrent somatic genomic mutations in FL will be present and may impact FL development, progression, transformation, and clinical outcomes. Methods: To address this, we performed exome sequencing (NimbleGen SeqCap EZ V2.0) of tumor and normal frozen tissue pairs from 24 patients in a discovery cohort with untreated FL (12), relapsed FL (6), or transformed FL/iNHL (6). We developed a custom capture assay (NimbleGen) that targets 7.05 MB corresponding to the coding, 5' and 3' UTR regions of 1717 genes. The custom capture genes included somatic mutations identified in our exome discovery cohort (898 genes) or somatic mutations previously published to be recurrently mutated in B cell NHL (819 genes). Instrument data from the discovery cohort exome and re-sequenced custom capture were combined and analyzed using the McDonnell Genome Institute (MGI) somatic caller pipeline (5 SNV callers, 3 indel callers), filtered (minimum 20X coverage, minimum 2.5% VAF, maximum 10% normal VAF) and manually reviewed. Additionally, the 1717 custom capture strategy was used to sequence an extension cohort consisting of FFPE tumor samples from 80 patients with FL, achieving >20x coverage for >75% of the targeted region. All discovery and extension samples have clinical annotations that include FLIPI prognostic score, treatment, and clinical outcomes. Results: Combined analysis of exome and custom capture data for the discovery cohort yielded a robust data set with good sequence coverage of >78% of the targeted regions with at least 20x depth in all samples and a mean depth of 89x. Based upon somatic mutations identified and manually reviewed using this approach, we conservatively estimate 0.98 mutations per MB in FL. 23 genes were recurrently mutated in 3 or more cases, and an additional 75 genes recurrently mutated in 2 cases in the discovery cohort. Consistent with recent publications (Li H et. al., Blood, 2014; Green MR, PNAS, 2015; Yildiz M et al, Blood, 2015) we confirmed a number of genes that were highly recurrently mutated in FL [TNFRSF14 (50%), Bcl2 (25%), IRF8 (13%), TP53 (13%)] including chromatin modifying genes consisting of histone methyl transferases [KMT2D/MLL2 (58%), EZH2 (13%)], histone acetyltransferases [CREBBP (42%), EP300 (17%)], histone linkers [HIST1H1C (13%), HIST1H1E (8%), HIST1H2BO (8%), HIST1H3G (8%), HIST2H2AC (8%); collectively 42%]. We also confirmed (ATP6V1B2, 13%) and found unreported (ATP6AP2, 8%; ATP6V0A1, 4%; ATP6V1F, 4%) mutations in vacuolar ATPase proton pump genes and P5 or Ca++ ATPase genes (ATP13A2, 4%; ATP13A4, 4%, ATP2B4, 4%;). We confirmed (CD79B, 13%; BCL10, 8%) and found unreported (CD22, 13%) mutations in components of the B cell receptor signaling pathway. The previously unreported recurrent mutations in CD22 were consistent with loss-of function (2 missense, 1 nonsense, 1 frame shift deletion). As a negative regulator of BCR signaling, mutation of CD22 may represent a strategy of to enhance BCR signals in malignant germinal center B cells. We also identified members of the SWI/SNF complex mutated in 33% of this FL cohort: ARID1B (8%), BCL11A (4%), SMARCB1 (4%) in addition to previously reported members BCL7A (12%), SMARCA4 (8%), ARID1A (4%). Somatic mutations were also identified in the Notch pathway: DTX1 (29%), Notch2 (4%), Notch3 (4%), Notch4 (4%). We identified several genes that have not been reported as highly recurrent in FL CXCR4 (42%, mutation calls primarily in RNA), DMD (13%), DNAH9 (13%), FLG (13%), GON4L (13%), PCDH7 (13%), RLTPR (13%), SCN7A (13%), ST6GAL1 (13%). Conclusions: FL genomes harbor a large number of recurrent mutations, consistent with a role in the development and progression of this malignancy. Analysis of the extension cohort and association of recurrently mutated genes and pathways with clinical outcomes is ongoing and will be presented. Disclosures Bartlett: Gilead: Consultancy, Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Millennium: Research Funding; Colgene: Research Funding; Medimmune: Research Funding; Kite: Research Funding; Insight: Research Funding; Seattle Genetics: Consultancy, Research Funding; MERC: Research Funding; Dynavax: Research Funding; Idera: Research Funding; Portola: Research Funding; Bristol Meyers Squibb: Research Funding; Infinity: Research Funding; LAM Theapeutics: Research Funding.

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Genomic Landscape of RUNX1-Familial Platelet Disorder with Myeloid Malignancies Reveals Rising Clonal Hematopoiesis

Blood ◽

10.1182/blood-2021-151781 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1090-1090

Author(s):

Kai Yu ◽

Matthew Merguerian ◽

Natalie Deuitch ◽

Erica Bresciani ◽

Joie Davis ◽

...

Keyword(s):

General Population ◽

Somatic Mutations ◽

Fusion Gene ◽

Snp Array ◽

Natural History Study ◽

Myeloid Malignancies ◽

Clonal Hematopoiesis ◽

Runx1 Gene ◽

Platelet Disorder ◽

Familial Platelet Disorder

Abstract Familial platelet disorder with associated myeloid malignancies (FPDMM) is a rare autosomal dominant disease caused by germline RUNX1 mutations. FPDMM patients have defective megakaryocytic development, low platelet counts, prolonged bleeding times, and a life-long risk (20-50%) of developing hematological malignancies. FPDMM is a rare genetic disease in need of comprehensive clinical and genomic studies. In early 2019 we launched a longitudinal natural history study of patients with FPDMM at the NIH Clinical Center and by May 2021 we have enrolled 98 patients and 100 family controls from 55 unrelated families. Genomic data have been generated from 56 patients in 24 families, including whole exome sequencing (WES), RNA-seq, and single-nucleotide polymorphism (SNP) array. We have identified 21 different germline RUNX1 variants among these 24 families, which include lost-of-function mutations throughout the RUNX1 gene, but pathogenic/likely pathogenic missense mutations are mostly clustered in the runt-homology domain (RHD). As an important form of RUNX1 germline mutations, five splice site variants located between exon 4-5 and exon 5-6 were identified in 6 families, which led to the productions of novel transcript forms that are predicted to generate truncated RUNX1 proteins. Large deletions affecting the RUNX1 gene are also common, ranging from 50 Kb to 1.5Mb, which were detected in 8 of the 55 enrolled families. Besides RUNX1, copy number variation (CNV) analysis from both SNP array and WES showed limited CNV events in non-malignant FPDMM patients. In addition, fusion gene analysis did not detect any in-frame fusion gene in these patients, indicating a relatively stable chromosome status in FPDMM patients. Somatic mutation landscape shows that the overall mutation burden in non-malignant FPDMM patients is lower than AML or other cancer types. However, in 13 of the 44 non-malignant patients (30%), somatic mutations were detected in at least one of the reported clonal hematopoiesis of indeterminate potential (CHIP) genes, significantly higher than the general population (4.3%). Moreover, 85% of our patients who carried CHIP mutations are under 65 years of age; in the general population, only 10% of people above 65 years of age and 1% of people under 50 were reported to carry CHIP mutations. Among mutated genes related to clonal hematopoiesis, BCOR is the most frequently mutated gene (5/44) in our FPDMM cohort, which is not a common CHIP gene among the general population. Mutations in known CHIP genes including SF3B1, TET2, and DNMT3A were also found in more than one patient. In addition, sequencing of 5 patients who already developed myeloid malignancies detected somatic mutations in BCOR, TET2, NRAS, KRAS, CTCF, KMT2D, PHF6, and SUZ12. Besides reported CHIP genes or leukemia driver genes, 3 unrelated patients carried somatic mutations in the NFE2 gene, which is essential for regulating erythroid and megakaryocytic maturation and differentiation. Two of the NFE2 mutations are nonsense mutations, and the other is a missense mutation in the important functional domain. NFE2 somatic mutations may play important roles in developing malignancy because 2 of the 3 patients already developed myeloid malignancies. For multiple patients in our cohort, we have sequenced their DNA on multiple timepoints. We have observed patients with expanding clones carrying FKBP8, BCOR or FOXP1 mutations. We have also observed a patient with relatively stable clone(s) with somatic BCOR, DNMT3A, and RUNX1T1, who have been sampled over more than four years. We will follow these somatic mutations through sequencing longitudinally and correlate the findings with clinical observations to see if the dynamic changes of CHIP clones harboring the mutations give rise to MDS or leukemia. In summary, the genomic analysis of our new natural history study demonstrated diverse types of germline RUNX1 mutations and high frequency of somatic mutations related to clonal hematopoiesis in FPDMM patients. These findings indicate that monitoring the dynamic changes of these CHIP mutations prospectively will benefit patients' clinical management and help us understand possible mechanisms for the progression from FPDMM to myeloid malignancies. Disclosures No relevant conflicts of interest to declare.

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