Comprehensive Identification Of Germline Alterations In Telomerase Complex Genes By Whole Exome Sequencing Of MDS and Related Myeloid Neoplasms

Abstract In addition to classical familiar forms of bone marrow failure, some cases of aplastic anemia (AA) have been linked to inherited germ line polymorphism/mutations of telomerase machinery, leading to excessive telomere shortening. Germline telomere maintenance machinery mutations have been also been found in a proportion of acute myeloid leukemia (AML) and Myelodysplastic syndromes (MDS) patients (pts). However, the molecular pathogenesis of adult MDS and AML is complex and determination of genetic risk factors in addition to established familial and congenital syndromes has been difficult. To date targeted sequencing has been used for mutational screens with the inherent limitations of limited exome coverage, empiric bias and labor intensity. New generation (NGS) whole genome approaches prioritize somatic mutations as initial discovery targets, but the availability of sequenced cohorts allows also for detection of germline lesions both in a targeted and an unbiased fashion. Using NGS we studied 136 pts (mean age, 68.8 years, range 41-85) with MDS and related myeloid neoplasms for the presence of non-synonymous polymorphisms (SNV), which could affect telomerase machinery. These genes included TERT, DKC1, SMG6, NOP10, POT1, WRAP53, NHP2, GAR1, TINF2. No somatic defects of the telomerase complex were detected. We focused on novel sequence alterations or those described in available databases with a population allelic frequency of less than 5%. We identified 45 non-synonymous germline sequence alterations in 39 cases (32%). Most frequent SNV were found in TERT (n=15), DKC1 (n=7), SMG6 (n=6), NOP10 (n=4), POT1 (n=4), WRAP53 (n=4), while observations of NHP2 (n=3), GAR1 (n=1), TINF2 (n=1) were less prevalent. These variants were distributed in an almost mutually exclusive manner. Out of 3 variants in TERT, p.H412Y (n=3) and p.A279T (n=9) were reported to be pathogenic in bone marrow failure syndromes. In addition, p.A999T found in 8 cases in our cohort could also be pathogenic since it is less frequent in healthy controls. Similarly, p.441_442del (n=1), located in the N-terminal region, is a completely novel germline variant not detected in 6500 samples publicly available in ESP6500. In the pAML cohort (TCGA; n=197), the observations of germline variants for these telomerase complex genes were SMG6 (n=21), POT1 (n=19), NHP2 (n=1), NOP10 (n=1) GAR1 (n=1). Next, we analyzed clinical characteristics, including treatment responsiveness as assessed per modified 2006 IWG response criteria. The mean age of the 39 patients with germline telomerase machinery alterations was 67 years, 24% (9/39) were younger (age<60 years) compared to 12% (12/97) of wild type (WT; p=.12). Of note, 58% of these cases had a family history of solid tumors including breast, gastrointestinal and prostate and 8% (3/36) had a family history of myeloid malignancies. 41% (16/39) of the telomerase mutants had higher-risk MDS/sAML at presentation compared to 23% in WT cases (23/97; p=.19). A higher percentage of mutants also had complex cytogenetics compared to WT (35% vs. 13%; p=.01). Response rates to common therapies, including hypomethylating agents were similar, but we noted that none of the carrier cases (n=16) treated with lenalidomide showed therapeutic responses (0% vs. 37%). The mean overall survival of the carrier cases was lower compared to the WT (36 vs. 39 months, p=.10). When we studied cases with telomerase alterations for the presence of coinciding somatic mutations, using a targeted deep sequencing panel of the 100 most common mutations acquired in pts with germline telomerase complex alterations, we found most common the acquisition of DNMT3 (18% vs. 6%, p.10) and cohesin mutations (13% vs. 4%,p=.11). In sum, unbiased NGS sequencing approaches in MDS and related myeloid neoplasms allowed for identification of genetic germline alterations in telomerase maintenance machinery at higher rates than previously detected using targeted screening approaches, suggesting that such genetic defects may more frequently than previously thought contribute to cryptic and likely complex genetic predisposition to these diseases. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding.

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Heterozygous RTEL1 Variants Are Associated with Bone Marrow Failure and Abnormal Clinical Phenotype

Blood ◽

10.1182/blood.v128.22.1043.1043 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1043-1043

Author(s):

Shreyans Gandhi ◽

Jie Jiang ◽

Mariam Ibanez ◽

Isabelle Callebaut ◽

Judith CW Marsh ◽

...

Keyword(s):

Bone Marrow ◽

Family History ◽

Bone Marrow Failure ◽

3D Structure ◽

Telomere Maintenance ◽

The Other ◽

Helicase Domain ◽

Cell Transplant ◽

Variable Regions ◽

History Of

Abstract Introduction Heterozygous RTEL1 mutations have recently been described in familial pulmonary fibrosis (PF) but are not known to be associated with cytopenias or bone marrow failure (BMF), in contrast to heterozygous mutations in other telomere maintenance genes TERT, TERC and TINF2. Constitutional BMF syndromes typically present with less severe pancytopenia and it is often unclear if they have hypocellular MDS (hypoMDS) or non-severe AA (NSAA) morphologically. Methods We screened 284 patients with idiopathic AA or uncharacterised BMF and 172 patients with MDS or acute myeloid leukemia (AML) for TL and RTEL1 variants, and for the other currently known telomere gene complex (TGC) mutations, after excluding patients with Fanconi anemia, DBA or other known inherited BMF syndrome. TL was measured using a monochrome multiplex quantitative PCR method on peripheral blood mononuclear cells. Illumina Nextera-amplicon sequencing was used to screen exons of the DC genes (DKC1, TERC, TERT, RTEL1, CTC1, NHP10, NOP2, USB1, WRAP53, TINF2, PARN and ACD) by MiSeq platform. Constitutional DNA was also analysed in 10 patients (skin 9, buccal swab 1) with RTEL1 variants. A targeted gene panel of 24 genes of an Illumina Tru-Seq Custom Amplicon workflow and platform was used to identify genes frequently mutated in MDS/AML. Impact of mutations was predicted based on 3D structure information from comparative modelling for the helicase domain, comprising the HD1 and HD2 subdomains, a Fe-S cluster and an ARCH domain, and for two harmonin-like (HML) domains and a RING finger domain, located in the C-terminal regulatory region of RTEL1. Results Heterozygous RTEL1 variants were identified in 20 (4.4%) patients. RTEL1 variant allele frequency (VAF) was 45-70% consistent with heterozygous inheritance in all cases. TL was short in 18 (90%) patients, being < 1st centile in 15 and <10th centile in 3. 2 patients had normal TL, <20th centile and >50th centile, respectively. Median age was 35 years (range 18-73). 15/20 (75%) had a hypocellular BM (7 hypoMDS, 5 non-severe AA, 3 ICUS), and 1 each with RAEB1, RAEB2, CMML1, AML and isolated macrocytosis. 3 patients had abnormal karyotype: +8 (hypoMDS), -Y,+1,del(1) (hypoMDS), del7q (RAEB1). 2 other patients with hypoMDS had somatic mutations: U2AF1 (30% VAF) with ASXL1 (27% VAF); U2AF1 (43% VAF). Lung abnormalities were early PF (1), interstitial lung disease (1), and abnormal lung function with reduced TLCO (1) and an obstructive picture (1). Liver fibrosis with portal hypertension and varices and reticulate skin pigmentation were present in the patient with ILD, 2 patients had dystrophic nails, and 1 unexplained mild hepato-splenomegaly. 2 patients had familial MDS, 5 had a family history of cancers affecting first-degree relatives, and 2 had skeletal and cartilage anomalies, associated with learning difficulties in 1 patient. 8/15 patients with hypocellular BM required no treatment (5 hypoMDS and 3 NSAA), one hypoMDS had CR with ciclosporin and another underwent successful unrelated donor stem cell transplant; for NSAA, 2 received ATG with CSA, with PR followed by relapse in one, the other was lost to follow up, and 1 was androgen responsive. 16/20 (80%) patients are alive; 3 patients with RAEB or AML died of progressive disease and 1 patient with ICUS and severe constitutional features died from lymphoma 10 years after presentation. Mutations were spread throughout the entire RTEL1 sequence (summarised in Figure). 3D structure analysis predicted the missense RTEL1 mutations would result in disturbance of the FeS cluster and/or interfere with DNA binding, destabilisation of the HD1, HD2 or the ARCH sub-domains of the helicase domain, or destabilisation of inter-domain interactions. One HML1 mutation occurred in a loop opposite the putative ligand binding site and the rest in the variable regions outside the conserved domains. RTEL1 variants were associated with TERT mutations in 4 patients, of which 3 were known pathogenetic and 1 novel TERT mutation with low telomerase activity on TRAP assay confirming its pathogenetic nature. Conclusions We show for the first time that heterozygous RTEL1 mutations occur in 4.4% of patients, most commonly in young patients with a hypocellular BM, and often a family history of BMF/malignancy, and less often with high risk MDS/AML. Abnormal clinical features were present in a third of patients, some similar to but others distinct from dyskeratosis congenita. Disclosures No relevant conflicts of interest to declare.

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Clinical and Molecular Features Of Young Patients With Myelodisplastic Syndromes (MDS)

Blood ◽

10.1182/blood.v122.21.1566.1566 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1566-1566

Author(s):

Bilori Bilori ◽

Hideki Makishima ◽

Bartlomiej P Przychodzen ◽

Mohamed Ashkar ◽

Rohan Garje ◽

...

Keyword(s):

Family History ◽

Research Funding ◽

Somatic Mutations ◽

Myeloproliferative Neoplasm ◽

Young Patients ◽

Blood Disorders ◽

Older Population ◽

Cytogenetic Abnormalities ◽

Significant Difference ◽

History Of

Abstract MDS typically affects older adults, and hereditary factors have been considered less contributory to disease pathogenesis. Moreover, their impact is obscured by the complexity of the clinical presentation and history. Similarly, familial MDS and pediatric MDS is rare and likely distinct from adult MDS occurring in younger adults. Younger MDS patients (pts), excluding those who present with treatment-related disease, may represent a distinct subtype of MDS characterized by a specific molecular pattern of lesions. We compared two groups of MDS pts focusing on pathological diagnosis at presentation, family history of solid malignancies and blood disorders (Leukemia and MDS) in first and second degree relatives, cytogenetic abnormalities and somatic mutations. Our analysis of 1030 MDS pts included MDS, MDS/myeloproliferative neoplasm (MPN) and secondary acute myeloid leukemia (sAML) pts. Overall the median age at presentation of this population was 71years (range 14-100); we classified the younger subset as those falling into the lower 8thpercentile of age to identify. Accordingly, the younger population was characterized by age less than 50 years (range 14-49; median age 41), and the older population age ≥ 50years (range 50-100; median age 75). Treatment-related MDS was excluded. Younger MDS pts more frequently presented with higher-risk disease compared to the older population (46% vs. 31%; P=.004). There was no significant difference between the two groups with regard to family history of cancers (40 vs. 47%; P=.21) and blood disorders (10 vs. 6%; P= .1). When we compared cytogenetic abnormalities between these patient subsets, there was no difference in detection of rate of abnormal cytogenetics (53% vs. 52%; P=.5) or complex karyotype (23 vs. 25%; P=.86). However, del 20q was more common in the older subset (19 vs. 6%; P=.03). We then investigated somatic mutational patterns using new generation deep sequencing for the 60 most commonly encountered MDS mutations (defined in the 200 MDS exome cohort presented in other abstract from our group). Data were available for 26 younger pts and 179 older pts. By analyzing comprehensive mutational spectrum, the average number of somatic mutational events (mean; 2.4/case) was significantly higher in the older subgroup compared to the younger (1.8/case; P<.001). RUNX1, PHF6, TP53 (12% each) are the most frequently affected genes in MDS associated with the younger population. Interestingly, germline mutations of these 3 genes are all associated with congenital syndromes, which lead to susceptibility for hematological neoplasms. Conversely, somatic mutations of TET2 (24%) and ASXL1 (15%) were most prevalent in the older MDS cohort. Notably, these 2 genes associated with older populations were less prevalent in younger MDS cases (<4%; P=.02 and < 4% P=.11 for TET2 and ASLX1 respectively). In contrast, there was no significant difference between these subgroups (old vs. young MDS) in the frequency of RUNX1 (9 vs. 12%; P=.67), U2AF1 (11 vs. 12%; P=.88), BCOR family (9 vs. 15%; P=.35), PRC2 family (10 vs. 4%; P= .34), RAS family (11 vs. 12%; P=.88), or many other gene mutations. In sum, 8% of MDS pts present at a younger age in our cohort. MDS in younger pts presents with more advanced disease and is less commonly affected by del20q- and TET2 mutations, consistent with less common myeloproliferative features in this population. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding.

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Germline and Acquired Genetic Variants in Myelodysplastic Syndromes in Young Adults without a Preexisting Disorder or Organ Dysfunction

Blood ◽

10.1182/blood-2018-99-116045 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4339-4339

Author(s):

Tzu Hua Chen-Liang ◽

Ana M Hurtado López ◽

Laura Palomo ◽

Teresa Bernal Del Castillo ◽

Mar Tormo ◽

...

Keyword(s):

Bone Marrow ◽

Young Adults ◽

Family History ◽

Board Of Directors ◽

Research Funding ◽

Bone Marrow Failure ◽

Advisory Committees ◽

Germline Variants ◽

Very High ◽

Congenital Syndrome

Abstract Background and Aim:It is increasingly recognized that patients with a de novomyelodysplastic syndrome (MDS) onset as young adults, lacking any other feature of a congenital disorder, may share a pathogenic overlap due to the presence of both germline and somatic variants. Identifying an inherited pathogenic variant has important therapeutic implications beyond family counselling: adapting the selection of sibling donor, the use of highly cytotoxic therapy and the monitoring for other cancer development. However, most studies have focused on patients with suspected inherited disorders based on the presence of physical abnormalities and/or family history. In addition, a mixture of pediatric and adult cases is usually reported. The aim of this study is to characterize the germline and tumor variants in a group of adult MDS patients without accompanying congenital physical anomalies and or family antecedent of bone marrow failure. Methods: We included 72 patients from 15 Spanish centers with a diagnosis of MDS between 18 and 60 years old (y.o). Patients with a previously diagnosed or suspected (one physical anomaly or family history) congenital syndrome were excluded. Diagnoses were made in accordance with the WHO 2016 classification. Whole-exome sequencing (WES) libraries were prepared using SureSelectXT Target Enrichment and sequenced on a HiSeq4000 platform (IlluminaInc.). Mean number of reads per sample was 138,726,017 with a Phred Quality Score >30 in 95.05% of bases. Read mapping sequence alignment and variant calling were performed using Biomedical Workbench (Qiagen). WES was performed on 72 tumor and 32 paired germinal DNA (buccal swab). To identify potential germline-causal mutations, a selection tool was implemented incorporating 239 genes associated with cause or predisposition to bone marrow failure or cancer. Variants with an ExAC, TOPMed and/or European 1000 Genomes minor allele frequency ≥0.01 were discarded. Results: The median age at diagnosis was 49 y.o. The cohort was categorised into two groups, less or equal 50 y.o. (62.5%) and between 50 and 60 y.o. (37.5%). In the first group, the frequency according to the WHO classification were 12% MDS with single lineage dyplasia (MDS-SLD), 8% MDS with ring sideroblasts (MDS-RS), 11% MDS with multilineage dyplasia (MDS-MLD), 24% MDS with excess blasts(MD-EB), 4% MDS with isolated del(5q)(MDS-del5q), 4% MDS unclassifiable and 4% chronic myelomonocytic leukemia (CMML). Meanwhile, in the group with age more than 50 y.o., the subtypes were 3.7% MDS-SLD, 7.4% MDS-RS, 29.6% MDS-MLD, 40.7% MD-EB, 3.7% MDS-del5q, and 14.8% CMML.Patients less or equal 50 y.o. were stratified based on IPSS-R as very low (4%), low (64%), intermediate (20%), high (12%) and very high (0%); and the group of more than 50 y.o. as very low (14.8%), low (33.3%), intermediate (29.6%), high (11.1%) and very high (11.1%).The mean number of somatic mutations was 0.68 in patients with less or equal 50 y.o. and 1.37 in those between 50 and 60 y.o., p=0.033 (U Mann-Whitney); and regarding germline variants, the first group mean number was 2.44 (p25-75, 1-3) and the second group showed a mean of 1.85 (QI 25-75, 1-3), p= 0,331.In the whole cohort, germline variants were found in 62 out of 72 patients, with the following frequencies: ATR(N=5, 6.9%), followed by BARD1(N=5, 6.9%), ERCC6L2(N=4, 5.6%), MSH6(N=4, 5.6%), TCIRG1(N=4, 5.6%), NBEAL2(N=4, 5.6%), ASXL1(N=3, 4.2%), ATM(N=3, 4.2%), MPL(N=3, 4.2%), NF1(N=3, 4.2%), RECQL4(N=3, 4.2%), SAMD9L(N=3, 4.2%), WRN(N=3, 4.2%).Among germline variants, those reported previously as pathogenic or likely pathogenic, or involving genes associated with familial MDS/AML included: ERCC6L2(N=4, 5.6%), SAMD9L(N=3, 4.2%), and one case mutated for DDX41, FANCC, JAK2, MSH6, SETBP1, MUTYH, BRCA1and RECQL4. In the whole cohort, somatic variants were found in 38 patients, with the following frequencies: TP53(N=7, 9.7%), ASXL1(N=7, 9.7%), SETBP1(N=5, 6.9%), NF1(N=5, 6.9%), SRSF2(N=4, 5.5%). Conclusion:In this subset of young adults with de novo MDS without congenital anomalies and/or familial history suggesting the presence of an undiagnosed congenital syndrome, 18% of the cohort harbored a likely causative germline variant. In addition, we noted a predominance of variants affecting genes with a cancer predisposition limited to the hematopoietic system, rather than classical telomere, DNA damage genes with an established mendelian link. Table. Table. Disclosures Díez-Campelo: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

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Pathogenic Relevance of Germ Line TET2 Alterations

Blood ◽

10.1182/blood.v128.22.3160.3160 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3160-3160 ◽

Cited By ~ 1

Author(s):

Cassandra M. Hirsch ◽

Aziz Nazha ◽

Kassy E Kneen ◽

Manja Meggendorfer ◽

Bartlomiej P Przychodzen ◽

...

Keyword(s):

Somatic Mutations ◽

Catalytic Domain ◽

Rare Variants ◽

Germ Line ◽

Bone Marrow Failure ◽

Genetic Syndromes ◽

Advisory Committees ◽

Myeloid Neoplasm ◽

Myeloid Neoplasms ◽

Biallelic Mutations

Abstract Several familial mutations predisposing to the development of leukemia have been identified, mostly in association with childhood or adolescent presentations of myeloid neoplasms or bone marrow failure states. However, many of these genetic syndromes have variable penetrance and latency. Thus, late presentations of familial diseases are possible as with DDX41 mutations associated with adult myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Growing evidence exists to support the notion that germ line (GL) alterations may play a greater than previously known role in otherwise typical spontaneous adult myeloid neoplasms. These alterations may include known predisposition leukemia genes (i.e.,. ETV6, RUNX1, CEBPa, and DDX41), heterozygous alterations known to generate recessive disorders, or new unidentified genes. The search for such predisposition genes is difficult but we hypothesized that genes frequently damaged by somatic mutations may also be affected by GL alterations, a similar principle as seen for CEBPa or TP53. Further, that a collection of rare variants (MAF<1%) within a gene could be associated with risk of MDS according to the allelic burden (AB) theory. To develop proof-of-principle of this theory we focused on the gene TET2. In comparisons of the AB of rare TET2 GL variants of patients vs. ethnically-matched controls (selected according to ethnic indicator SNPs), MDS patients examined by whole exome sequencing (WES, N=368) had more rare coding-sequence variants than controls (p<.001). On average, patients had 3 TET2 rare variants, while controls had only 1.5 variants. This suggests that the burden of TET2 rare variants could increase risks of myeloid neoplasms. This finding is in agreement with several other observations; 1.) TET2 is frequently mutated and extremely polymorphic; 2.) it is often associated with biallelic mutations, of which some may be a combination of GL and somatic lesions; and 3.) GL alterations in TET2 may predispose to acquisition of additional lesions. We analyzed a cohort of 4159 patients with myeloid neoplasms using WES and targeted deep next generation sequencing (NGS). Extensive scoring algorithms, along with mutational databases were used to detect GL variants. Serial tumor samples were examined to discriminate GL from somatic defects. We identified 2518 TET2 SNPs in 1475 patients. Multiple SNPs were seen in the same patient, 28% of patients had 2 SNPs, 11% had 3 and 6% had 4 or more SNPs. Of those 52% were found in proximity to the catalytic domain, and 10% were homozygous. TET2 SNPs irrespective of their population frequency were found in 21% of MDS, 27% of MDS/MPN, 31% of MPN, and 33% of AML patients. We focused our analysis on the top 10 canonical SNPs with the highest odds ratio (OR) when comparing frequencies in our population to that of healthy controls derived from the ExAC base of 60,000 individuals. The SNPs with the highest OR was present in 2% of patients (p.Val1718Leu) vs. 0.5% of controls. Combinations of SNPs (biallelic configuration) were then investigated; SNPs p.Leu1721Trp and p.Pro363Leu co-occurred in 8% of patients, and p.Val218Met, p.Leu34Phe and p.His1778Arg co-occurred in 3%. We also queried whether GL TET2 SNPs create a haplotype (i.e., increased the risk for acquisition of somatic TET2 mutations) or are mutually exclusive. Overall, 32% of cases with any TET2 SNP also acquired a TET2 somatic mutation vs the WT form for these (68%). Conversely, among the somatic mutant carriers, for example SNP p.Met1701Ile, SNP p.Tyr864His and SNP p.Gly355Asp had an OR vs. controls of 1.1. In particular, 55% of all TET2 mutants had SNP p.Leu1721Trp in a biallelic fashion vs. 46% of somatic WT cases. This suggests a predisposition haplotype. We also examined the relationship between unrelated somatic mutations and "top OR" TET2 SNPs. No correlation was seen between SNPs and other somatic mutations. In sum, several GL TET2 variants or their combination may constitute complex, likely low penetrance predisposing factors for myeloid neoplasm that also interact with somatic lesions and lead to cancer decades later. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Maciejewski:Celgene: Consultancy, Honoraria, Speakers Bureau; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau.

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Clonal hematopoiesis in the inherited bone marrow failure syndromes

Blood ◽

10.1182/blood.2019000990 ◽

2020 ◽

Author(s):

Frederick D Tsai ◽

R. Coleman Lindsley

Keyword(s):

Bone Marrow ◽

Tumor Suppressor ◽

Malignant Transformation ◽

Somatic Mutations ◽

Bone Marrow Failure ◽

Telomere Maintenance ◽

Clonal Hematopoiesis ◽

Bone Marrow Failure Syndromes ◽

Increased Risk ◽

Somatic Alterations

Inherited bone marrow failure syndromes (IBMFS) are characterized by ineffective hematopoiesis and increased risk of developing myeloid malignancy. The pathophysiologies of different IBMFS are variable, and can relate to defects in diverse biological processes, including DNA damage repair (Fanconi anemia), telomere maintenance (dyskeratosis congenita), and ribosome biogenesis (Diamond-Blackfan anemia, Shwachman-Diamond syndrome). Somatic mutations leading to clonal hematopoiesis have been described in IBMFS, but the distinct mechanisms by which mutations drive clonal advantage in each disease and their associations with leukemia risk are not well understood. Clinical observations and laboratory models of IBMFS suggest that the germline deficiencies establish a qualitatively impaired functional state at baseline. In this context, somatic alterations can promote clonal hematopoiesis by improving the competitive fitness of specific hematopoietic stem cell clones. Some somatic alterations relieve baseline fitness constraints by normalizing the underlying germline deficit through direct reversion or indirect compensation, while others do so by subverting senescence or tumor suppressor pathways. Clones with normalizing somatic mutations may have limited transformation potential due to retention of functionally intact fitness-sensing and tumor suppressor pathways, while those with mutations that impair cellular elimination may have increased risk of malignant transformation due to subversion of tumor suppressor pathways. Since clonal hematopoiesis is not deterministic of malignant transformation, rational surveillance strategies will thus depend on the ability to prospectively identify specific clones with increased leukemic potential. We describe a framework by which an understanding of the processes that promote clonal hematopoiesis in IBMFS may inform clinical surveillance strategies.

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The Significance of the Next Generation Targeted Sequencing in the Precise Diagnosis of Pediatric Acquire Aplastic Anemia and Inherited Bone Marrow Failure Syndromes

Blood ◽

10.1182/blood.v128.22.3908.3908 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3908-3908 ◽

Cited By ~ 1

Author(s):

Wenbin An ◽

Ye Guo ◽

Yumei Chen ◽

Yao Zou ◽

Xiaojuan Chen ◽

...

Keyword(s):

Bone Marrow ◽

Family History ◽

Bone Marrow Failure ◽

Gene Mutations ◽

Chromosome Breakage ◽

Targeted Sequencing ◽

Genetic Mutations ◽

Bone Marrow Failure Syndromes ◽

Precise Diagnosis ◽

History Of

Abstract Background Diagnosis of inherited bone marrow failure syndromes (IBMFs) depend on classic clinical manifestation including early onset, physical anomalies, family history of cancer and/or bone marrow failure and chromosome breakage testing (MMC and/or DEB), mutation analyses and bone marrow chromosome analyses. At present, more than 70 pathogenic gene mutations had been identified. However, in some patients, physical anomalies is absent or delayed, and were misdiagnosed as acquired aplastic anemia(AA). Genetic analysis is very important to establish a precise diagnosis, predict cancer risk, direct treatment and genetic counseling. In this study, we focus on the application of next generation targeted sequencing in precise diagnosis of pediatric acquired AA/IBMFs, and the association between genetic abnormalities and clinical and laboratory characteristics. Methods We designed a targeted sequencing assay to test a panel of 417 genes. The panel contain reported gene associated with IBMFs and other diseases need be differentiated. Pediatic patients (≤14 year old) with suspected diagnosis of AA/IBMFs were enrolled. Peripheral blood (PB) DNA was used to genetic analysis and oral epithelia cells or PB DNA from their parents were used to identify somatic mutations and unreported polymorphism. All the results were validated by Sanger sequencing. Results The average coverage of targeted region was 98.15%, and the average sequencing depth was 315.9×. Totally, 283 patients were enrolled, including 176 clinically diagnosed acquired AA, 51 Fanconi anemia (FA), 8 dyskeratosis congenital(DKC), 30 Diamond-Blanckfan anemia(DBA), 15 congenital neutropenia(CN), and 3 congenital thrombocytopenia. Totally, 19% subjects had IBMFs related genetic mutations. In the patients who were clinically diagnosed as acquired AA patients, about 7% had IBMFs related disease-causing genetic mutations. Finally, 7 patients were genetically diagnosed as FA, 2 were DKC, 1 was WAS and 1 was SDS. In patients who were clinically diagnosed as FA, 33.4% had FANC related gene mutations. Telomere associated gene mutations were detected in 75% of clinical diagnosed DKC. For patients clinically diagnosed as DBA and CN, 36.7% and 20% were detected disease-causing mutations. After genetic screening, 2 patients who had been diagnosed as FA were modified as WAS and 1 DBA was modified as SDS. Only 26% genetic diagnosed IBMFs patients had family history of bone marrow failure, leukemia, tumor or physical anomalies. Compared with acquired AA, patients with genetic diagnosed FA were more likely to have physical anomalies of short stature and development retardation, Cafe au lait spots and finger or toe malformation(P<0.001).However, 46% patients with IBMFs did not have any type of physical anomalies. Moreover, there were only 24% patients with genetic diagnosed IBMFs had positive results of MMC induced chromosome breakage test or SCGE, and both the examinations could not differentiate subtype of IBMFs. FANCD2 mono-ubiquitination test were performed recently. However, even in the genetically confirmed FA, the positive rate was only 18% (2/11). And, there were positive results in some acquired AA patients. For FA patients with definitely genetic mutations, 62.5%(15/24) were compound heterozygous mutations,37.5%(9/24) were homozygous mutations. Mutational frequencies of FANC were: FANCA 65%, FANCD2 23%, FANCG 9%, FANCI 9% and FANCB 4%. For the mutated type, the frequencies of missense, frameshift, nonsense, splicing mutation were 42%, 26%, 16%, 16%. In our study, there were 4 undetermined patients met the clinical diagnostic criteria of FA, and having heterozygous damaged mutations in FANC genes. Conclusion In conclusion, our IBMFs associated genes targeted sequencing assay is an effective strategy for precise diagnosis of bone marrow failure diseases, especially for those without family history or physical anomalies. However, nearly half of the clinically diagnosed IBMFs patients in our study were not detected the disease-causing mutations. This may be due to the mutations in the intron area, or large fragment deletion, which cannot be detected by targeted sequencing. And the novel gene involved in IBMFs need further study. Disclosures No relevant conflicts of interest to declare.

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Androgen Derivatives Improve Blood Counts and Elongate Telomere Length in Patients with Dyskeratosis Congenita

Blood ◽

10.1182/blood-2018-99-118197 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2585-2585 ◽

Cited By ~ 1

Author(s):

Martin Kirschner ◽

Monica Sofia Ventura Ferreira ◽

Anne-Sophie Bouillon ◽

Marcin W. Wlodarski ◽

Michaela Schwarz ◽

...

Keyword(s):

Telomere Length ◽

Peripheral Blood ◽

Research Funding ◽

Somatic Mutations ◽

Bone Marrow Failure ◽

Dyskeratosis Congenita ◽

Telomere Maintenance ◽

Treatment Start ◽

Increased Risk

Abstract Introduction: Classical Dyskeratosis Congenita (DKC) is a systemic disorder characterized mainly by mucocutaneous features and bone marrow failure. DKC is caused by mutations affecting proper telomere maintenance leading to premature telomere shortening. Clinically, assessment of telomere length (TL) is being used for screening and diagnosis of DKC. Previous studies showed that androgen derivatives (AD) such as danazol or oxymetholone can improve blood counts and reduce transfusion frequency in patients with DKC. Reports from in vitro studies suggest that AD can increase the expression of telomerase and elongate telomeres reversing at least partially the mutation-related haploinsufficiency of the telomerase complex. However, whether telomere elongation can be observed in vivo is still controversial. Patients with DKC have an increased risk of developing solid tumors and acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Malignant transformation occurs mostly by chromosomal instability mediated by critical short telomeres and not via clonal hematopoiesis (CHIP) and eventual selection for MDS-related somatic mutations. The question whether increased telomerase activity by AD increases the risk for additional MDS-related mutations is unclear. In our study, we aimed to investigate TL and MDS-related somatic mutations in DKC patients undergoing treatment with AD. Methods and Patients: 5 patients enrolled in the Aachen Telomeropathy Registry (ATR) that underwent AD treatment were included in the analysis. All patients had molecularly confirmed DKC (4 patients having mutations in TERC, 1 patient in TERT). TERC mutated patients received danazol treatment (mean dosage 625 mg per day) while the patient with TERT mutation was treated with low dose oxymetholone (0.22mg/kg) per day. Patients were at a median age of 43.1 (range from 21.7 to 53.8) years. Median duration of treatment with AD was 14 months (3 to 29 mo) and is actually ongoing in all patients treated with danazol. Follow-up for blood counts and TL length assessment was carried out after median 14 months after treatment start with AD. TL assessment and blood counts of the patient receiving oxymetholone was carried out at the end of AD treatment after 29 months. All patients underwent next-generation sequencing (NGS) analysis using custom NGS-panel including frequent genes implicated in MDS development. Quality parameters of the NGS analysis were satisfactory (Q30>85%) and 95% of the expected area was covered at minimum 300x. To minimize risk of detecting sequencing errors, a threshold of 10 (absolute) and 5% (relative) variant allele frequency (VAF) was chosen. TL assessment of peripheral blood granulocytes and lymphocytes was carried out by Flow-FISH and all results are given in kb. Results: Analysis of the peripheral blood counts revealed a significant increase in platelets counts from mean 56/nl ±50 S.D. before treatment to 88/nl ±49 (p=0.03) during treatment. Similar results were observed for leukocyte counts increasing significantly from 3.83/µl±1.86 to 4.70/µl±2.88 (p=0.04). Hemoglobin counts showed a non-significant increase from 8.9 g/dl ±2.6 to 10.2 g/dl ±2.9 (p=0.13, all student paired t-test). Focusing on TL, lymphocyte TL increased significantly from 4.32kb±0.47 to 5.13kb ±0.57 (p=0.001). TL in the granulocyte subpopulation increased from 4.73kb±0.33 before treatment start to 6.10kb±0.50 under treatment (p=0.026). Calculated median increase in TL per months for lymphocytes and granulocytes was 0.092 kb (0.019 to 0.223 kb) and 0.166 kb (0.019kb to 0.513kb). Finally, NGS analysis for possible MDS-related mutations did not reveal any mutations before and under AD treatment. Conclusions: Based on our data in this genetically homogenous cohort of 5 patients with mutations in the telomerease genes TERC and TERT and short TL, AD significantly improve blood counts and elongate telomeres in granulocytes and lymphocytes. No MDS-related somatic mutations were observed during telomerase activation with AD. Pending longer follow up, treatment with AD seems to represent an efficient and safe therapy for patients with TERT or TERC mutations. Whether AD persistently elongate telomeres in DKC patients and how much this is dependent on the underlying DKC-related mutation requires further investigation. Disclosures Kirschner: Basilea Pharmaceutica: Other: travel support; BMS: Consultancy; Bayer: Consultancy; Roche: Consultancy. Wilop:Medizinwelten-Services GmbH: Honoraria; Amgen: Consultancy; Celgene: Consultancy, Honoraria, Other: Travel grant; Bristol-Myers Squibb: Honoraria. Brümmendorf:Pfizer: Consultancy, Research Funding; Janssen: Consultancy; Novartis: Consultancy, Research Funding; Takeda: Consultancy; Merck: Consultancy. Beier:Gilead: Other: travel support; Celgene: Other: travel support.

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Paroxysmal nocturnal hemoglobinuria

Blood ◽

10.1182/blood-2014-02-522128 ◽

2014 ◽

Vol 124 (18) ◽

pp. 2804-2811 ◽

Cited By ~ 194

Author(s):

Robert A. Brodsky

Keyword(s):

Monoclonal Antibody ◽

Bone Marrow ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Somatic Mutations ◽

Germ Line ◽

Bone Marrow Failure ◽

Multiple Congenital Anomalies ◽

Gpi Anchor ◽

Class A ◽

Terminal Complement

AbstractParoxysmal nocturnal hemoglobinuria (PNH) is a rare bone marrow failure disorder that manifests with hemolytic anemia, thrombosis, and peripheral blood cytopenias. The absence of two glycosylphosphatidylinositol (GPI)-anchored proteins, CD55 and CD59, leads to uncontrolled complement activation that accounts for hemolysis and other PNH manifestations. GPI anchor protein deficiency is almost always due to somatic mutations in phosphatidylinositol glycan class A (PIGA), a gene involved in the first step of GPI anchor biosynthesis; however, alternative mutations that cause PNH have recently been discovered. In addition, hypomorphic germ-line PIGA mutations that do not cause PNH have been shown to be responsible for a condition known as multiple congenital anomalies-hypotonia-seizures syndrome 2. Eculizumab, a first-in-class monoclonal antibody that inhibits terminal complement, is the treatment of choice for patients with severe manifestations of PNH. Bone marrow transplantation remains the only cure for PNH but should be reserved for patients with suboptimal response to eculizumab.

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Heterozygous CTC1 Variants in Acquired Bone Marrow Failure

Blood ◽

10.1182/blood-2018-99-115193 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3866-3866

Author(s):

Wenyi Shen ◽

Cassandra M. Hirsch ◽

Bartlomiej P. Przychodzen ◽

Reda Z. Mahfouz ◽

Tomas Radivoyevitch ◽

...

Keyword(s):

Family History ◽

Board Of Directors ◽

Germ Line ◽

Index Case ◽

Bone Marrow Failure ◽

Compound Heterozygous ◽

Advisory Committees ◽

Tier 1 ◽

History Of ◽

History Of Cancer

Abstract Germ line (GL) alterations of telomerase machinery genes may lead to inherited telomeropathies, but recent analysis of large control populations revealed that some of the previously assumed pathologic variants are present in comparable frequencies in healthy individuals. Pathogenic telomerase gene variants can be found, but are rare in idiopathic aplastic anemia (AA) and are associated with excessive telomere attrition. Previously, in a cohort of patients with MDS, pathogenic germ line variants of telomerase genes were very extremely rare. Recently a patient with bone marrow failure and liver cirrhosis presented with a biallelic CTC1 gene mutation. CTC1 is a member of the CTC complex, located on Chr 17p13.1, and critical for telomere replication. GL alterations of this gene were found to cause inherited disease, including Coats plus (CP), dyskeratosis congenital (DC) and cerebroretinal microangiopathy with calcifications and cysts (CRMCC) in a compound heterozygous manner. Our patient (age 28) had compound heterozygous CTC1 germline mutations (p.Lys242Leufs*41 and p.Cys985del) resulting in early childhood presentation of DC, subsequent severe AA at the age of 19, and a significant shortening of telomeres. Both parents as confirmed CTC1 variant carriers showed normal blood counts. Based on this index case and literature reports, manifest CTC1 diseases follow a recessive trait, however, but it is possible that heterozygous carriers may indeed have also an increased, albeit attenuated risk of BMF leading to a later manifestation or incomplete penetrance. To test this possible scenario we screened a cohort of acquired BMF (n=538) with AA/PNH (172), MDS/AML (n=366), with deep NGS of all coding regions of CTC1. In total, we identified 10 heterozygous CTC1 variants in 10 unrelated patients (8 AA/PNH and 2 MDS); 4/10 variants (1 stop gain R1202X and 3 frameshift deletions D405fs, P999fs, E454fs) were fulfilled the criteria of Tier-1 lesions and were found in AA/PNH patients (4/172, 2.3%), The remaining 6 missense variants were of uncertain significance or likely benign(Tier-2), 2 of which (H1092G and E1136K) were found in a compound heterozygous configuration in a AA/PNH patient. K438N, as a novel missense variant, was recurrently present in 2 MDS pts. Given the expected frequency of the CTC1 variants found in controls (104/33370, 0.3%), CTC1 variants appear to be enriched in AA/PNH subgroup (p<0.001). Of note is that none of the carriers of pathogenic CTC1 mutations showed any physical signs of inherited congenital BMF syndromes or any family history of leukemia, BMF and any other cancers. Interestingly, patients with sAA/PNH syndrome and biallelic CTC1 variant eventually evolved to MDS, while the other monoallelic CTC1 carriers showed stable disease and responsed to immunosuppression. Flow cytometric telomere length measurement (adjusted for age) showed a markedly shortened telomere in the index case, as well as 1 SAA case carrying biallelic CTC1 variants and 1 AA case with co-concurrent POT1 and CTC1 variants when compared with age matched controls. A significant difference was seen when comparing telomere length between CTC1 variants carriers and age-matched normal controls, while no difference was seen among CTC1 variants carriers, AA/PNH group without any variants. Because of the curious co-incidence of heterozygous CTC1 variants and AA/PNH, we further analyzed this subgroup for the presence of other telomerase gene mutations, 11/172 Tier-1 variants, while 6 Tier-1 GLVs detected in MDS. Most frequent SNVs were found in POT1 with 5/172 (3%) in AA/PNH and 3/366 (0.8%) in MDS/AML. Notably, in one AA/PNH patient POT1 stop gain variant (Q364X) was co-concurrent with CTC1 heterozygous frame shift deletion (P999fs). The enrichment of variants in TELO-related genes in AA/PNH subgroup was found in comparison to MDS/AML (p<.01) and ExAC database controls (p<.01). Further clinical phenotype analysis indicated that 3 AA/PNH cases harboring POT1, TINF2 and TERT Tier-1 variants had family history of cancer. Family history of cancer was also found in a MDS patient with a TINF2 variant. In sum, our results indicate that heterozygous CTC1 variants were associated with otherwise typical AA with clonal outgrowth of PNH clone and the presence of this variant does not seem to preclude response to immunosuppression. Disclosures Maciejewski: Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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Mutational and Clonal Landscape of Acute Myeloid Leukemia with Myelodysplastic Related Changes

Blood ◽

10.1182/blood-2018-99-119899 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1514-1514

Author(s):

Guillermo Montalban Bravo ◽

Rashmi Kanagal-Shamanna ◽

Christopher B. Benton ◽

Simona Colla ◽

Irene Ganan-Gomez ◽

...

Keyword(s):

Bone Marrow ◽

Myeloid Leukemia ◽

Research Funding ◽

Goodness Of Fit ◽

Somatic Mutations ◽

Prior History ◽

Cytogenetic Abnormalities ◽

Genetics Research ◽

History Of ◽

Acute Myeloid

Abstract INTRODUCTION: Acute myeloid leukemia (AML) with myelodysplasia-related changes (AML-MRC) is a subtype of AML within the WHO classification system defined by morphologic, cytogenetic and clinical features. Although cytogenetic abnormalities define this group, there is little knowledge on the mutational landscape and clonal architecture of this heterogeneous group of leukemias. METHODS: We evaluated all patients (pts) with AML-MRC diagnosed and treated at The University of Texas MD Anderson Cancer Center from April 2017 to May 2018. All patients underwent conventional metaphase karyotyping. Somatic mutation analysis was done by use of an 81-gene targeted amplicon-based next generation sequencing (NGS) platform using whole bone marrow mononuclear cells. Previously described somatic mutations registered at the Catalogue of Somatic Mutations in Cancer (COSMIC) and other databases, as well as literature were considered as potential drivers. Variant allele frequency (VAF) estimates were used to evaluate clonal variant relationships. In mutations with likely loss of heterozygosity (VAF >60%), VAFs were adjusted according to zygosity. Clonal relationships were tested using Pearson goodness-of-fit tests with heterogeneity being defined in pts with goodness-of-fit p values <0.05. Mutations with significantly higher VAF in pts with p<0.05 where defined as dominant and those with significantly lower VAF as minor. RESULTS: A total of 95 pts with AML-MRC were included. Median age at diagnosis was 70 years (range 28-84). Diagnosis of AML-MRC was based on MDS-defining cytogenetic abnormalities in 30 (32%) patients, presence of >50% dysplasia in at least 2 lineages in 21 (22%) pts and due to history of prior myelodysplastic syndrome (MDS) or myelodysplastic/myeloproliferative neoplasm (MDS/MPN) in 44 (46%) pts. Among pts with a prior history of MDS or MDS/MPN, 23 (52%) had received therapy with hypomethylating agents, 1 (2%) with lenalidomide and 3 (7%) with ruxolitinib. Median bone marrow blast percentage on aspirate was 33% (range 1-94%). A total of 55 (58%) pts had complex karyotype, with 19 (20%) having monosomy 5 or del(5q), 14 (15%) having monosomy 7 or del(7q) and 18 (19%) having both. A total of 260 mutations were identified among 90 pts. The most frequently mutated gene was TP53, present in 43% of pts, followed by ASXL1, NRAS, DNMT3A, SRSF2, TET2 and U2AF1, all present in >10% pts (Figure A). The median number of detectable mutations was 2 (range 0-8) with 21 (22%) pts having 1 mutation, 28 (30%) 2, 15 (16%) 3, 9 (10%) 4, 7 (7%) 5, 6 (6%) 6, 1 (1%) 7 and 3 (3%) 8 mutations. Mutations in TP53 were more commonly observed in pts in whom the diagnosis of AML-MRC was due to cytogenetic abnormalities (p=0.001). In addition, mutations in RUNX1 were more commonly observed in pts with a known prior history of MDS (p=0.038). Mutations in ASXL1 were significantly associated with NRAS (r=0.338, p=0.01), SETBP1 (r=0.471, p<0.001), STAG2 (r=0.54, p<0.001) and SRSF2 (r=0.337, p=0.001) mutations. A significant association was found between STAG2 and U2AF1 mutations (r=0.438, p<0.001). Variant allele frequencies of identified mutations in genes found to be mutated in at least 4 pts are shown in Figure B. Clonal relationships were studied among pts with 2 or more detectable mutations (n=69). Among these, 44 pts (64%) were found to have clonal heterogeneity with presence of multiple clones. Clonal dominance of identified mutations is shown in Figure C. Mutations in ASXL1, BCOR, IDH1, SF3B1, SRSF2, TP53 and U2AF1 tended to appear in dominant clones while mutations In IKZF1, JAK2, KRAS, NRAS and PTPN11 were more commonly observed within minor clones. CONCLUSION: AML-MRC is a heterogeneous sub-type of AML with diverse mutational abnormalities. Further characterization of molecular abnormalities and their clonal context may define distinct subgroups within this WHO entity. Figure. Figure. Disclosures Colla: Abbvie: Research Funding. Sasaki:Otsuka Pharmaceutical: Honoraria. Ravandi:Amgen: Honoraria, Research Funding, Speakers Bureau; Abbvie: Research Funding; Orsenix: Honoraria; Abbvie: Research Funding; Sunesis: Honoraria; Xencor: Research Funding; Seattle Genetics: Research Funding; Amgen: Honoraria, Research Funding, Speakers Bureau; Macrogenix: Honoraria, Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Seattle Genetics: Research Funding; Sunesis: Honoraria; Macrogenix: Honoraria, Research Funding; Jazz: Honoraria; Astellas Pharmaceuticals: Consultancy, Honoraria; Xencor: Research Funding; Orsenix: Honoraria; Jazz: Honoraria; Bristol-Myers Squibb: Research Funding; Bristol-Myers Squibb: Research Funding. Kadia:Novartis: Consultancy; Amgen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Takeda: Consultancy; Abbvie: Consultancy; Jazz: Consultancy, Research Funding; Abbvie: Consultancy; BMS: Research Funding; Celgene: Research Funding; Amgen: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; BMS: Research Funding; Novartis: Consultancy; Takeda: Consultancy; Celgene: Research Funding. Cortes:novartis: Research Funding. Daver:Kiromic: Research Funding; Pfizer: Consultancy; Karyopharm: Consultancy; Novartis: Consultancy; Daiichi-Sankyo: Research Funding; ImmunoGen: Consultancy; Incyte: Consultancy; ARIAD: Research Funding; Alexion: Consultancy; Novartis: Research Funding; Sunesis: Consultancy; Karyopharm: Research Funding; Otsuka: Consultancy; Sunesis: Research Funding; BMS: Research Funding; Pfizer: Research Funding; Incyte: Research Funding. DiNardo:Medimmune: Honoraria; Abbvie: Honoraria; Karyopharm: Honoraria; Agios: Consultancy; Celgene: Honoraria; Bayer: Honoraria. Jabbour:novartis: Research Funding. Konopleva:Stemline Therapeutics: Research Funding; cellectis: Research Funding; Immunogen: Research Funding; abbvie: Research Funding.

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