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Rampersaud, Evadnie, David S. Ziegler, Ilaria Iacobucci, Debbie Payne-Turner, Michelle L. Churchman, Kasmintan A. Schrader, Vijai Joseph, et al. "Germline deletion of ETV6 in familial acute lymphoblastic leukemia." Blood Advances 3, no. 7 (April 2, 2019): 1039–46. http://dx.doi.org/10.1182/bloodadvances.2018030635.
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Abstract Recent studies have identified germline mutations in TP53, PAX5, ETV6, and IKZF1 in kindreds with familial acute lymphoblastic leukemia (ALL), but the genetic basis of ALL in many kindreds is unknown despite mutational analysis of the exome. Here, we report a germline deletion of ETV6 identified by linkage and structural variant analysis of whole-genome sequencing data segregating in a kindred with thrombocytopenia, B-progenitor acute lymphoblastic leukemia, and diffuse large B-cell lymphoma. The 75-nt deletion removed the ETV6 exon 7 splice acceptor, resulting in exon skipping and protein truncation. The ETV6 deletion was also identified by optimal structural variant analysis of exome sequencing data. These findings identify a new mechanism of germline predisposition in ALL and implicate ETV6 germline variation in predisposition to lymphoma. Importantly, these data highlight the importance of germline structural variant analysis in the search for germline variants predisposing to familial leukemia.
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Moriyama, Takaya, Monika Metzger, Gang Wu, Rina Nishii, Maoxiang Qian, Meenakshi Devidas, Wenjian Yang, et al. "Germline Genetic Variation in ETV6 and Predisposition to Childhood Acute Lymphoblastic Leukemia." Blood 126, no. 23 (December 3, 2015): 695. http://dx.doi.org/10.1182/blood.v126.23.695.695.
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Abstract Acute lymphoblastic leukemia (ALL) is the most common cancer in children, and the etiology of this aggressive cancer is not fully understood. Common germline polymorphisms in lymphoid development genes and tumor suppressor genes have been associated with ALL susceptibility, although most have modest effects. Only a small fraction of ALL cases are thought to be related to congenital genetic disorders and consequently hereditary predisposition is rarely considered in clinical practice. However, a growing number of rare germline genetic mutations have been discovered in familial ALL (e.g., PAX5, TP53), raising the possibility that the proportion of ALL attributable to inherited predisposition may be higher than currently proposed. In particular, germline ETV6 variations were recently reported in families with hereditary thrombocytopenia and dramatically increased susceptibility to hematologic malignancies (Nat Genet 2015 47: 180 and 535). ETV6 is a transcriptional repressor essential for hematopoiesis and is frequently targeted by somatic genomic aberrations in childhood ALL (e.g., the ETV6-RUNX1 fusion). Therefore, we sought to comprehensively identify ALL predisposition variants in ETV6 and to determine the extent to which these variants contribute to childhood ALL risk in general. We first identified a family with three cases of childhood ALL at St. Jude Children's Research Hospital. Whole exome sequencing of this family (mother and 2 daughters with ALL, the unaffected father and 1 unaffected daughter) identified a single variant in ETV6 (p.R359X) in the 3 cases with ALL and also in the healthy daughter. This nonsense variant is predicted to create a stop codon within the ETS domain of ETV6, resulting in a truncated protein without DNA-binding function. This highly damaging variant is likely to be responsible for the ALL predisposition in this family with a high albeit incomplete penetrance. To comprehensively determine the prevalence of ALL-predisposing alleles in ETV6, we performed targeted sequencing of this gene in 4,405 children with newly-diagnosed ALL enrolled on the Children's Oncology Group (COG) AALL0232, P9904, P9905 and P9906 protocols and St. Jude Total Therapy XIIIA, XIIIB and XV studies. We identified a total of 43 germline variants in the exonic regions of ETV6. Thirty-one of the 43 ETV6 variants were defined as "ALL-related" because they were not found or extremely rare in non-ALL populations (N=60,706). These ALL risk variants included 4 nonsense, 21 missense, 1 splice site, and 5 frameshift variants occurring in 35 children (0.79% of ALL cases studied). Fifteen of the 31 ALL-relatedvariants (48.4%) were clustered in the ETS DNA-binding domain of ETV6. We used the combined annotation dependent depletion algorithm (CADD) to predict deleterious effects of each variant. ALL-related ETV6 variants were significantly more likely to be damaging compared to germline variants observed in the non-ALL population (mean CADD phred-like score of 25.6 vs 15.2, respectively, p<0.0001). Interestingly, of the 18 most deleterious ETV6 variants, 10 (55.6%) resided in the ETS domain although none were located within the helix directly interacting with target DNA. Instead, 7 of the 10 variants in ETS domain were between the first and second helices. We next analyzed the relationship between germline risk variants in ETV6 and clinical features of ALL in a subset of 2,021 cases enrolled on St. Jude and COG frontline ALL trials. These cases were comprehensively evaluted for ALL charateristics and representative of the US childhood ALL population. Children with ALL-related ETV6 variants were significantly older at the time of diagnosis than those without these variants (9.5 years vs 6.4 years; P=0.009). The hyperdiploid leukemia karyotype was strikingly overrepresented in ALL cases harboring germline ETV6 risk variants compared to the wildtype group (64.3% vs 26.8%; P=0.0045). In contrast, the frequency of somatic ETV6 -RUNX1 fusion was much lower in cases with ETV6 germline risk variants, compared to cases with wildtype ETV6 (7.1% vs 22.7%), even though this difference did not reach statistical significance. Of note, there was also a trend towards overrepresentation of females in carriers of ALL-related ETV6 variants (71.4% vs 45.7%; P=0.063). In conclusion, our findings indicate that germline ETV6 variations are important determinants for genetic predisposition to childhood ALL. Disclosures Martin: Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gentium SpA/Jazz Pharmaceuticals: Research Funding. Evans:Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping. Hunger:Spectrum Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Merck: Equity Ownership; Sigma Tau: Consultancy.
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Järviaho, Tekla, Benedicte Bang, Vasilios Zachariadis, Fulya Taylan, Jukka Moilanen, Merja Möttönen, C. I. Edvard Smith, Arja Harila-Saari, Riitta Niinimäki, and Ann Nordgren. "Predisposition to childhood acute lymphoblastic leukemia caused by a constitutional translocation disrupting ETV6." Blood Advances 3, no. 18 (September 13, 2019): 2722–31. http://dx.doi.org/10.1182/bloodadvances.2018028795.
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Key PointsWe report the first known family with a constitutional translocation disrupting ETV6 predisposing to ALL. Germline monoallelic expression of ETV6 contributes to leukemia predisposition without thrombocytopenia.
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Nishii, Rina, Rebekah Baskin, Takaya Moriyama, Keito Hoshitsuki, Monika L. Metzger, Gang Wu, Meenakshi Devidas, et al. "Comprehensive Functional Characterization of Germline ETV6 Variants Associated with Inherited Predisposition to Acute Lymphoblastic Leukemia in Children." Blood 128, no. 22 (December 2, 2016): 1085. http://dx.doi.org/10.1182/blood.v128.22.1085.1085.
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Abstract There is increasing evidence for inherited susceptibility to acute lymphoblastic leukemia (ALL) in children. Both common and rare germline genetic variants can significantly influence the risk of developing ALL, often in an age- and/or leukemia subtype-dependent manner (e.g., ARID5B, IKZF1, GATA3, PIP4K2A, PAX5, and TP53). Recently, we and others reported a novel genetic syndrome characterized by damaging germline ETV6 mutations that co-segregate with hereditary thrombocytopenia and increased propensity for hematologic malignancies (especially ALL). Sequencing 4,405 childhood ALL cases, we subsequently identified 30 germline coding ETV6 variants that were potentially responsible for disease predisposition in 35 patients (Lancet Oncol 2015). These findings unequivocally point to ETV6 as a critical ALL risk gene; however, the exact functional consequences of the ALL-related ETV6 variants are largely unknown and the mechanism by which they contribute to leukemogenesis is unclear. To this end, we first systematically evaluated these 30 ETV6 risk variants for their transcriptional repressor activity, ability to bind to target DNA sequences, localize to the nucleus, and also to homo-dimerize. Using PF4 and MMP3 promoter-driven luciferase transcription in HEK293T cells as the model system, we showed that 18 of these 30 ETV6 variants had loss of transcriptional repression activity (median of 53.2% of wildtype ETV6 activity [range 7.1% to 83.9%]). These include 4 of the 5 frameshift variants, all 4 nonsense variants, and also 10 of 11 missense variants in the ETS/DNA binding domain. None of the missense variants outside the ETS domain had significant effects on transcriptional repression function, and surprisingly the E44fs variant showed enhanced activity. Co-expression of wildtype and each of the 18 loss-of-function variants consistently impaired the normal repressor activity of wildtype ETV6, indicating dominant-negative effects. In cells expressing both wildtype and variant ETV6, immunoprecipitation of variant protein pulled down wildtype ETV6, in line with the dominant negative mode of action of variant ETV6 via dimerization with wildtype protein. Cellular fractionation showed significantly increased cytoplasmic localization of 18 loss-of-function ETV6 variants when expressed in HEK293T cells with concomitant reduction in nuclear ETV6. Analysis using EMSA revealed that all 14 variants in the ETS domain resulted in impaired binding to the consensus sequence in ETV6 target genes in vitro, as was also true for all 4 loss-of-function frameshift variants. To examine the role of ETV6 variants on hematopoietic stem and progenitor cell function, we lentivirally transduced murine LSK cells with wildtype or variant murine Etv6 (R355X or R395C). These cells were cultured ex vivo and analyzed for myeloid and pre-B cell differentiation, using colony formation assay. We found no change in the formation of myeloid colonies, whereas the number of pre-B colonies increased slightly with mutant Etv6, although we observed no effects on cell cycle or apoptosis. Focusing on the 18 ETV6 variants with experimentally confirmed deleterious effects, we then evaluated the association of germline ETV6 status with ALL clinical features in 3,906 children from frontline ALL protocols at St. Jude Children's Research Hospital and Children's Oncology Group. Patients with these ETV6 variants were significantly older at ALL diagnosis than those without (13.3 [2.6-21.7] vs 6.8 [0.08-30.7] years, P=0.026) but were more likely to have leukocyte count <50x109/L at presentation (100% vs. 68.6%, P=9.5x10-4). 80% of patients with deleterious ETV6 variants had hyperdiploid ALL compared to 22.2% of those with normal ETV6 (P=9.3x10-8), strongly suggesting interactions between germline and somatic variations during leukemogenesis. Finally, whole genome sequencing of matched ALL blast and germline samples from 5 familial ALLs in 2 kindreds with pathologic ETV6 variants identified somatic lesions as plausible second events to promote leukemogenesis in these cases. In conclusion, we comprehensively characterized 30 ALL risk variants in ETV6, the majority of which function by disrupting DNA binding and impairing its transcriptional repressor activity in a dominant negative fashion. ETV6 variants also affect hematopoiesis but most likely require additional somatic lesions for overt leukemogenesis. Disclosures Martin: Novartis: Other: Support of clinical trials; Jazz Pharmaceuticals: Other: One time discussion panel. Evans:Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping.. Relling:Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping. Hunger:Patent: Patents & Royalties: Dr. Hunger is a co-inventor of a patent (#8658,964) for the identification of novel subgroups in high risk B-ALL and outcome correlations and diagnostic methods related to the same; Erytech: Honoraria; Sigma Tau Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Honoraria; Merck: Equity Ownership; Pfizer: Equity Ownership; Amgen: Equity Ownership; Spectrum Pharmaceuticals: Honoraria. Mullighan:Loxo Oncology: Research Funding; Amgen: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees.
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Rio-Machin, Ana, and Jude Fitzgibbon. "Germline ETV6 variants: not ALL created equally." Blood 137, no. 3 (January 21, 2021): 288–89. http://dx.doi.org/10.1182/blood.2020008190.
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Romero, Diana. "ETV6 germline mutation — a risk for ALL." Nature Reviews Clinical Oncology 13, no. 1 (November 17, 2015): 4. http://dx.doi.org/10.1038/nrclinonc.2015.211.
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Jones, Courtney L., Gregory Kirkpatrick, Courtney Fleenor, Welsh Seth, Leila J. Noetzli, Susan Fosmire, Dmitry Baturin, et al. "ETV6 Regulates Pax5 Expression in Early B Cell Development." Blood 128, no. 22 (December 2, 2016): 2655. http://dx.doi.org/10.1182/blood.v128.22.2655.2655.
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Abstract Recent studies from our group and others have revealed a role for ETV6 germline mutations in the predisposition to ALL. Although ETV6 is among the most commonly mutated genes in ALL, its mechanistic role in leukemogenesis remains unclear. ETV6 is an ETS family transcription factor. ETV6 regulates gene transcription through homo- and hetero- oligomerization with other ETS family members and transcriptional repressors. The germline mutation (P214L amino acid change) identified by our group and others impairs the transcriptional activity and nuclear localization of ETV6 in a dominant negative fashion. The goal of this project is to determine the role of ETV6 in early B cell development and define how germline ETV6 mutations result in predisposition to leukemia. To identify functions of ETV6 in B cell development, we queried the gene expression commons database for evidence of Etv6 expression during B cell development. Etv6 is highly expressed in hematopoietic stem and lymphoid progenitor cells through the pre-pro-B stage (FrA), but its expression is significantly reduced in fraction B and thereafter (P<0.0001). To confirm relative patterns of Etv6 and Pax5 expression in developing B cells, we isolated bone marrow (BM) from wild type (WT) mice and fractionated cells committed to the B cell lineage via B220+ and CD43+ staining by flow cytometry and then separated into the following fractions: Fraction A (CD24low, CD19-), Fraction B (CD19+, CD24+, BP1-) and Fraction C (CD19+ CD24+ BP1+). Etv6 expression decreases as B cells develop and is negatively correlated with Pax5 expression (r2=.9993; P= 0.0167). We next confirmed the expression patterns of ETV6 and PAX5 during B cell development in human samples. We found that ETV6 expression was higher in the early B cell fraction (CD10+, CD34+, CD19-, and CD20-) compared to the preB cell fraction (CD10+, CD34-, CD19+, CD20-). Conversely, we observed that PAX5 expression was higher in the preB cell fraction compared to the early B cell fraction. To determine if a function relationship exists between ETV6 and Pax5 we overexpressed an empty vector (MiG), wild type (WT) ETV6 and ETV6 P214L in a murine lymphoid progenitor line (Ba/F3). ETV6, but not ETV6 P214L overexpression significantly decreased Pax5 expression (P≤0.05). To further interrogate the role of ETV6 in regulating Pax5 transcription we measured the association of ETV6 with putative ETS factor binding sites (GGAA sequence) within the Pax5 transcription start site (TSS) using ChIP-PCR. ETV6 is associated with the proximal GGAA site 72 base pairs upstream of the Pax5 TSS, but not GGAA sites further from the TSS. In addition, the transcriptional repressors SIN3A and HDAC3 were detected on the same regions of the Pax5 locus. We next determined the consequences of ETV6 mutation on the recruitment of ETV6, SIN3A, and HDAC3 to the Pax5 locus by performing ChIP-PCR in Ba/F3 cells that express a FLAG-tagged WT ETV6 or ETV6 P214L. We detected association of ETV6, SIN3A and HDAC3 with the proximal GGAA site upon expression of WT ETV6, but not ETV6 P214L. We conclude that ETV6, SIN3A and HDAC3 are responsible for the repression of Pax5 transcription. Moreover, mutant ETV6 inhibits the ability of normal ETV6 to bind and recruit SIN3A and HDAC3 to the Pax5 locus. Finally, we determined if the recruitment of SIN3A and HDACs to the Pax5 locus was essential to repression of Pax5 by WT ETV6 by knocking out SIN3A and inhibiting HDACs using pan HDAC inhibitor, SAHA and measuring Pax5 expression by RT-PCR. We found that upon SIN3A knockout or HDAC inhibition Pax5 expression was no longer repressed upon WT ETV6 overexpression. To determine the consequences of ETV6 P214L expression on B cell development, we generated a transgenic mouse expressing the P214L mutation in the endogenous ETV6 gene. Preliminary data suggests that these mice have thrombocytopenia, similar to patients with germline ETV6 mutation. In addition, mice with the ETV6 P214L mutation displayed reduced level of cKIT expression on the FrA B cell population. Further studies will be necessary to understand the consequences of reduced cKIT expression to overall B cell development and if this cKIT reduction is linked to aberrant Pax5 expression. In conclusion, ETV6 regulates Pax5 expression through the recruitment of SIN3A and HDAC3 to the Pax5 locus. These findings are significant because Pax5 misregulation results in a B cell development halt, lineage infidelity and leukemogenesis. Disclosures No relevant conflicts of interest to declare.
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Kirkpatrick, Greg, Courtney Jones, Susan Fosmire, Christopher Porter, and Jorge DiPaola. "2485." Journal of Clinical and Translational Science 1, S1 (September 2017): 65–66. http://dx.doi.org/10.1017/cts.2017.234.
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OBJECTIVES/SPECIFIC AIMS: The goal of this project is to determine the role of ETV6 in early B-cell development and define how germline ETV6 mutations result in predisposition to leukemia. METHODS/STUDY POPULATION: Gene expression commons were queried for expression levels of Etv6 and Pax5 at different stages of hematopoiesis. Mouse bone marrow was isolated and fractioned into cells committed to the B cell lineage via B220+ and CD43+ staining by flow cytometry and then separated into the following fractions: Fraction A (CD24low, CD19−), Fraction B (CD19+, CD24+, BP1−), and Fraction C (CD19+ CD24+ BP1+). Wild-type or germline mutant P214L ETV6 were cloned in an MiG vector and expressed in Ba/F3 cells. ChIP-PCR was performed by cross-linking proteins to DNA with 1% formaldehyde for 10 minute at room temperature, followed by cell lysis with RIPA buffer. Lysates were sonicated to shear DNA to a length of 200–1000 base pairs, then Protein A agarose beads were used to clean and immunoprecipitate chromatin. RESULTS/ANTICIPATED RESULTS: We observed that Etv6 is highly expressed in hematopoietic stem and lymphoid progenitor cells through the pre-pro-B stage (FrA), but its expression is significantly reduced in fraction B and thereafter (p<0.0001). Etv6 expression decreases as B cells develop and is negatively correlated with Pax5 expression (r2=0.9993; p=0.0167). We next confirmed the expression patterns of ETV6 and PAX5 during B cell development in human samples. We found that ETV6 expression was higher in the early B cell fraction (CD10+, CD34+, CD19−, and CD20−) compared to the pre-B cell fraction (CD10+, CD34−, CD19+, CD20−). Conversely, we observed that PAX5 expression was higher in the preB cell fraction compared with the early B cell fraction. In Ba/F3 cells expressing ETV6 constructs, ETV6, but not ETV6 P214L overexpression significantly decreased Pax5 expression (p≤0.05). ETV6 is associated with the proximal GGAA site 72 base pairs upstream of the Pax5 TSS, but not GGAA sites further from the TSS. In addition, the transcriptional repressors SIN3A and HDAC3 were detected on the same regions of the Pax5 locus. We detected association of ETV6, SIN3A, and HDAC3 with the proximal GGAA site upon expression of WT ETV6, but not ETV6 P214L. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results provide a mechanism of interaction for ETV6 and PAX5, 2 genes often disrupted in B-cell leukemia. These findings are significant because PAX5 misregulation results in a B cell development halt, lineage infidelity, and leukemogenesis. In continuing our studies, we have generated a transgenic mouse endogenously expressing the ETV6 P214L mutation by CRISPR/Cas9 editing, and these mice appear to have a thrombocytopenic phenotype similar to that observed in patients carrying the ETV6 P214L mutation. These animals will be the focus of our continued investigation of the mechanism by which ETV6 germline mutation results in a predisposition to leukemia. Our ultimate goal is a comprehensive understanding of how this process may be targeted more efficiently in patients with both heritable and sporadic forms of leukemia involving ETV6.
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Feurstein, Simone, and Lucy A. Godley. "Germline ETV6 mutations and predisposition to hematological malignancies." International Journal of Hematology 106, no. 2 (May 29, 2017): 189–95. http://dx.doi.org/10.1007/s12185-017-2259-4.
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Bernardi, Simona, Mirko Farina, Camilla Zanaglio, Federica Cattina, Nicola Polverelli, Francesca Schieppati, Federica Re, et al. "ETV6: A Candidate Gene for Predisposition to “Blend Pedigrees”? A Case Report from the NEXT-Famly Clinical Trial." Case Reports in Hematology 2020 (January 11, 2020): 1–7. http://dx.doi.org/10.1155/2020/2795656.
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Background. The identification of germline mutations in familial leukemia predisposition genes by next generation sequencing is of pivotal importance. Lately, some “blend pedigrees” characterized by both solid and hematologic malignancies have been described. Some genes were recognized as related to this double predisposition, while the involvement of others is still a matter of debate. ETV6 was associated with hematologic malignancies, in particular myeloid malignancies, and recently described as mutated also in oncologic patients. No clear evidences in its involvement in blend pedigrees are known. Case Presentation. We present our recent experience in the identification of an ETV6-mutated “blend pedigree,” suggesting the involvement of ETV6 in the predisposition to both solid and hematologic neoplasia. The pedigree recognition started with a MDS case enrolled in the NEXT-Famly protocol. The patient presented 9 relatives affected by solid tumors and hematological malignancies. Following the clinical trial protocol, the patient underwent NGS analysis, which confirmed the presence of a mutation on the noncoding region of ETV6 both on tumor and on germline DNA. The mutation resulted was shared by the still alive affected relatives. Conclusion. This evidence supports the involvement of ETV6 in the predisposition to both solid and hematologic neoplasia and the importance of the investigation of the noncoding regions of the genes as recently suggested by different expert groups.
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Bernardi, Simona, Camilla Zanaglio, Elif Dereli Eke, Federica Cattina, Mirko Farina, Stefania Masneri, Benedetta Rambaldi, et al. "Identification of a Novel Mutation Predisposing to Familial AML and MDS Syndrome By a NGS Approach." Blood 132, Supplement 1 (November 29, 2018): 4387. http://dx.doi.org/10.1182/blood-2018-99-109927.
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Abstract Introduction In AML and MDS cases, the genetic lesions inherited or acquired by the hematopoietic stem cells are considered as starting events. Familial AML and MDS, recently recognized in the revised WHO classification (2016) provide a useful model for investigation of predisposing genetic mutations. Genetic analysis of several pure familial leukemia pedigrees led to the discovery of well defined syndromes associated with inherited de novo mutations on germline DNA. Growing clinical awareness as well as a widespread use of NGS have led to an enlarged description of familial MDS/AML cases, and the number of mutations involved, suggesting they are more frequent than those previously recognized. Despite the recent discovery of well-established causative gene mutations (RUNX1, GATA2, ETV6, TERT, TERC, SRP72, ANKRD26, DDX41, CEBPA), many cases remain unexplained (about 80%), suggesting that other inherited mutations could predispose to MDS/AML. It is expected that new sequencing approaches will help to the identification of more cases, more genes as well as novel syndromes. In 2017, we started a multicentric prospective study (Clinical trial.gov NCT03058588) aiming to look for predisposing mutations in patients and relatives affected by Familial AML and MDS syndromes (FAMS) by NGS and to screen for old and new mutations potentially associated with the disease. Methods At present, 12 AML/MDS patients have been enrolled. Leukemic (bone marrow) and germline (buccal swab) DNA were analyzed by NGS gene panel approach based on a 28 genes associated to myeloid leukemogenesis, including the 9 above mentioned genes associated to FAMS. NGS libraries were performed by a Nimblegen (Roche) custom panel based on gene capture strategy and the sequencing was performed by MiSeq (Illumina). Results Ten patients did not reveal any germline mutations and the candidates are undergoing to whole exome sequencing. One presented a germline mutation on RUNX1, and the analysis of the affected relatives is on going. One revealed a new mutation. She was a 70 years old woman affected by RARS and her pedigree was characterized by 9 relatives affected by hematologic and solid neoplasia and trombocytopenia (fig 1). The NGS analysis revealed the mutation c.*514C>T in 3'UTR of ETV6 with VAF of 50% on tumor DNA. The variant has never been described before, while ETV6 has been already associated with FAMS. Sanger sequencing confirmed the mutation on the germline DNA in heterozygosis. The screening of 2 affected relatives still alive confirmed the presence of the variant in heterozygosis. In silico analysis performed on PolymiRST Database revealed that c.*514C>T in 3'UTR of ETV6 results in a gain of miRNA binding site: hsa-miR- 4717-3p and hsa-miR- 942-3p. Discussion The variant c.*514C>T in 3'UTR of ETV6 seems to repress ETV6 due to RNA interference. The new binding miRNAs have been already described as over-expressed in solid and hematologic tumors. Moreover, the down-regulation of ETV6 is associated with alteration of cell growth and hematopoiesis. Due to these evidences, c.*514C>T in 3'UTR of ETV6 could be considered as a new mutation involved in FAMS predisposition. Disclosures No relevant conflicts of interest to declare.
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Karastaneva, Anna, Karin Nebral, Axel Schlagenhauf, Marcel Baschin, Raghavendra Palankar, Herbert Juch, Ellen Heitzer, et al. "Novel phenotypes observed in patients with ETV6-linked leukaemia/familial thrombocytopenia syndrome and a biallelic ARID5B risk allele as leukaemogenic cofactor." Journal of Medical Genetics 57, no. 6 (November 8, 2019): 427–33. http://dx.doi.org/10.1136/jmedgenet-2019-106339.
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Background. The phenotypes of patients with the recently discovered, dominant, ETV6-linked leukaemia predisposition and familial thrombocytopenia syndrome are variable, and the exact mechanism of leukaemogenesis remains unclear.Patients and Methods. Here, we present novel clinical and laboratory phenotypes of seven individuals from three families with ETV6 germline mutations and a refined genetic analysis of one child with additional high-hyperdiploid acute lymphoblastic leukaemia (HD-ALL), aiming to elucidate second oncogenic hits.Results. Four individuals from two pedigrees harboured one novel or one previously described variant in the central domain of ETV6 (c.592C>T, p.Gln198* or c.641C>T, p.Pro241Leu, respectively). Neutropenia was an accompanying feature in one of these families that also harboured a variant in RUNX1 (c.1098_1103dup, p.Ile366_Gly367dup), while in the other, an autism-spectrum disorder was observed. In the third family, the index patient suffered from HD-ALL and life-threatening pulmonary mucor mycosis, and had a positive family history of ‘immune’ thrombocytopenia. Genetic analyses revealed a novel heterozygous mutation in the ETS domain of ETV6 (c.1136T>C, p.Leu379Pro) along with absence of heterozygosity of chromosome (10)(q21.2q21.3), yielding a biallelic leukaemia risk allele in ARID5B (rs7090445-C). The neutrophil function was normal in all individuals tested, and the platelet immune histochemistry of all three pedigrees showed delta-storage-pool defect-like features and cytoskeletal defects.Conclusions. Our clinical observations and results of high-resolution genetic analyses extend the spectrum of possible phenotypes cosegregating with ETV6 germline mutations. Further, we propose ARID5B as potential leukaemogenic cofactor in patients with ETV6-linked leukaemia predisposition and familial thrombocytopenia syndrome.
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Dirse, Vaidas, Rimvydas Norvilas, Egle Gineikiene, Rėda Matuzevičienė, Laimonas Griskevicius, and Egle Preiksaitiene. "ETV6 and NOTCH1 germline variants in adult acute leukemia." Leukemia & Lymphoma 59, no. 4 (August 4, 2017): 1022–24. http://dx.doi.org/10.1080/10428194.2017.1359742.
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Zhang, Michael Y., Jane E. Churpek, Siobán B. Keel, Tom Walsh, Ming K. Lee, Keith R. Loeb, Suleyman Gulsuner, et al. "Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy." Nature Genetics 47, no. 2 (January 12, 2015): 180–85. http://dx.doi.org/10.1038/ng.3177.
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Gocho, Yoshihiro, and Jun J. Yang. "Genetic defects in hematopoietic transcription factors and predisposition to acute lymphoblastic leukemia." Blood 134, no. 10 (September 5, 2019): 793–97. http://dx.doi.org/10.1182/blood.2018852400.
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Abstract Recent genome-wide studies have revealed a plethora of germline variants that significantly influence the susceptibility to acute lymphoblastic leukemia (ALL), thus providing compelling evidence for genetic inheritance of this blood cancer. In particular, hematopoietic transcription factors (eg, ETV6, PAX5, IKZF1) are most frequently implicated in familial ALL, and germline variants in these genes confer strong predisposition (albeit with incomplete penetrance). Studies of germline risk factors for ALL provide unique insights into the molecular etiology of this leukemia.
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Fisher, Marlie H., Greg Kirkpatrick, Courtney L. Jones, Brett M. Stevens, Michael Callaghan, Eric Pietras, Kenneth L. Jones, Christopher C. Porter, and Jorge Di Paola. "Germline Mutations in ETV6 are Associated with In Vivo cytoplasmic Localization of ETV6 and Cause Transcriptional Dysregulation of Interferon Response Genes." Blood 134, Supplement_1 (November 13, 2019): 2316. http://dx.doi.org/10.1182/blood-2019-132032.
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ETV6, an ETS family transcription factor, has been characterized as a master regulator of hematopoiesis and megakaryocyte development. In this work we identified a novel role for ETV6 as a key repressor of peripheral inflammation and interferon response in humans. Flow cytometry based immunophenotyping of peripheral blood mononuclear cells (PBMC) from 5 patients carrying a germline mutation in ETV6 (p.Pro214Leu) of various ages demonstrated normal frequencies of peripheral cell populations, including B and T cells (CD3+ and CD19+, respectively), progenitor populations, including CLP (CD19- CD3- CD34+ CD10+) and MEP (CD10- CD34+ CD38+ CD135- CD45RA-), but significantly increased circulating HSCs (CD34+ CD38- CD90+) as compared to healthy related and unrelated controls. Immunofluorescence studies in these PBMC showed cytoplasmic localization of ETV6 in all affected individuals. Furthermore, 10X genomics single cell RNA sequencing of these peripheral blood cells demonstrated profound transcriptomic dysregulation as compared to PBMCs of related and unrelated healthy controls. We initially detected a total of 204 differentially expressed transcripts in monocytes, NK cells and T and B cell populations, demonstrating a cell-type specific upregulation of inflammatory genes. We then analyzed the intersection of the significantly increased transcripts across these populations, identifying a highly specific suite of 22 genes, which depending on cell type were upregulated 4 to 400-fold. These significantly upregulated 22 transcripts in the affected individuals were highly enriched for the interferon response pathway, consisting of pro-inflammatory transcripts such as CCL4, CCL4L2, CCL3, CCL3L3, IFIT2, ISG15, MX1, IFIT3, IRF1, and NFKBIA among others. Ingenuity Pathway Analysis revealed HDAC3 as a highly predicted upstream master regulator of this group of genes. We then demonstrated by protein immunoprecipitation that both wild-type and P214L ETV6 form a complex with HDAC3, suggesting that the ETV6-HDAC3 complex is being removed from the nucleus, disrupting homeostatic transcriptional repression of interferon response genes. In summary, we have determined a new role for ETV6 as a repressive regulator of interferon response genes likely via its interaction with HDAC3. These findings have potential implications for the development of myelodysplasia and hematological malignancies observed in individuals with ETV6 germline mutations. Disclosures Callaghan: Novonordisk: Consultancy, Speakers Bureau; Octapharma: Consultancy; Pfizer: Research Funding; Roche: Research Funding; Biomarin, Bioverativ, Grifols, Kedrion, Pfizer, Roche/Genentech, Shire, and Spark Therapeutics: Consultancy; Alnylum: Equity Ownership; Bayer: Consultancy, Speakers Bureau; Takeda: Consultancy, Research Funding; Sanofi: Consultancy; Global Blood Therapeutics: Consultancy; Shire/Takeda: Speakers Bureau; Roche/Genentech: Speakers Bureau.
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Difilippo, Emma Catherine, Alejandro Ferrer, Laura Schultz-Rogers, Naseema Gangat, Shakila P. Khan, Aref Al-Kali, Abhishek A. Mangaonkar, et al. "Spectrum of Hematological Malignancies in 130 Patients with Germline Predisposition Syndromes - Mayo Clinic Germline Predisposition Study." Blood 136, Supplement 1 (November 5, 2020): 34–35. http://dx.doi.org/10.1182/blood-2020-139050.
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Introduction Germline predisposition syndromes (GPS) are inherited disorders associated with germinal aberrations that increase the risk of malignancies. While aberrations in certain genes increase the risk for all types of malignancies (Tp53, ATM, CDKN2A, CHEK2), there is a growing list of genes associated specifically with hematological malignancies (GATA2, RUNX1, DDX41, ETV6, ANKRD26). At our institution, we have established a hematology GPS clinic to diagnose and manage GPS and with this report, detail our experience with 130 patients. Methods GPS were investigated in pediatric and adult patients with one or more first degree relatives with hematological/visceral malignancies or in those with antecedent thrombocytopenia (ANKRD26, RUNX1, ETV6), or with specific syndromic features (short telomere syndromes/STS, GATA2 haploinsufficiency, Fanconi anemia/FA, Shwachman-Diamond syndrome/SDS). Depending on the phenotype, specific functional assays such as flow-FISH for telomere length assessment and chromosomal breakage assays were ordered. After informed consent and genetic counselling, germline testing was carried out on peripheral blood mononuclear cell, skin fibroblast, or hair follicle-derived DNA. A custom-designed marrow failure NGS panel (200 genes) was used in most cases and interrogation of variants, in silico studies, and functional assays were carried out as previously described (Mangaonkar et al MC Proc 2019). Copy number variations were identified by aCGH. At the time of progression/worsening cytopenias, bone marrow/lymph node biopsies and NGS (next generation sequencing) were carried out where indicated. Results 130 patients with germline predisposition have been identified to date. The spectrum of disorders seen include STS 29 (22%), FA 17 (13%), GATA2 16 (12%), Diamond Blackfan anemia/DBA 13 (10%), RUNX1-FPD 12 (9%), ATM deletions/mutations 11 (8%), ANKRD26 6 (5%), SDS 5 (4%), DDX41 4 (3%), MPL 3 (2%), CHEK2, MECOM, Tp53 mutations 2 (2%) each, and CBL, CEPBA, ELANE, NF1, CDKN2A, CSF3R, ETV6, and GATA1 mutations, 1 (1%) each. Evidence for clonal evolution (CCUS) and hematological malignancies were seen in 51 (39%) patients, involving all the aforementioned genes/syndromes with the exception of DBA, CBL, ETV6, MPL, CSF3R, and GATA1. Seven (64%) of 11 patients with germline ATM deletions/mutations developed lymphoid malignancies; homozygous ATM (Follicular NHL-1, Burkitt lymphoma-1, T-ALL-1, T-LPD-1) and heterozygous ATM (T-PLL-1, DLBCL-1, CLL-1). Clonal evolution occurred in 11 (69%) of 16 GATA2 haploinsufficient patients (CCUS-2, MDS-3, CMML-1, AML-5) and in 7 (58%) of 12 RUNX1-FPD patients (CCUS-1, MDS-1, MDS/MPN-3, AML-2). Five of 29 (17%) STS patients had clonal progression (CCUS-2, MDS-2, AML-1), and 5 (29%) of 17 FA patients progressed to MDS-2 or AML-3. JMML was seen in one patient with a germline NF1 mutation, while 1 (20%) of 5 SDS patients progressed to AML. NGS data at progression was available in 24 (55%) of 44 myeloid/CCUS progressions, with somatic truncating ASXL1 mutations being most frequent (29%), followed by RAS pathway mutations (15%). AML/MDS progressions in STS, FA, and SDS were universally associated with complex/monosomal karyotypes, translating to refractory disease. Seventeen (39%) of 44 patients with myeloid predisposition underwent allogenic HCT (GATA2-7, FA-3, RUNX1-FPD-3, STS-2, NF1-1, Tp53-1), with 10 (59%) being alive at last follow up (Table 1). Conclusion We demonstrate the spectrum of germline aberrations associated with predisposition to hematological malignancies and outline the phenotypic heterogeneity of clonal transformation. The advent of NGS allows identification of clonal progression earlier than morphological changes, with mutations in ASXL1 and RAS pathway genes being commonly implicated. This study supports the universal development of dedicated germline predisposition clinics. Disclosures Pruthi: CSL Behring: Honoraria; Genentech Inc.: Honoraria; Bayer Healthcare: Honoraria; HEMA Biologics: Honoraria; Instrumentation Laboratory: Honoraria; Merck: Honoraria.
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Ho, Thanh, Juliana Perez Botero, William J. Hogan, Saad S. Kenderian, Naseema Gangat, Ayalew Tefferi, Roshini S. Abraham, et al. "Clinical Spectrum of Germline Mutations with Predisposition to Myeloid Neoplasms- 2016 World Health Organization Classification Update." Blood 128, no. 22 (December 2, 2016): 300. http://dx.doi.org/10.1182/blood.v128.22.300.300.
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Abstract Background: The 2016 revision to the World Health Organization (WHO) classification of myeloid neoplasms has specifically categorized germline mutations that are associated with myeloid clonal evolution (Arber et al. Blood 2016). This group consists of myeloid neoplasms with an isolated germline predisposition (CEBPA, DDX41), myeloid neoplasms associated with congenital thrombocytopenia (ETV6, RUNX1, ANKRD26) and germline myeloid neoplasms with multi-organ dysfunction (GATA2, chromosomal breakage disorders, telomere biology disorders etc). We carried out this study to describe the clinical spectrum of germline disorders with predisposition to myeloid neoplasms as categorized by the 2016 WHO classification revision. Methods: After Institutional Review Board (IRB) approval, the adult and pediatric bone marrow failure syndrome database (1990-2016) and the electronic medical record were queried for germline disorders involving GATA2, CEBPA, DDX41, ETV6, RUNX1, ANKRD26, Down syndrome and Noonan syndrome. Chromosomal breakage assays (Diepoxybutane/Mitomycin-C), flow-fluorescent in-situ hybridization (FISH) for telomere length assessment, Fanconi anemia complementation assays and Sanger/Next Generation sequencing (NGS) for the aforementioned germline disorders with myeloid predisposition were carried out in Clinical Laboratory Improvement Amendments (CLIA)-certified laboratories. These disorders were then classified based on the 2016 WHO classification revision. Results : 54 individuals (37 families) were included in the study. Eleven (20%) patients belonging to 5 families were identified as having germline mutations with a preexisting platelet disorder: ETV6 (n=1), ANKRD26 (n=5), RUNX1 (n=5). Forty-three (79%) patients (32 families) had inherited syndromes with multi-organ dysfunction: GATA2 (n=11, 26%), bone marrow failure syndromes (n=14, 33%) and telomere biology disorder (n=14, 33%). There was one patient with neurofibromatosis with a germline PTPN11 mutation who developed juvenile myelomonocytic leukemia, while there were three patients with Down syndrome; 2 with transient abnormal myelopoiesis and one who developed acute megakaryocytic leukemia. The clinical phenotype, prevalence and characteristics of myeloid clonal evolution and outcomes are presented in Table 1. No patients with germline CEBPA or DDX41 mutations were identified. Patients with germline platelet disorders did not have any prominent non-hematological manifestations. Erythrocytosis (20%) with long-standing thrombocytopenia (100%) was a unique feature associated with ANKRD26 mutations. Non-hematologic clues such as human papillomavirus (HPV)-driven warts, primary lymphedema (Emberger syndrome) and frequent atypical infections with monocytopenia were seen in patients with germline GATA2 mutations, and preceded myeloid clonal evolution (morphologic, cytogenetic and molecular). Notably, the age at presentation and penetrance of myeloid transformation was variable, with individuals from the same family developing symptoms during the first decade of life and others remaining asymptomatic to date (fifth decade). Somatic ASXL1 mutations were detected in all 3 (100%) patients with GATA2 mutations and in one patient with ANKRD26 mutation that developed myeloid clonal evolution. In our study myeloid clonal evolution was seen in 40% with RUNX1 mutations, 27% with GATA2 mutations, and 20% with ANKRD26 mutations. We could not calculate the same for bone marrow failure syndromes as the total number of cases seen are still being assessed. Outcomes with allogeneic stem cell transplantation were favorable in appropriately selected patients (Table 1). Conclusion : The 2016 WHO revision to the classification of myeloid neoplasms highlights the importance of recognition and molecular characterization of germline mutations (syndromic and non-syndromic) with risk for myeloid clonal evolution. While some of these disorders (GATA2, Fanconi anemia, telomere biology disorders) may have important non-hematological clues, many present with isolated thrombocytopenia (RUNX1, ETV6). The age and frequency of myeloid evolution is highly variable. Acquisition of somatic ASXL1 mutations at the time of clonal myeloid transformation highlights the role of epigenetic dysregulation in disease evolution. Disclosures Kenderian: Novartis: Patents & Royalties, Research Funding.
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Di Paola, Jorge. "Novel Congenital Platelet Disorders." Blood 128, no. 22 (December 2, 2016): SCI—39—SCI—39. http://dx.doi.org/10.1182/blood.v128.22.sci-39.sci-39.
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The processes of megakaryocyte differentiation, proplatelet formation, and the daily release of 1011 platelets into the bloodstream are tightly regulated. Genetic disturbances can lead to a cascade of downstream molecular alterations that markedly affect the function of megakaryocytes and platelets. Therefore, identifying new genes and their function in megakaryocytes and platelets is critical for understanding how these unique cells contribute to health and disease. Over the last decade advances in genomics, specifically next generation sequencing, have allowed for the discovery of several mutations and genetic variants that cause disease or influence associated hematological traits. By performing platelet RNA-Seq we were among the first to identify NBEAL2 as the causative gene for gray platelet syndrome (GPS) and showed that NBEAL2 regulates megakaryocyte development and platelet function.1-3 Mice carrying targeted Nbeal2 null alleles not only replicated the thrombocytopenia and lack of alpha granules observed in humans, but also provided new information about the role of platelets in thromboinflammation, wound healing, myelofibrosis and metastasis dissemination.4-7 More recently, we and others found that germline mutations in ETV6 lead to thrombocytopenia, red cell macrocytosis, and predisposition to lymphoblastic leukemia.8,9ETV6 encodes an ETS family transcriptional repressor, which exerts its activity by binding a consensus sequence in the promoter regions of DNA. Mice with conditional Etv6 knockout in megakaryocytic-erythroid cells are thrombocytopenic indicating the involvement of Etv6 in thrombopoiesis.10 Several of the families recently described have a missense mutation in the central domain of ETV6 (p.P214L). This mutation results in aberrant cellular localization of ETV6, decreased transcriptional repression, and impaired megakaryocyte maturation. The bone marrow of individuals affected by this mutation show hyperplasia of immature megakaryocytes suggesting a differentiation arrest. Deep sequencing of the platelet transcriptome also revealed significant differences in mRNA expression levels between patients with the ETV6 p.P214L mutation and non-affected family members, indicating that ETV6 is critically involved in defining the molecular phenotype and function of platelets. Consistent with this notion, individuals with the ETV6 p.P214L mutation experience bleeding that is disproportionate to their mild thrombocytopenia. We have also used CRISPR/Cas9 technology to generate a mouse colony where the human p.P214L ETV6 mutation was inserted into the conserved site of Etv6. Mice with this mutation (Etv6H.P214L) have reduced platelet counts. In summary, advances in human genetics that led to the discovery of novel congenital platelet disorders coupled with relevant animal models will likely contribute to our understanding of megakaryopoiesis and platelet function. References 1. Kahr WH, Hinckley J, Li L, et al. Mutations in NBEAL2, encoding a BEACH protein, cause gray platelet syndrome. Nature genetics. 2011;43(8):738-740. 2. Gunay-Aygun M, Falik-Zaccai TC, Vilboux T, et al. NBEAL2 is mutated in gray platelet syndrome and is required for biogenesis of platelet alpha-granules. Nature genetics. 2011;43(8):732-734. 3. Albers CA, Cvejic A, Favier R, et al. Exome sequencing identifies NBEAL2 as the causative gene for gray platelet syndrome. Nature genetics. 2011;43(8):735-737. 4. Deppermann C, Cherpokova D, Nurden P, et al. Gray platelet syndrome and defective thrombo-inflammation in Nbeal2-deficient mice. The Journal of clinical investigation. 2013. 5. Kahr WH, Lo RW, Li L, et al. Abnormal megakaryocyte development and platelet function in Nbeal2(-/-) mice. Blood. 2013;122(19):3349-3358. 6. Guerrero JA, Bennett C, van der Weyden L, et al. Gray platelet syndrome: proinflammatory megakaryocytes and alpha-granule loss cause myelofibrosis and confer metastasis resistance in mice. Blood.2014;124(24):3624-3635. 7. Tomberg K, Khoriaty R, Westrick RJ, et al. Spontaneous 8bp Deletion in Nbeal2 Recapitulates the Gray Platelet Syndrome in Mice. PLoS One. 2016;11(3):e0150852. 8. Noetzli L, Lo RW, Lee-Sherick AB, et al. Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia. Nature Genetics. 2015;47(5):535-538. 9. Zhang MY, Churpek JE, Keel SB, et al. Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy. Nature genetics. 2015;47(2):180-185. 10. Wang LC, Swat W, Fujiwara Y, et al. The TEL/ETV6 gene is required specifically for hematopoiesis in the bone marrow. Genes & development. 1998;12(15):2392-2402. Disclosures Di Paola: CSL BEhring: Consultancy; Biogen: Consultancy.
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Topka, Sabine, Joseph Vijai, Michael F. Walsh, Lauren Jacobs, Ann Maria, Danylo Villano, Pragna Gaddam, et al. "Germline ETV6 Mutations Confer Susceptibility to Acute Lymphoblastic Leukemia and Thrombocytopenia." PLOS Genetics 11, no. 6 (June 23, 2015): e1005262. http://dx.doi.org/10.1371/journal.pgen.1005262.
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Fisher, Marlie H., Leila J. Noetzli, Michael Callaghan, Walter H. Kahr, Jesse W. Rowley, and Jorge Di Paola. "Studying the Role of ETV6 in Megakaryopoiesis and Thrombopoiesis Using a Novel CRISPR-Cas9 Halotag Genome Editing Strategy." Blood 134, Supplement_1 (November 13, 2019): 2468. http://dx.doi.org/10.1182/blood-2019-131967.
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Emerging data indicate that germline mutations in transcription factors involved in hematopoiesis can lead to a cascade of downstream molecular alterations that modify the function of megakaryocytes (MK) and platelets. Our group and others have found that mutations in ETV6 lead to mild thrombocytopenia with a bleeding diathesis, red cell macrocytosis, and predisposition to lymphoblastic leukemia. The mechanisms responsible for thrombocytopenia and propensity for bleeding in patients with ETV6 mutations are unknown. We described families with missense mutations in the central domain (p.Pro214Leu) and the ETS DNA binding domain (p.Arg418Gly) of ETV6 that result in aberrant cellular localization of ETV6, decreased transcriptional repression, and impaired MK maturation. Deep sequencing of the platelet transcriptome revealed significant differences in mRNA expression levels between patients with the ETV6 p.Pro214Leu mutation and non-affected family members, indicating that ETV6 is critically involved in defining the molecular phenotype and function of platelets. We hypothesize that normal regulation and function of ETV6 is essential for the transcriptional machinery that controls megakaryocyte differentiation and formation of platelets that function normally under homeostatic conditions. We have successfully generated a CRISPR-Cas9 model to edit the genome of ETV6-expressing iPSC derived megakaryocyte cell line (imMKCL) to characterize the role of wild-type ETV6 in megakaryocyte development and elucidate the molecular mechanism driving mutant ETV6 mislocalization, transcriptional dysregulation, and subsequent dysmegakaryopoiesis and thrombocytopenia. In this imMKCL model, we have genetically engineered the cells to express wild-type, P214L, and the DNA binding domain mutations R418G and R369Q ETV6 fused to HALOtag, a reporter protein that can react with ligands carrying a variety of functionalities, including fluorescent labels, affinity handles, and attachment to solid phase, making this novel reporter conducive to immunofluorescence imaging, biochemical pulldown, and ChIPSeq. This system allows us to express wild type and mutant forms of ETV6 in appropriate allele ratios in imMKCL cells and various hematopoietic-relevant cell lines. Using this approach, we detected nuclear localization of wild-type ETV6 and altered cytoplasmic localization of both P214L and R418G ETV6 mutants. We have also demonstrated dimerization between both wild-type and mutant ETV6 in this cell model. Importantly, we have used HALOtag protein immunoprecipitation to demonstrate ETV6 binding to FLI1, another ETS family member and key transcriptional regulator of megakaryocyte development, suggesting that ETV6 and FLI1 cooperate to regulate megakaryopoiesis under homeostatic conditions. Altogether, these data suggest that mutant ETV6 functions as a dominant negative, sequestering wild type ETV6 in the cytoplasm, de-regulating key transcriptional targets for homeostatic megakaryocyte development. Ongoing studies will define the full repertoire of protein interactions and transcriptional targets of wild-type and mutant ETV6. Discoveries from this novel tool will further advance our understanding of normal megakaryocyte and platelet biology, and will provide potential therapeutic targets for disorders of platelet number and function to optimize the clinical approach to these patients. Disclosures Callaghan: Bayer: Consultancy, Speakers Bureau; Alnylum: Equity Ownership; Biomarin, Bioverativ, Grifols, Kedrion, Pfizer, Roche/Genentech, Shire, and Spark Therapeutics: Consultancy; Takeda: Consultancy, Research Funding; Sanofi: Consultancy; Global Blood Therapeutics: Consultancy; Novonordisk: Consultancy, Speakers Bureau; Octapharma: Consultancy; Pfizer: Research Funding; Roche: Research Funding; Shire/Takeda: Speakers Bureau; Roche/Genentech: Speakers Bureau.
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rodriguez-Hernandez, Guillermo, Julia Hauer, Alberto Martín-Lorenzo, Daniel Schaefer, Christoph Bartenhagen, Idoia Garcia-Ramirez, Franziska Auer, et al. "A New ETV6-RUNX1 In Vivo Model Produces a Phenocopy of the Human Pb-ALL." Blood 126, no. 23 (December 3, 2015): 3658. http://dx.doi.org/10.1182/blood.v126.23.3658.3658.
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Abstract Introduction The ETV6-RUNX1 fusion gene,the most common subtype of childhood pB-ALL, is acquired in utero, producing a persistent and hidden preleukemic clone. However, the underlying mechanism explaining how the preleukemic clone evolves to pB-ALL remains to be identified. The lack of genetically engineered human-like ETV6-RUNX1 pB-ALL models has hampered our understanding of the pathogenesis of this disease. Methods We have used a novel experimental approach to generate a murine strain that mimics the human ETV6-RUNX1 pB-ALL. We expressed ETV6-RUNX1 specifically in hematopoietic stem cells (HSC) of C57BL/6 x CBA mice by placing ETV6-RUNX1 under the control of the Sca1 promoter. Two founder mice were obtained for the Sca1-ETV6-RUNX1 transgene, which had normal gestation, were viable and developed normally. Sca1-ETV6-RUNX1 transgenic mice were characterized with respect to clinical, immunephenotypic and genetic characteristics. For the detection of shared secondary genomic alterations we analyzed three murine Sca1-ETV6-RUNX1 and 11 ETV6-RUNX1 positive human pB-ALL and corresponding germline by whole-exome (WES) and whole-genome sequencing using a HiSeq 2500 (Illumina) platform. Results In our transgenic murine model Sca1-ETV6-RUNX1 transgene expression was detected in HSCs, while there was no detectable expression in pro B cells or later stages of B-cell development, which mimics human ETV6-RUNX1 preleukemic biology. Sca1-ETV6-RUNX1 mice developed exclusively pB-ALL at a low penetrance (7.5%; 3 out of 40) with a CD19+ B220+ IgM- cell surface phenotype. Overall survival was not significantly reduced compared to wild-type mice (P value = 0.7901). pB-ALL in Sca1-ETV6-RUNX1 mice manifested with splenomegaly, disruption of splenic architecture, and appearance of blast cells in the peripheral blood (PB). All leukemic cells displayed clonal immature BCR rearrangement. Tumor pro B cells grew independent of IL-7 and were able to propagate the disease when transplanted into sub-lethally irradiated syngeneic recipient mice. Whole-exome sequencing of murine pB-ALL revealed in one mouse a deletion of three amino acids in the B-cell differentiation factor EBF1, which is well known in the context of human ETV6-RUNX1 leukemia. Additionally we found mutations in genesimplicated in histone modification, i.e. in KDM5C causing a premature translation stop. We compared the genomic alterations detected in the mouse model to published genomic data of pediatric ETV6 -RUNX1 pB-ALL and identified multiple copy number variations, which are shared between the murine and human ETV6 -RUNX1 pB-ALL. Among them were copy number gains and losses including i.e. the tumorsuppressor locus CDKN2A/B with a well-known role in human and mouse pB-ALL. A high proportion of genes implicated in histone modification was also mutated in published data of human ETV6-RUNX1 positive pB-ALL. We validated this novel finding of recurrent alterations of histone modifying genes in both the murine model and the human disease using an independent human ETV6-RUNX1 cohort of 11 patients. In this cohort were able to reproduce this finding. Similar to the murine model, we also detected a missense mutation in the methyltransferase KDM5C in one patient of our cohort of ETV6-RUNX1 positive patients. Conclusion In summary, we have characterized a new Sca1-ETV6-RUNX1 mouse model and this is, to our knowledge the first model, which represents a phenocopy of the human pB-ALL. Sca1-ETV6-RUNX1 mice develop exclusively pB-ALL at a very low penetrance as it is the case in human ETV6-RUNX1 positive pB-ALL. The acquisition of secondary mutations in pB-ALL with a high proportion in histone modifying genes confers the second hit for the conversion of a preleukemic clone into the clinically overt ETV6-RUNX1 positive pB-ALL disease. These findings are important for encouraging novel interventions that might help to prevent or treat ETV6-RUNX1 positive childhood leukemias. Disclosures No relevant conflicts of interest to declare.
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Junk, Stefanie V., Norman Klein, Sabine Schreek, Martin Zimmermann, Anja Möricke, Kirsten Bleckmann, Gunnar Cario, Martin Schrappe, Christian P. Kratz, and Martin Stanulla. "TP53, ETV6 and RUNX1 Germline Variants in Patients Developing Secondary Neoplasms after Treatment for Childhood Acute Lymphoblastic Leukemia." Blood 130, Suppl_1 (December 7, 2017): 884. http://dx.doi.org/10.1182/blood.v130.suppl_1.884.884.
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Abstract Introduction: Today most of the children treated for acute lymphoblastic leukemia (ALL) can be cured by the application of intensive combination chemotherapy regimens. However, up to 10% develop a secondary malignancy (SMN) after undergoing ALL treatment with cure rates being often dismal. Thus, strategies are needed for an early identification of patients at risk for SMN development, an improved understanding of the underlying pathobiology and, ideally, the development of preventive actions. Analyzing three candidates associated with predisposition to neoplastic diseases - TP53, ETV6 and RUNX1 - we aimed to determine the frequency of single nucleotide variants (SNV) in these genes in a cohort of SMN patients. Methods: Using targeted sequencing, we analyzed a cohort of childhood ALL patients with SMN after treatment on one of seven consecutive ALL-BFM protocols - ALL-BFM 79 to AIEOP-BFM ALL 2000.In the observation period from 1984 to 2008, 168 SMN patients were identified. The median follow-up for the entire patient cohort was 10.6 years as of April, 2013. With the exception of ALL-BFM 79, treatment was stratified into 3 branches, mainly according to the initial leukemic cell load, adverse genetic aberrations and treatment response. DNA isolated from remission bone marrow smears was analyzed employing two different multiplex PCR-based Ion AmpliSeq™ Panels (Life Technologies, Darmstadt, Germany) according to the manufacturer's instructions. The first panel, covering all coding exons of TP53, was employed in 49 patients with adequate material available for analysis. The second panel, interrogating the complete coding regions of ETV6 and RUNX1, was applied in 38 patients with sufficient material available after TP53 analysis. SPSS (IBM Deutschland GmbH, Ehningen, Germany) was used for computerized calculations. Results: In our TP53 analyses, only 1/49 (2%) genotyped SMN patients carried a heterozygous non-synonymous SNV within the coding region. This rare missense variant, p.Asn235Ser (rs144340710), was detected in a patient developing a small round cell sarcoma after treatment for ALL. The in silico scores from Polyphen and Sift algorithms indicated a benign effect for this change. Regarding ETV6, 3/38 (8%) patients carried a heterozygous non-synonymous SNV; two of them developed a hematologic and one patient a solid SMN. One frameshift insertion (p.Ile48Glyfs*2) and a missense variant (p.Leu79Pro) were observed in exon 2, part of the pointed domain. A further missense variant (p.Arg399Gly) was detected in exon 7 within the C-terminal DNA-binding ETS domain. Although ETV6 p.Arg399 is a hotspot mutation site for recurrent somatic mutations in malignancies, none of these variants was recorded in the databases dbSNP, the 1000Genomes Project, the NHLBI GO Exome Sequencing Project, Exome Aggregation Consortium or the Catalogue of Somatic Mutations in Cancer. For both missense variants in silico scores from Sift and Polyphen algorithms predicted a probably damaging effect. Molecular modeling of p.Arg399Gly suggests that the change might directly modulate the DNA binding qualities of the ETS domain. Furthermore, 2/38 patients (5%) had coding variants in RUNX1, both developed a hematologic SMN. One patient carried a nonsense insertion (Leu253Argfs*3). A second patient had a missense SNV (p.Leu56Ser, rs111527738) within the N-terminal RUNT domain, with a probably damaging effect (Polyphen). The described ETV6 and RUNX1 SNV were mutually exclusive. Compared to published data on childhood ALL patients (T. Moriyama et al. Lancet Oncol. 2015 and J. Zhang et al. N Engl J Med. 2015), we found approximately 10-fold and 4-fold higher frequencies of ETV6 and RUNX1 SNV, respectively, in patients developing a SMN after ALL treatment on ALL-BFM protocols. Conclusions: The low frequency of TP53 SNV in our case series suggests that alterations in this frequently mutated tumor suppressor gene may not play a major role in the pathobiology of SMN associated with the treatment of ALL. However, our findings in ETV6 and RUNX1 analyses suggest that genetic variation in these genes may be involved in the development of SMN after undergoing ALL treatment and warrant additional studies employing appropriate control groups for further clarification. Disclosures Schrappe: Novartis: Consultancy, Research Funding; Medac: Consultancy, Research Funding; SigmaTau: Consultancy, Research Funding; JAZZ Pharma: Consultancy, Research Funding; Baxalta: Consultancy, Research Funding.
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Ellinghaus, E., M. Stanulla, G. Richter, D. Ellinghaus, G. te Kronnie, G. Cario, G. Cazzaniga, et al. "Identification of germline susceptibility loci in ETV6-RUNX1-rearranged childhood acute lymphoblastic leukemia." Leukemia 26, no. 5 (November 11, 2011): 902–9. http://dx.doi.org/10.1038/leu.2011.302.
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Maierhofer, Anna, Constance Baer, Christian Pohlkamp, Manja Meggendorfer, Wolfgang Kern, Claudia Haferlach, and Torsten Haferlach. "Putative Germline Variants in the Predisposition Genes DDX41, ETV6 and GATA2 investigated in 1,228 Patients with Sporadic AML or MDS." Blood 136, Supplement 1 (November 5, 2020): 17–18. http://dx.doi.org/10.1182/blood-2020-140742.
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Background: In the latest revision of WHO classification (2017) the new category `myeloid neoplasms with germline predisposition´ was introduced. Pathogenic germline (GL) mutations predisposing to hereditary hematological malignancies (HHMs) have been described in various genes and have largely been identified in families, but may also occur sporadically (Godley, Blood Adv 2019; Rio-Machin et al., Nat Commun 2020). It is estimated that 4-9% of adults with myeloid malignancies have GL predisposition and it is discussed that HHMs could even be more common (Sung and Babushok, Blood 2020). Here, we performed a screening for putative GL variants in the predisposition genes DDX41, ETV6 and GATA2 in a large cohort of 1,228 patients with sporadic AML or MDS. Aim: To determine the frequency of genetic variants in the predisposition genes DDX41, ETV6 and GATA2 in patients with AML or MDS and to characterize the mutational patterns in patients carrying putative GL variants in these genes. Patients and Methods: Between 02/2019 and 01/2020 a total of 1,228 patients were diagnosed with AML or MDS by cytomorphological analysis (475 de novo AML, 647 MDS, 60 s-AML from MDS, 46 MDS/AML; M: 753, F: 475; median age: 74 [20-96]) and analyzed by next-generation sequencing for DDX41, ETV6, GATA2 and additional genes associated with AML or MDS (Figure 1). DNA was isolated from bone marrow or peripheral blood; sequencing was performed on NovaSeq after NextFlex library preparation (Illumina, ILMN, San Diego, CA) and hybrid capture according to manufacturer's protocol (IDT Inc. Coralville, IA). Data was analyzed with Pisces and Pindel (for FLT3-ITD) (available via BaseSpace, ILMN) using a minimum of 3% sensitivity. All changes except for synonymous mutations and known polymorphisms were declared as genetic variants. Results: We identified 73/1,228 patients (5.9%) with genetic variants in DDX41, 28 (2.3%) with variants in ETV6 and 50 (4.1%) with variants in GATA2. Aiming to estimate the proportion of patients carrying a putative GL variant, a variant allele frequency (VAF) higher than 0.3 was considered to be presumptive of a GL origin. We identified 64/1,228 patients (5.2%) with a putative GL variant in DDX41. 37/64 patients harbored DDX41 variants that were previously described as causal GL variants, like for example p.M1I (12/37) or p.D140fs (4/37), and consistent with other studies 43/64 (67.2%) had an additional DDX41 mutation with a lower VAF (<0.3) suggesting somatic acquisition of the second mutation (27/43: p.R525H) (Sébert et al., Blood 2019). 13/1,228 patients (1.1%) were found to have a putative GL variant in ETV6. Two of them carried a p.R353Q mutation which was recently presumed to be a rare GL variant contributing to myeloid malignancy susceptibility (Li et al., Leukemia 2020). 26/1,228 patients (2.1%) carried a putative GL variant in GATA2, including the already in the context of HHMs described mutation p.P41A (3/26) (Holme et al., Br J Haematol 2012). In total, 102/1,228 patients (8.3%) diagnosed with AML or MDS carried a putative GL variant in one of the predisposition genes DDX41, ETV6 or GATA2. Patients with presumed GL variants were about the same age as patients without (mean: 72 vs. 71 years). The mutational patterns for the 102 patients are illustrated in Figure 1. Genes that were most often additionally mutated were ASXL1, DNMT3A, SRSF2 and TET2. For ETV6 and GATA2, all but one of the patients carrying a putative GL variant harbored at least one genetic variant in an additional gene. In contrast, 16/64 patients (25.0%) carrying a presumed GL variant in DDX41 (and in 12 cases a second somatic DDX41 mutation) had no further genetic variants in other well-known AML- or MDS-related genes indicating that DDX41 and especially the gain of a second somatic mutation drives leukemogenesis, while for ETV6 and GATA2 additional hits are required. Conclusions: Our data support the hypothesis that GL mutations in predisposition genes could be more common in sporadic cases of AML or MDS than anticipated highlighting the importance of systematic testing for these variants, irrespective of family history or age. Identifying GL mutations can be important for clinical management, including donor choice for allogeneic stem cell transplantation. Routine workflows should be adapted to improve the identification of variants in predisposition-associated genes and to facilitate confirmation of GL origin, e.g. by analyzing reference material. Figure 1 Disclosures No relevant conflicts of interest to declare.
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Baskin, Rebekah, Rina Nishii, Javad Nadaf, Katherine Verbist, Paige Tedrick, Keito Hoshitsuki, Maoxiang Qian, et al. "Functional analysis of germline ETV6 variants associated with familial thrombocytopenia and acute lymphoblastic leukemia." Experimental Hematology 53 (September 2017): S126—S127. http://dx.doi.org/10.1016/j.exphem.2017.06.320.
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de Smith, Adam J., Geneviève Lavoie, Kyle M. Walsh, Sumeet Aujla, Erica Evans, Helen M. Hansen, Ivan Smirnov, et al. "Germline GAB2 Mutations in Childhood Acute Lymphoblastic Leukemia." Blood 132, Supplement 1 (November 29, 2018): 388. http://dx.doi.org/10.1182/blood-2018-99-119235.
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Abstract Recent studies using next-generation sequencing of selected individuals, such as those with familial leukemia or congenital syndromes, have identified rare and highly penetrant germline mutations that predispose to childhood acute lymphoblastic leukemia (ALL). High hyperdiploidy (HD), the most common cytogenetic subtype of childhood ALL, is enriched in children with RASopathies who develop ALL and, similarly, a high proportion of ALL patients with germline ETV6 or IKZF1 mutations presented with the HD subtype. Here, we aimed to identify novel predisposition genes in a selected group of HD-ALL patients. Targeted sequencing of 538 cancer-relevant genes was carried out using the UCSF500 Cancer Gene Panel in diagnostic bone marrow (i.e. tumor) DNA from 57 HD-ALL patients from the California Childhood Leukemia Study (CCLS). HD-ALL patients were selected based on absence of somatic KRAS or NRAS hotspot mutations detectable by Sanger sequencing, and absence of somatic copy number deletions from multiplex ligation-dependent probe amplification (MLPA) assays. After filtering out likely somatic mutations (mutant allele fraction <0.44), and restricting to variants with low frequency in unselected individuals (allele frequency <0.01% in the Exome Aggregation Consortium, ExAC) and with predicted functional effects (Combined Annotation Dependent Depletion, CADD score ≥20), we identified 151 putative predisposing mutations. Of 41 mutations of interest selected for validation, 37 (90.2%) were confirmed as germline in origin via Sanger sequencing of remission or newborn bloodspot DNA. Rare and predicted functional germline mutations in known (NBN, SH2B3, ETV6, CREBBP, MSH6) or suspected (MLL, ABL1, FLT3, MYH9) ALL predisposition genes were identified in nine out of 57 patients (15.8%). Three additional patients harbored germline mutations in the GRB2-associated binding protein 2 (GAB2), a known binding partner of PTPN11-encoded SHP2 and activator of the ERK/MAPK and PI3K/AKT pathways. Two GAB2 mutations, a missense mutation S592F and frameshift mutation P621fs, were predicted to be highly functional (CADD scores = 34 and 36 respectively) and absent in ExAC. Frequency of rare and damaging GAB2 mutations was significantly higher in our patient set (2.6%) than in ExAC (0.28%, P = 2.70 x 10-6). We replicated this finding in sequencing data from 309 ALL patients in the TARGET (Therapeutically Applicable Research to Generate Effective Treatments) project (0.81% vs. 0.28%, P = 0.015). Patient GAB2 mutations were cloned into HEK293 cells and, following EGF stimulation, we found that the P621fs mutation reduced SHP2 binding and ERK1/2 phosphorylation but increased AKT phosphorylation. This suggested possible Ras-independent leukemogenic effects, supported by a lack of somatic Ras pathway mutations in the three GAB2 mutant patients. Additional functional analyses and sequencing of larger patient cohorts will be required to elucidate the role of germline GAB2 mutations in childhood ALL. Disclosures No relevant conflicts of interest to declare.
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Okpanyi, Vera, Christoph Bartenhagen, Michael Gombert, Vera Binder, Hans-Ulrich Klein, Silja Roettgers, Jochen Harbott, Martin Dugas, and Arndt Borkhardt. "Global Identification of Genomic Structural Variants In Childhood ETV6/RUNX1 (TEL/AML1) Acute Lymphoblastic Leukemias by Mate-Pair Massively Parallel Sequencing." Blood 116, no. 21 (November 19, 2010): 3364. http://dx.doi.org/10.1182/blood.v116.21.3364.3364.
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Abstract Abstract 3364 Introduction Chimeric fusion genes generated by chromosomal translocations are highly prevalent in childhood acute lymphoblastic leukemia (ALL) and mainly represent early, prenatal events. The t(12;21)(p13;q22) translocation generates the ETV6/RUNX1 fusion gene and is the most frequent gene recombination in childhood B-lineage ALL, occurring in approximately 25% of the cases. It is associated with favorable prognosis even though a substantial proportion of the cases relapse. The fusion gene itself initiates the pre-leukemic process but additional genetic hits are needed to trigger a full blown leukemia. Being one of the best-characterized childhood leukemias, both nature and mechanisms of the events that cooperate with the chimeric protein are still poorly understood. Furthermore, studies addressing the genetic origin of relapse demonstrated a clonal relationship between relapse and diagnostic sample, assuming the existence of an ancestral, pre-leukemic clone. Objective Second-generation sequencing of both ends of huge numbers of DNA fragments allows comprehensive characterization of patterns of somatic rearrangements on an unprecedented, high-resolution level. By using the Illumina mate-pair massively parallel sequencing technology and intra-individual side-by-side comparison of leukemic and normal germline DNA, we aim to elucidate the cooperating genetic events in leukemogenesis in ETV6/RUNX1-ALLs as well as the clonal relationship between relapse and diagnostic sample. Methods We investigated diagnostic and relapse samples as well as non-leukemic germline material from one pediatric patient, diagnosed with ETV6/RUNX1-ALL in Germany. Mate-pair genomic sequencing libraries with an insert size of approximately 2-kb were constructed and paired-end sequence reads of 36-bp each were generated on the Illumina Genome Analyzer IIx from randomly created ~500-bp DNA fragments. Data were filtered and aligned to the human reference genome (GRCh37) using BWA. Reads considered PCR duplicates were removed and detection as well as clustering of structural variants (translocations, deletions, inversions) was subsequently carried out with GASV. Discordantly mapping read pairs defined potential structural variations and cluster sizes of at least 4 uniquely and correctly mapping read pairs were included in further analyses. In order to confirm breakpoints and resolve them to base-pair level, areas of putative chromosomal rearrangements were amplified from genomic DNA of tumor and matched normal sample and were conventionally sequenced. Results An average of ~73,000,000 read pairs were generated for each sample and after alignment, the whole genome was sequenced with a mean fragment coverage of 18.8X. A substantial variation in prevalence of structural variants could be detected between paired diagnostic and relapse samples. Within the diagnostic sample we could in total observe 739 deletions, 66 inversions and 107 translocations while the relapse sample exhibited 26 deletions, 14 inversions and 240 translocations. In a first analysis we focused on translocations, presuming the high impact of chromosomal rearrangements on leukemogenesis. Subtracting translocations being of germline origin, 73 translocations at diagnosis and 207 translocations in relapse could be detected, of which 183 (74%) were identified being intragenic. Remarkably, both samples shared only 16 translocations (6%), while 57 (22%) uniquely appear in the diagnostic sample and 191 (72%) could only be observed in the relapse sample. Intragenic, shared translocations in diagnostic and relapse samples include the t(12;24) PDE3A/RN18S1, the t(2;17) PID1/UNC45B as well as the t(1;6) HFM1/EYA4 fusion gene products. Detection of the known ETV6/RUNX1 translocation in diagnostic and relapse sample as well as confirmation of selected breakpoints via Sanger sequencing validated our methodological approach. Conclusion Mate-pair sequencing of leukemic samples in comparison to germline material provides a powerful tool to identify genome-wide chromosomal structural variations and will allow analysis of clonality between diagnostic and relapse samples. The low percentage of shared translocations gives a first hint probably objecting the thesis of a common pre-leukemic clone, but will only be elucidated by analysis of further patient samples. Disclosures: No relevant conflicts of interest to declare.
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Moriyama, Takaya, Monika L. Metzger, Gang Wu, Rina Nishii, Maoxiang Qian, Meenakshi Devidas, Wenjian Yang, et al. "Germline genetic variation in ETV6 and risk of childhood acute lymphoblastic leukaemia: a systematic genetic study." Lancet Oncology 16, no. 16 (December 2015): 1659–66. http://dx.doi.org/10.1016/s1470-2045(15)00369-1.
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Noetzli, Leila, Richard W. Lo, Alisa B. Lee-Sherick, Michael Callaghan, Patrizia Noris, Anna Savoia, Madhvi Rajpurkar, et al. "Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia." Nature Genetics 47, no. 5 (March 25, 2015): 535–38. http://dx.doi.org/10.1038/ng.3253.
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Duployez, Nicolas, Wadih Abou Chahla, Sophie Lejeune, Alice Marceau-Renaut, Guillaume Letizia, Thomas Boyer, Sandrine Geffroy, et al. "Detection of a new heterozygous germline ETV6 mutation in a case with hyperdiploid acute lymphoblastic leukemia." European Journal of Haematology 100, no. 1 (November 9, 2017): 104–7. http://dx.doi.org/10.1111/ejh.12981.
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Poggi, Marjorie, Matthias Canault, Marie Favier, Ernest Turro, Paul Saultier, Dorsaf Ghalloussi, Veronique Baccini, et al. "Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34 + progenitors." Haematologica 102, no. 2 (September 23, 2016): 282–94. http://dx.doi.org/10.3324/haematol.2016.147694.
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Varghese, Anna M., Isha Singh, Rituraj Singh, Siddharth Kunte, Joanne F. Chou, Marinela Capanu, Winston Wong, et al. "Early-Onset Pancreas Cancer: Clinical Descriptors, Genomics, and Outcomes." JNCI: Journal of the National Cancer Institute 113, no. 9 (March 23, 2021): 1194–202. http://dx.doi.org/10.1093/jnci/djab038.
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Abstract Background Recent evidence suggests a rising incidence of cancer in younger individuals. Herein, we report the epidemiologic, pathologic, and molecular characteristics of a patient cohort with early-onset pancreas cancer (EOPC). Methods Institutional databases were queried for demographics, treatment history, genomic results, and outcomes. Overall survival from date of diagnosis was estimated using Kaplan-Meier method. Results Between 2008 and 2018, 450 patients with EOPC were identified at Memorial Sloan Kettering. Median overall survival was 16.3 (95% confidence interval [CI] = 14.6 to 17.7) months in the entire cohort and 11.3 (95% CI = 10.2 to 12.2) months for patients with stage IV disease at diagnosis. Of the patients, 132 (29.3% of the cohort) underwent somatic testing; 21 of 132 (15.9%) had RAS wild-type cancers with identification of several actionable alterations, including ETV6-NTRK3, TPR-NTRK1, SCLA5-NRG1, and ATP1B1-NRG1 fusions, IDH1 R132C mutation, and mismatch repair deficiency. A total of 138 patients (30.7% of the cohort) underwent germline testing; 44 of 138 (31.9%) had a pathogenic germline variant (PGV), and 27.5% harbored alterations in cancer susceptibility genes. Of patients seen between 2015 and 2018, 30 of 193 (15.5%) had a PGV. Among 138 who underwent germline testing, those with a PGV had a reduced all-cause mortality compared with patients without a PGV controlling for stage and year of diagnosis (hazard ratio = 0.42, 95% CI = 0.26 to 0.69). Conclusions PGVs are present in a substantial minority of patients with EOPC. Actionable somatic alterations were identified frequently in EOPC, enriched in the RAS wild-type subgroup. These observations underpin the recent guidelines for universal germline testing and somatic profiling in pancreatic ductal adenocarcinoma.
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Baskin-Doerfler, Rebekah, Mackenzie Bloom, Ninad Oak, Katherine Verbist, Paige Tedrick, Rachel Bassett, Rina Nishii, et al. "ASSESSING THE IMPACT OF A B-ALL-ASSOCIATED GERMLINE ETV6 VARIANT ON MURINE HEMATOPOIESIS AND STEM CELL FUNCTION." Experimental Hematology 76 (August 2019): S58. http://dx.doi.org/10.1016/j.exphem.2019.06.328.
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Kansal, Rina. "Germline Predisposition to Myeloid Neoplasms in Inherited Bone Marrow Failure Syndromes, Inherited Thrombocytopenias, Myelodysplastic Syndromes and Acute Myeloid Leukemia: Diagnosis and Progression to Malignancy." Journal of Hematology Research 8 (June 2, 2021): 11–38. http://dx.doi.org/10.12974/2312-5411.2021.08.3.
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The diagnosis of any genetic predisposition to any malignancy carries profound significance for the patient and the family, with implications for clinical management that differ from when there is no identifiable heritable cause. The presence of a genetic predisposition to develop hematologic neoplasms is under-recognized. Therefore, such genetic predisposition was added as a separate diagnosis in the diagnostic World Health Organization classification in 2016. Such genetic predisposition may occur in the absence of syndromic or physical signs; even a familial history may be absent in some individuals. Also, currently, surveillance guidelines for individuals who may harbor such a genetic predisposition but have not developed a malignancy are mostly limited to expert opinion. The application of genomic sequencing methods in clinical laboratories has allowed increased recognition of such germline predisposition. Very recently, evidence is beginning to emerge that sheds light on possible steps for progression to a myelodysplastic syndrome or acute myeloid leukemia. This article provides an overview of the clinical aspects of the inherited forms of bone marrow failure syndromes, myelodysplastic syndromes, and acute myeloid leukemia, including for germline mutated CEBPA, RUNXI, ANKRD26, ETV6, DDX41, GATA2, and SAMD9/9L genes. Considerations for diagnosis are discussed for individuals and families who harbor a genetic or familial predisposition to developing a myeloid malignancy with future perspectives.
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Trevino, Lisa R., Wenjian Yang, Stephen Hunger, William L. Carroll, Meenakshi Devidas, Cheryl L. Willman, James Downing, William E. Evans, Ching-Hon Pui, and Mary V. Relling. "Children with t(12;21)/TEL-AML1-Positive Acute Lymphoblastic Leukemia Exhibit a Distinct Germline Genomic Signature." Blood 110, no. 11 (November 16, 2007): 760. http://dx.doi.org/10.1182/blood.v110.11.760.760.
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Abstract High-throughput genome-wide studies have identified significant associations between inherited genetic variations and complex diseases. To delineate the heterogeneous nature of pediatric acute lymphoblastic leukemia (ALL), we performed a genome-wide interrogation of approximately 600K single nucleotide polymorphisms (SNPs) in 470 Caucasian children (using > 99% European ancestry genetic markers to define race) with newly diagnosed ALL to identify the contribution of germline SNP genotypes to ALL subtype. Our patient population included children enrolled on St Jude Children’s Research Hospital Total XIIIB and Total XV ALL protocols and the COG9906 ALL protocol. The study cohort included 47 cases with the t(12;21)/TEL-AML1 (aka ETV6/RUNX1) translocation, 116 with hyperdiploidy > 50 chromosomes, 46 with T-lineage ALL, and 139 with non-hyperdiploid B-lineage ALL (without known translocations). Using genotype data from the Affymetrix 100K and 500K Mapping Array sets and logistic regression analyses, we found that only patients with TEL-AML1-positive ALL demonstrated a distinct germline SNP signature (p = 0.043, 1000 permutations) compared to patients with other ALL subtypes. We identified 1,046 germline SNPs whose allele frequencies discriminated TEL-AML1 ALL from non-TEL-AML1 ALL (p < 0.005). For example, the odds ratio associated with the risk of TEL-AML1 vs. other ALL subtypes for the T vs. C allele at the ITPR2 SNP (rs12814812) was 3.5 (p=0.000019), and that for the G vs. A allele at the IL26 SNP (rs11570906) was 3.1 (p = 0.000111). These 1,046 SNPs represent 224 unique genes. Among these 1,046 SNPs, 28 cis SNPs were significantly associated with the mRNA expression of 19 genes in diagnostic leukemic lymphoblasts. Of these 19 genes, the gene expression of the growth factor, ANGPT2;, coagulation factor, F13A1; and the enzyme, AGA, differed significantly between TEL-AML1-positive and negative cases. We also identified one intronic SNP in each of the translocation target genes (RUNX1 and ETV6) whose genotype frequencies differed between TEL-AML1-positive and negative cases (p = 0.01 and 0.03 respectively). Conversely, SNPs in high linkage disequilibrium with previously hypothesized risk alleles in MTHFR and NQO1 did not differ in allele frequencies between the two subgroups (p > 0.05). It is well recognized that TEL-AML1-positive ALL represents a fourth of childhood cases but is exceedingly rare in adults, and hence, it is not surprising that the TEL-AML1-positive ALL subtype may be more likely to be affected by inherited genomic variability compared to other subtypes. We conclude that inherited genetic variation may contribute to risk of or pathogenesis of the TEL-AML1 subtype of childhood ALL.
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Király, Péter Attila, Krisztián Kállay, Dóra Marosvári, Gábor Benyó, Anita Szőke, Judit Csomor, and Csaba Bödör. "Familiáris myelodysplasiás szindróma és akut myeloid leukaemia klinikai és genetikai háttere." Orvosi Hetilap 157, no. 8 (February 2016): 283–89. http://dx.doi.org/10.1556/650.2016.30375.
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Myelodysplastic syndrome and acute myeloid leukaemia are mainly sporadic diseases, however, rare familial cases exist. These disorders are considered rare, but are likely to be more common than currently appreciated, and are characterized by the autosomal dominant mutations of hematopoietic transcription factors. These syndromes have typical phenotypic features and are associated with an increased risk for developing overt malignancy. Currently, four recognized syndromes could be separated: familial acute myeloid leukemia with mutated CEBPA, familial myelodysplastic syndrome/acute myeloid leukemia with mutated GATA2, familial platelet disorder with propensity to myeloid malignancy with RUNX1 mutations, and telomere biology disorders due to mutations of TERC or TERT. Furthermore, there are new, emerging syndromes associated with germline mutations in novel genes including ANKRD26, ETV6, SRP72 or DDX41. This review will discuss the current understanding of the genetic basis and clinical presentation of familial leukemia and myelodysplasia. Orv. Hetil., 2016, 157(8), 283–289.
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Yoshino, Hiroshi, Yohei Nishiyama, Hiroshi Kamma, Tomohiro Chiba, Masachika Fujiwara, Takehiro Karaho, Yasunao Kogashiwa, et al. "Functional characterization of a germline ETV6 variant associated with inherited thrombocytopenia, acute lymphoblastic leukemia, and salivary gland carcinoma in childhood." International Journal of Hematology 112, no. 2 (May 4, 2020): 217–22. http://dx.doi.org/10.1007/s12185-020-02885-y.
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Medford, Arielle J., Lauren Oshry, Baris Boyraz, Lesli Kiedrowski, Sofia Menshikova, Anna Butusova, Charles S. Dai, et al. "TRK inhibitor in a patient with metastatic triple-negative breast cancer and NTRK fusions identified via cell-free DNA analysis." Therapeutic Advances in Medical Oncology 15 (January 2023): 175883592311528. http://dx.doi.org/10.1177/17588359231152844.
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Tissue-agnostic indications for targeted therapies have expanded options for patients with advanced solid tumors. The Food and Drug Administration approvals of the programmed death-ligand 1 inhibitor pembrolizumab and the TRK inhibitors larotrectinib and entrectinib provide rationale for next-generation sequencing (NGS) in effectively all advanced solid tumor patients given potential for clinical responses even in otherwise refractory disease. As proof of concept, this case report describes a 64-year-old woman with triple-negative breast cancer refractory to multiple lines of therapy, found to have a rare mutation on NGS which led to targeted therapy with meaningful response. She initially presented with metastatic recurrence 5 years after treatment for a localized breast cancer, with rapid progression through four lines of therapy in the metastatic setting, including immunotherapy, antibody–drug conjugate-based therapy, and chemotherapy. Germline genetic testing was normal. Ultimately, NGS evaluation of cell-free DNA via an 83-gene assay (Guardant Health, Inc.) identified two NTRK3 fusions: an ETV6-NTRK3 fusion associated with the rare secretory breast carcinoma, and CRTC3-NTRK3, a novel fusion partner not previously described in breast cancer. Liver biopsy was sent for whole exome sequencing and RNA-seq analysis of tissue (BostonGene, Inc., Boston, MA, USA), which provided orthogonal confirmation of both the ETV6-NTRK3 and CRTC3-NTRK3 fusions. She was started on the TRK inhibitor larotrectinib with a marked clinical and radiographic response after only 2 months of therapy. The patient granted verbal consent to share her clinical story, images, and data in this case report. This case demonstrates the significant potential benefits of NGS testing in advanced cancer and the lessons we may learn from individual patient experiences.
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Mullighan, Charles G., Xiaoping Su, Jing Ma, Wenjian Yang, Mary V. Relling, William L. Carroll, Gregory Reaman, et al. "Genome-Wide Profiling of High-Risk Pediatric Acute Lymphoblastic Leukemia (ALL): The ALL Pilot Project for the Therapeutically Applicable Research To Generate Effective Treatments (TARGET) Initiative." Blood 110, no. 11 (November 16, 2007): 229. http://dx.doi.org/10.1182/blood.v110.11.229.229.
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Abstract Recent studies have identified a high frequency of recurrent acquired DNA copy number abnormalities in pediatric ALL, most commonly involving genes regulating lymphoid development (Nature2007;446:758). The majority of B-progenitor ALL cases in this study harbored recurring chromosomal abnormalities such as hyperdiploidy and recurring translocations, many of which are associated with favorable outcome. Data from large cohorts of poor risk B-ALL have not been reported. Here we report genomic profiling data from a cohort of 221 pediatric ALL cases treated on the Children’s Oncology Group P9906 study from 2000–2003 with an augmented BFM regimen (N Engl J Med1998;338:1663). The study targeted poor prognosis ALL cases, using age/WBC criteria designed to identify a subset of patients with NCI high risk ALL that historically had a very poor outcome. Cases with favorable (trisomy 4 + 10; ETV6-RUNX1) or unfavorable (BCR-ABL1 or hypodiploid) genetic features were excluded. The cohort included 25 TCF3-PBX1 and 19 MLL-rearranged cases. Profiling of DNA copy number abnormalities was performed using Affymetrix 250k Sty and Nsp arrays, reference normalization, dChipSNP, and circular binary segmentation. Germline SNP array data was available for 210 cases. The most frequent abnormalities were deletions of CDKN2A in 101 cases (45.7%), the B-lineage transcription factors PAX5 (N=68, 30.5%) and IKZF1 (Ikaros, N=32, 14.5%), ETV6 (N=29, 13.1%), RB1 (N=25, 11.3%), BTG1 (N=23, 10.4%), the tumor suppressor candidate TSC22D1 (N=20, 9%), the microRNA cluster at 13q14 (N=19, 8.6%), and deletion (N=3) or amplification (N=20, 4 focal, 16 broad) of the retinoic acid pathway gene CCDC26. Other recurring copy number abnormalities with relevance to leukemogenesis included deletions of BTLA, EBF, IL3RA, ERG, TOX, RAG1/2, KRAS, NRAS, NR3C2 and the adenosine deaminase pathway genes ADAR and ADARB2. An unexpected finding was focal deletion involving DMD (dystrophin) at Xp21.1 in 15 (6.8%) cases. Copy number abnormalities were uncommon in MLL rearranged leukemias, suggesting that fewer secondary mutations are needed for leukemogenesis in this genetic subtype of ALL. Clustering of copy number data using non-negative matrix factorization identified 11 clusters of cases, including clusters driven by isochromosome 7q, deletions of 9p, gains of 1q, multiple whole chromosomal gains, and intrachromosomal amplification of chromosome 21 (iamp21). These findings confirm the high frequency of deletions involving genes regulating B cell development and cell cycle such as PAX5 and CDKN2A in both standard and poor risk B-ALL. Furthermore ETV6 (TEL) deletions are shown to be common in B-ALL cases lacking the ETV6-RUNX1 translocation. The identification of novel recurring abnormalities (IL3RA, KRAS, NRAS) emphasizes the importance of high resolution copy number analysis in leukemia. These data are being integrated with gene expression data to select genes for resequencing as part of the TARGET project.
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Braunstein, Evan M., Ruijuan Li, Nara Sobreira, Christopher Gocke, Robert A. Brodsky, David Valle, and Linzhao Cheng. "A Germline Mutation in ERBB3 Predisposes to Inherited Erythroid Myelodysplasia/Erythroleukemia." Blood 126, no. 23 (December 3, 2015): 4105. http://dx.doi.org/10.1182/blood.v126.23.4105.4105.
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Abstract Background: Myelodisplastic syndrome (MDS) and acute myeloid leukemia (AML) are usually sporadic diseases. However, rare familial cases have helped to identify human disease genes and provided insight into hematopoiesis and leukemogenesis. Further, the ability to identify familial MDS/AML disease genes has improved due to advances in next generation sequencing technology. In addition to RUNX1, GATA2, CEBPA, germline mutations in both ETV6 and DDX41 were recently shown to predispose to myeloid hematologic malignancies. Methods: We investigated a large family with an inherited MDS/AML marked by erythroid hyperplasia in all affected individuals. A family pedigree was consistent with an autosomal dominant mode of inheritance with high penetrance. Whole exome sequencing was performed on germline DNA of the proband and multiple unaffected family members in order to identify the inherited genetic mutation predisposing to disease. Candidate single nucleotide variants (SNVs) were obtained after excluding all known SNVs with a variant allele frequency greater than 0.01, then prioritized and screened using DNA from a second affected individual. Results: An Ala1337Thr missense variant in the ERBB3 gene, a member of the EGFR tyrosine kinase receptor family, was identified as the potential pathologic germline mutation in this family. Further sequencing identified the presence of this variant in two additional affected individuals and its absence in eight unaffected family members, indicating that it co-segregates with disease. Functional validation of this ERBB3 variant was performed using an erythroleukemia cell line. Overexpression of the ERBB3 A1337T mutant in TF1 cells conveyed a block in erythroid differentiation compared to cells expressing unmutated ERBB3, measured by the lack of upregulation of Glycophorin A expression in the presence of erythropoietin. This activity was dependent upon the presence of Neuregulin-1β, the ERBB3 receptor ligand, and expression of the co-receptor ERBB2. In addition, ERBB3 mutated cells displayed a 1.3-fold growth advantage over unmutated ERBB3 in the presence of the Neuregulin-1β. Conclusions: This work has identified a novel mutation that predisposes to MDS/AML and adds ERBB3 to the growing list of disease predisposition genes in hematologic malignancies. It will allow for the early diagnosis of individuals with inherited erythroid MDS/AML prior to the onset of disease. In addition, it allows for the characterization of a gene with a likely crucial role in erythropoiesis as well as hematologic disease. Figure 1. Figure 1. Disclosures Brodsky: Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.
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Bloom, Mackenzie, Rebekah Baskin-Doerfler, Ninad Oak, Patrick Ozark, Katherine Verbist, Alexa Stroh, Chunliang Li, Rina Nishii, Jun Yang, and Kim Nichols. "3049 – THROMBOCYTOPENIA 5-ASSOCIATED GERMLINE ETV6 VARIANT IMPAIRS THE HEMATOPOIETIC STEM AND PROGENITOR CELL STRESS RESPONSE AND INDUCES AN INFLAMMATORY GENE SIGNATURE." Experimental Hematology 100 (August 2021): S66. http://dx.doi.org/10.1016/j.exphem.2021.12.268.
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Junk, Stefanie V., Norman Klein, Sabine Schreek, Martin Zimmermann, Anja Möricke, Kirsten Bleckmann, Julia Alten, et al. "TP53, ETV6 and RUNX1 germline variants in a case series of patients developing secondary neoplasms after treatment for childhood acute lymphoblastic leukemia." Haematologica 104, no. 9 (July 9, 2019): e402-e405. http://dx.doi.org/10.3324/haematol.2018.205849.
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Kwok, Brian, Renee Mohrmann, Kim Janatpour, Yin Xu, Matthew McGinniss, Aine Yung, Hong Drum, et al. "Next-Generation Sequencing Of ASXL1, TP53, RUNX1, EZH2, and ETV6 Identifies a Significant Proportion Of Lower-Risk Myelodysplastic Syndromes With Poor Prognostic Indicators." Blood 122, no. 21 (November 15, 2013): 1552. http://dx.doi.org/10.1182/blood.v122.21.1552.1552.
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Abstract Introduction Management of patients with myelodysplastic syndrome (MDS) is dependent in part on risk stratification by the International Prognostic Scoring System (IPSS). Patients with lower-risk disease as defined by low and intermediate-1 IPSS are predicted to have a relatively indolent clinical course and are usually offered less aggressive therapies, including transfusions, growth factors, or observation without treatment. In contrast, patients with higher-risk disease as defined by intermediate-2 and high IPSS are predicted to have a relatively aggressive clinical course and are usually treated with disease-modifying hypomethylating agents or considered for stem cell transplantation. However, despite IPSS risk stratification a subset of patients with lower-risk MDS will have more aggressive disease with shorter overall survival and increased risk of transformation to acute myeloid leukemia. Somatic mutations are common in MDS, but are not currently included in either the IPSS or revised IPSS (IPSS-R). Recent literature (Bejar et al. N Engl J Med 2011;364:2496-2506) revealed that mutations in ASXL1, TP53, RUNX1, EZH2, and ETV6 are indicators of poor overall survival in patients with MDS independent of IPSS and other established risk factors, and that mutations in one or more of these 5 genes essentially up-scores the IPSS to the next highest risk group. The aim of this study is to evaluate the frequency of mutations in these 5 genes using a clinically validated and sensitive next-generation sequencing (NGS) assay, and to determine the percentage of lower-risk MDS patients with a worse than expected prognosis who may potentially benefit from earlier treatment with disease-modifying therapy. Methods DNA sequences of key exons in ASXL1, TP53, RUNX1, EZH2, and ETV6 were determined from the bone marrow aspirates of 200 MDS patients (29% low, 52% intermediate-1, 18% intermediate-2, and 1% high IPSS) using a clinically validated and sensitive (5% limit of detection) NGS assay on the Illumina MiSeq platform. Mutations were compared between duplicate samples and annotated using software that queried databases containing known somatic mutations and germline variants. Results Somatic mutations in one or more of the 5 genes were detected in 50.5% of MDS patients in this study. Mutations in ASXL1 were the most frequent (29.5%), followed by mutations in TP53 (15.5%), RUNX1 (9.5%), EZH2 (7%), and ETV6 (7%). While mutant allele percentage varied among the genes, 64% of MDS patients with ETV6 mutations had <20% mutant allele, followed by 37% for ASXL1, 29% for TP53, 21% for RUNX1, and 7% for EZH2. Mutations were detected in 45% (46/102) of MDS patients with normal cytogenetics and 60% (37/92) with abnormal cytogenetics. When stratifying by IPSS, 46% of MDS patients with lower-risk disease had a mutation, compared with 72% with higher-risk disease. When stratifying by IPSS-R, 41% of MDS patients with lower-risk disease (very low and low) had a mutation, compared with 77.5% with higher-risk disease (high and very high). Conclusions Somatic mutations in ASXL1, TP53, RUNX1, EZH2, and ETV6 have previously been shown to be indicators of poor overall survival in patients with MDS independent of IPSS and other established risk factors. Using a clinically validated and sensitive NGS assay, 50.5% of MDS patients in this study were shown to have a mutation in one or more of the 5 genes. This percentage is higher than previously reported by Bejar et al (31.2%), and is likely explained by the higher sensitivity of NGS (5% limit of detection) as compared to Sanger sequencing (typically 20% limit of detection), which was utilized in the aforementioned study for ASXL1, EZH2, and ETV6. Mutations in one or more of the 5 genes were detected in 46% of MDS patients with lower-risk disease defined by IPSS, and a similar 41% with lower-risk disease defined by IPSS-R. Mutational profiling using NGS can thus help identify a significant proportion of lower-risk MDS patients with a worse than expected prognosis who may potentially benefit from earlier treatment with disease-modifying therapy. Disclosures: No relevant conflicts of interest to declare.
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Voshtina, Ensi, Arun K. Singavi, Amanda Jacquart, Lyndsey Runaas, Ehab L. Atallah, Laura C. Michaelis, and Sameem Abedin. "Systematic Screening for Familial Leukemia Based on Somatic Genomic Profiling Results." Blood 132, Supplement 1 (November 29, 2018): 2253. http://dx.doi.org/10.1182/blood-2018-99-117020.
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Abstract The incidence of familial acute leukemias (AL) and myelodysplastic syndrome (MDS) in the adult population is not well characterized, though recent estimates report that up to 4% of newly diagnosed individuals have a familial syndrome. Recognizing these syndromes is critical to proper clinical management of patients with an inherited susceptibility, and for genetic screening of family members. Within our tertiary care academic institution, less than 1% of AL/MDS cases are referred to genetic counseling, presenting an opportunity for improvement in practice. With the integration of next generation sequencing into standard clinical practice, we recently initiated a bi-monthly meeting to review these sequencing results, with the intent to detect possible familial AL/MDS syndromes and increase appropriate genetic counseling referrals. Here, we describe the potential value of this approach, through a retrospective analysis of somatic genomic profiling results in AL/MDS patients. We performed a retrospective, single-center analysis of all patients who underwent somatic genomic profiling with FoundationOne Heme for AL and MDS between May 2015 and July 2018. Genomic alterations implicated in familial leukemias or familial cancers and included in the FoundationOne Heme panel were as follows: RUNX1, CEBPA, ETV6, GATA2, TERC/TERT, PAX5, CHEK2, and TP53. We recorded baseline characteristics including age, sex, and diagnosis. The presence of the suspected germline variant and up 6 other genomic alterations were recorded. We described whether a comprehensive family history, defined as whether a family history of bleeding tendency, low blood counts, or cancers, was documented for all patients. All patients with a positive family history had the malignancies and blood disorders reported. Finally, we observed if a genetic counseling referral was placed. A total of 108 patients underwent genomic profiling during the study period. Pathogenic variants implicated in familial AL/MDS or familial cancers were detected in 41 of those patients. The number of patients under the age of 50 was 7. Twenty-nine patients had a diagnosis of AML and 12 patients had MDS. Of the reported relevant pathogenic variants, TP53 was seen in 20 patients, RUNX1 in 14 patients, CEBPA in 4 patients, ETV6 in 4 patients, and GATA2 in 3 patients. There were 5 patients that had 2 pathogenic variants noted on their genetic testing. Among the patients with positive pathogenic variant, 22/41 had a comprehensive family history performed. Family history was positive for malignancy in 26/41 patients. Of those 26, 9 patients had a first degree relative with a history of hematologic malignancy including leukemia. Only 2 patients overall were referred to genetic counseling. In AL/MDS patients who underwent somatic genomic profiling at our institution, nearly half of patients with suspected germline variants for familial AML-MDS syndromes had either a family history of malignancy or development of their malignancy at an earlier age, warranting genetic counseling referral. There also is room to improve comprehensive family history collection. Beginning in March 2018, we initiated a bi-monthly meeting to review somatic genomic profiling results in AL/MDS patients with a licensed geneticist. If a suspected germline variant is discovered, we now issue a statement to the primary oncologist to clarify family history if needed, and recommend a referral for formal genetic counseling in the presence of a suggestive family history or on the basis of age. In future investigations, we plan to study how this changes the rate of genetic counseling referrals, and whether this results in an increase in the detection of familial AL/MDS or familial cancer syndromes among this patient group. Disclosures Atallah: Abbvie: Consultancy; Pfizer: Consultancy; BMS: Consultancy; Jazz: Consultancy; Novartis: Consultancy.
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Trottier, Amy M., Ira L. Kraft, Lawrence J. Druhan, Amanda Lance, Belinda R. Avalos, and Lucy A. Godley. "New Germline Syndrome Discovery: Heterozygous CSF3R Mutations May Predispose to Myeloid and Lymphoid Malignancies." Blood 134, Supplement_1 (November 13, 2019): 2543. http://dx.doi.org/10.1182/blood-2019-129492.
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Introduction Colony stimulating factor 3 receptor (CSF3R) plays an important role in granulocyte proliferation and differentiation. Acquired mutations in CSF3R are found in the majority of cases of chronic neutrophilic leukemia and in a minority of atypical chronic myeloid leukemia and acute myeloid leukemia patients, whereas inherited biallelic mutations have been identified as a cause of severe congenital neutropenia (SCN). CSF3R variants have also been detected in lymphoid cells in patients with SCN. However, whether germline monoallelic CSF3R variants predispose to myeloid and/or lymphoid malignancies is unknown. Methods We analyzed variants detected by a clinical next generation sequencing (NGS) panel of about 150 genes important in oncogenesis (OncoPlus) performed on peripheral blood and/or bone marrow of patients with hematopoietic malignancies at The University of Chicago Medical Center from June 2017 to February 2019. We prioritized genes for which variants were likely of germline origin based on variant allele fraction (VAF) > 0.4 and persistence over multiple tests. Variant germline status was determined by extracting DNA from cultured bone marrow-derived mesenchymal stromal cells and/or from cultured skin fibroblasts. Variants in CSF3R [transcript NM_000760.3] were ranked by predicted pathogenicity using standard American College of Medical Genetics criteria. Variants of uncertain significance (VUS) were evaluated using population frequency data, in silico functional prediction, and REVEL scores. Results A total of 620 OncoPlus tests were conducted on 496 patients (ages 1 month - 96 years; median 64 years), and 824 unique variants were identified within 33 genes. Among these patients, 89 (17.9%) had an invariant variant, one that was found at similar VAFs across multiple testing time points. These invariant variants involved 31 different genes with a total of 203 unique variants. Variants with a VAF less than 0.4 were excluded from further study, leaving 81 patients with 164 invariant variants among 30 genes (Figure 1). The genes with the most invariant variants included known germline predisposition genes, such as ATM, BRCA2, DDX41, ETV6, and TP53, as well as CSF3R, which was not previously recognized as an autosomal dominant germline cancer susceptibility gene. Among the 81 patients, 8 (9.9%) were found to have invariant variants in CSF3R, with 7 unique variants identified. Including patients for whom only a single OncoPlus test was conducted in addition to those with invariant variants, there were a total of 43 patients with 20 unique variants in CSF3R among the entire 496 patient cohort (Figure 2). Of these 20 CSF3R variants, 8 (40%) were found to be germline, and among those, one was classified as likely pathogenic, one was classified as a VUS, and six were classified as likely benign. We focused our attention on the likely pathogenic and VUS germline variants. These two variants were among those with the highest predicted pathogenicity scores. The likely pathogenic variant, W547*, is a nonsense mutation in the extracellular domain of CSF3R that was identified in a patient with a history of bladder cancer treated with surgery and chemotherapy who subsequently developed therapy-related myelodysplastic syndrome. W547* has previously been found to be pathogenic in a compound heterozygous state in an infant with SCN. The germline VUS, P784T, is a missense variant identified in a patient with multiple myeloma. This variant localizes to the cytoplasmic domain of CSF3R and is not listed in the COSMIC or gnomAD databases. Functional studies are underway to assess the oncogenic potential of the W547* and P784T variants. Conclusion Detection of invariant variants on clinical, tumor-based NGS panels can be used as a way to identify potential germline mutations to aid in new germline syndrome discovery. Using this approach, CSF3R was identified as a candidate gene for which monoallelic pathogenic variants may predispose to both myeloid and lymphoid malignancies. Future work is ongoing to assess the functional consequences of germline CSF3R variants and the frequency of such mutations. Disclosures Avalos: Best Practice-Br Med J: Patents & Royalties: receives royalties from a coauthored article on evaluation of neutropenia; Juno: Membership on an entity's Board of Directors or advisory committees. Godley:Invitae, Inc.: Membership on an entity's Board of Directors or advisory committees; UpToDate, Inc.: Patents & Royalties: receives royalties from a coauthored article on inherited hematopoietic malignancies .
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Qian, Maoxiang, Heng Xu, Virginia Perez-Andreu, Kathryn G. Roberts, Hui Zhang, Wenjian Yang, Shouyue Zhang, et al. "Novel susceptibility variants at the ERG locus for childhood acute lymphoblastic leukemia in Hispanics." Blood 133, no. 7 (February 14, 2019): 724–29. http://dx.doi.org/10.1182/blood-2018-07-862946.
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Abstract Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Characterized by high levels of Native American ancestry, Hispanics are disproportionally affected by this cancer with high incidence and inferior survival. However, the genetic basis for this disparity remains poorly understood because of a paucity of genome-wide investigation of ALL in Hispanics. Performing a genome-wide association study (GWAS) in 940 Hispanic children with ALL and 681 ancestry-matched non-ALL controls, we identified a novel susceptibility locus in the ERG gene (rs2836365; P = 3.76 × 10−8; odds ratio [OR] = 1.56), with independent validation (P = .01; OR = 1.43). Imputation analyses pointed to a single causal variant driving the association signal at this locus overlapping with putative regulatory DNA elements. The effect size of the ERG risk variant rose with increasing Native American genetic ancestry. The ERG risk genotype was underrepresented in ALL with the ETV6-RUNX1 fusion (P < .0005) but enriched in the TCF3-PBX1 subtype (P < .05). Interestingly, ALL cases with germline ERG risk alleles were significantly less likely to have somatic ERG deletion (P < .05). Our results provide novel insights into genetic predisposition to ALL and its contribution to racial disparity in this cancer.
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Wu, Hsin-Ta, Ekaterina Kalashnikova, Samay Mehta, Raheleh Salari, Himanshu Sethi, Bernhard Zimmermann, Paul R. Billings, and Alexey Aleshin. "Characterization of clonal hematopoiesis of indeterminate potential mutations from germline whole exome sequencing data." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): 1525. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.1525.
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1525 Background: Clonal hematopoiesis of Indeterminate Potential (CHIP) is an age-related phenomenon where somatic mutations accumulate in cells of the blood or bone marrow. It is a source of biological noise that causes false-positives in ctDNA analysis and is present in up to 20% of individuals over the age of 70. The presence of CHIP has been linked to an increased risk of hematologic cancers and cardiovascular disease. The Signatera assay filters CHIP mutations through tumor tissue and germline sequencing thereby reducing false-positive results and focuses on tumor-specific mutations for each patient. Methods: Whole exome sequencing data (average depth ~250x) analyzed from patients’ buffy coat (n = 159) was used to characterize CHIP mutations. Variant calling was performed using Freebayes variant caller with allele frequency threshold between 1% and 10%. Following which variant annotation and selection was performed based on the top 54 genes that are most implicated in myeloid disorders. The selected variants were further screened based on the reported variants in the literature and/or the Catalog of Somatic Mutations in Cancer (COSMIC). Results: The analysis revealed an average of 0.14 (0-2) CHIP mutations per patient with an average variant allele frequency of 3.49% (1%-8.5%). The most common CHIP mutations were observed in DNMT3A, (n = 17), TET2 (n = 7) and TP53 (n = 7) genes. The percentage of patients with at least 1 mutation found in DNMT3A, TET2, and TP53 were 4.2%, 1.94%, and 1.38%, respectively. Other genes containing CHIP mutation included CEBPA, ETV6, HRAS, PDGFRA, NRAS, KMT2A, EZH2, GATA2, GNAS at a frequency below 1%. CHIP mutations were not observed in patients younger than 40 years, but they increased in frequency with every decade of life thereafter. The incidence of CHIP increased from 0.04 for the 40-50 yrs age group to 0.18 for individuals older than 60. Further analysis of associations between incidence of CHIP and cancer type, prior exposure to chemotherapy as well as longitudinal evolution of CHIP mutations during cytotoxic treatment are underway and will be presented. Conclusions: CHIP, a common finding in the elderly population is an important factor to consider in ctDNA analysis and most frequently involves DNMT3A, TET2, and TP53 genes. The frequency of CHIP can be impacted by a number of other factors such as cytotoxic chemo- or radiotherapy.
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de Smith, Adam J., Kyle M. Walsh, Ivan Smirnov, Sumeet Aujla, Erica Sanders, Hansen M. Helen, Catherine Metayer, and Joseph L. Wiemels. "Somatic and Germline Mutational Heterogeneity in High Hyperdiploid Acute Lymphoblastic Leukemia." Blood 128, no. 22 (December 2, 2016): 1727. http://dx.doi.org/10.1182/blood.v128.22.1727.1727.
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Abstract High hyperdiploidy (HD), the most common cytogenetic subtype of acute lymphoblastic leukemia (ALL), is characterized by a nonrandom gain of chromosomes and is thought to arise from a single abnormal mitosis. However, the causes of this leukemia-initiating event remain unknown. A recognized enrichment of HD-ALL among children with RASopathies and with germline ETV6 mutations suggests that germline predisposition underlies a component of HD-ALL risk, in addition to the increased risk of the HD subtype with ALL-associated heritable risk variants in PIP4K2A, ARID5B, and CEBPE. Though cure rates of HD-ALL are high, the significant treatment-related morbidities and mortality warrant more etiologic investigations which may reveal molecularly-targeted therapies for this disease. We carried out deep-sequencing of 538 cancer-related genes using the UCSF500 Cancer Gene Panel in 57 HD-ALL tumors from California Childhood Leukemia Study patients. Selected patients lacked overt KRAS and NRAS hotspot mutations (assessed by Sanger sequencing) and common ALL deletions (assessed by MLPA), to enrich for discovery of novel driver genes. A Combined Annotation Dependent Depletion (CADD) Phred score ≥20 was used to filter predicted damaging mutations. To remove polymorphisms, we retained only mutations with allele frequency <0.01% in the Exome Aggregation Consortium (ExAC). We adjusted the mutant allele fraction (MAF) of each mutation in relation to chromosome copy-number, as determined using the CNVkit tool. Sanger sequencing of remission DNA was used to validate a subset of predicted germline mutations (adjusted MAF≥0.45) of interest, including in known ALL predisposition genes. Novel damaging somatic mutations were discovered in epigenetic regulatory genes, including DOT1L (n=4), with 33% of patients harboring mutations in this pathway. Somatic mutations in the receptor tyrosine kinase (RTK)/Ras/MAPK signaling pathway were found in two thirds of patients, including mutations in ROS1, which mediates phosphorylation of the PTPN11-encoded protein SHP2. An extraordinary level of tumor heterogeneity was detected, with microclonal (mutant allele fraction <10%) hotspot mutations in KRAS, NRAS, FLT3 or PTPN11 identified in 31/57 (54.4%) patients. Multiple microclonal mutations at KRAS and NRAS codons 12 and 13 significantly co-occurred within tumor samples (P=4.8x10-4), suggesting ongoing formation of, and selection for, Ras mutation. Moreover, 7 patients had multiple microclonal mutations at the same Ras hotspot locus, in adjacent codon 12/13 nucleotides or in adjacent codons. The adjacent mutations occurred on different sequencing reads in all 7 patients (P=0.016), indicating they were part of distinct tumor subclones. We also detected an unexpectedly high frequency of putatively causal germline mutations, which were validated in remission DNA samples by Sanger sequencing. At least 25% of HD-ALL patients carried one or more rare (<0.01% allele frequency in ExAC) and predicted-damaging germline mutations in known ALL predisposition genes, DNA repair genes, or within known hotspot mutation loci that had previously been reported mutated only in tumor genomes. Future work is required to investigate whether tumor microheterogeneity should impact therapeutic regimens and to elucidate the biologic function of epigenetic dysregulation in development of HD-ALL. Whole-exome sequencing of more patients and functional analysis of novel mutations are required to understand the contribution of germline predisposition to HD-ALL etiology, which may be much larger than previously realized. Disclosures No relevant conflicts of interest to declare.
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Hosono, Naoko, Hasan Rehman, Bartlomiej Przychodzen, Ines Gomez-Segui, Kathryn M. Guinta, Kenichi Yoshida, Satoru Miyano, et al. "Various Germline Congenital Disorder Genes Are Somatically Mutated in Myeloid Malignancies." Blood 120, no. 21 (November 16, 2012): 1405. http://dx.doi.org/10.1182/blood.v120.21.1405.1405.
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Abstract Abstract 1405 Genes involved in congenital genetic cancer susceptibility syndromes are also targets of somatic mutations in various tumors. Examples include WT1, NF1, CBL, TP53 and MLL2 affected both in germ line as well as somatic mutations present in malignant disorders. To apply this approach to investigation of pathogenic mutations in myeloid malignancies, we selected 183 congenital disorders in which germline mutations of disease specific genes are reported to be pathogenic. Their main clinical presentations are skeletal abnormalities (N=54 disorders), skin abnormality (N=24), mental retardation (N=17) and hematological disorders (N=12). In total, we searched for mutations in 204 genes associated with these congenital disorders. We analyzed whole exome of various myeloid malignancies, including 60 cases with myelodysplastic syndromes (MDS), 29 MDS/MPN, 5 with MPN and 122 with acute myeloid leukemia (AML) and found somatic mutations in 62 genes, which also mutated in germ line in various congenital syndromes. Of those, the most frequently mutated genes were TP53 (25 cases) and WT1 (16 cases), associated with germline mutation of Li-Fraumeni syndrome and Wilms tumor, respectively. Some somatic mutations, for example, NF1 (R1276Q) and PTPN11 (D61N), were exactly the same as observed in corresponding congenital disorders (Neurofibromatosis or Noonan syndrome). One of the novel findings is that somatic SET binding protein 1 (SETBP1) mutations (D868N, G870S and I871T) were commonly observed in sAML and CMML, and were identical to germline mutations in Schinzel-Giedion syndrome (see designated abstract). We found recurrent somatic SETBP1 mutations in 15% of each CMML and sAML. Moreover, multiple genes pathogenic in Usher syndrome (congenital hearing and vision loss, complicated by vasoproliferative retinal tumor), were somatically mutated in various myeloid neoplasms. Out of 9 genes which are causative for this syndrome, 15 mutations of 6 genes (MYO7A, USH1C, CDH23, PCDH15, USH2A, and GPR98) were observed in 13 cases, including 2 frameshift and 13 missense mutations. These genes coordinate with each other to form a functional network. CDH23 and PCDH15 are cadherins and act as cell adhesion molecules. MYO7A are actin-based motor molecules with a variety of functions. USH1C serves as an anchor and codes for a scaffolding protein to form a complex with all the other proteins. Through the PDZ binding site, USH1C forms a complex with CDH23, which was the most frequently mutated gene in this family (1 frame shift and 3 misssense mutations). CDH23 mutations were observed in 2 cases with primary AML, sAML and MDS. Specifically, a somatic missense mutation G2771S of CDH23 in a secondary AML case was identical to germline of Usher syndrome. The second most frequently mutated gene, GPR98, is located in 5q14.3 locus; a small hemizygous clone found in del5q of an MDS case. In a serial sample analysis, this mutation increased to become the larger main clone during AML evolution. Moreover, in this case, an additional CDH23 mutation was acquired in the course of leukemic expansion. In such cases with Usher syndrome gene mutations, U2AF1, ZRSR2, EZH2, IDH2 and ETV6 mutations were also observed, suggesting pathogenic cooperation with these well-known tumor suppressor genes and oncogenes. Disclosures: Maciejewski: NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding. Makishima:Scott Hamilton CARES Initiative: Research Funding.