Genetic Background of Idiopathic Bone Marrow Failure Syndromes in Children

Abstract Introduction Pancytopenia with a severe decrease in bone marrow (BM) cellularity in children may be caused by a broad variety of underlying disorders. The appropriate classification of bone marrow failure syndromes in children is challenging, particularly with respect to the histological distinction between aplastic anemia (AA), refractory cytopenia of childhood (RCC), and refractory cytopenia with multilineage dysplasia (RCMD). Inherited bone marrow failure syndromes (IBMFS) further defy accurate diagnosis. Clonal hematopoiesis in AA is indicated by the presence of paroxysmal nocturnal hemoglobinuria (PNH) cells and by the identification of uniparental disomies involving chromosome 6p. In addition, "clonal transformation," as defined by the development of myelodysplastic syndromes (MDS) or acute myelogenous leukemia has been noted in about 15% of AA patients. In adult patients with AA, somatic mutations were frequently detected in myeloid malignancy-related genes such as DNMT3A, BCOR, and ASXL1. We aimed to characterize the genetic background of childhood AA/RCC/RCMD. Patients and Methods We studied 168 patients with idiopathic AA/RCC/RCMD in children. Diagnosis with AA, RCC, and RCMD was made on the basis of the 2008 World Health Organization classification criteria. Blood, bone marrow, and buccal samples were obtained from the patients after written informed consent was received according to protocols approved by the ethics committee of Nagoya University Graduate School of Medicine. Target sequencing (n = 168) was performed for 88 IBMFS-associated genes and 96 myeloid malignancy-related genes. Furthermore, whole-exome sequencing (WES, n = 25) was performed with matched tumor/normal samples. The mean depth for targeted sequencing was 451x, and the mean depth for WES was 103x. Somatic mutations were detected with the use of a frequency threshold of 0.07 (WES) or 0.02 (targeted sequencing) for variant allele frequency and were individually validated with the use of deep sequencing of polymerase-chain-reaction-amplified targets. Results Only one germline mutation that was diagnostic of IBMFS was detected in our cohort (0.6%). It was a RTEL1 mutation, which supported the diagnosis of dyskeratosis congenita. Telomere length of the patient with a RTEL1 mutation was shorter compared with that of age-matched healthy individuals (−3.2 Standard Deviation). WES, performed in 25 patients, detected only three somatic mutations, all of which affected BCOR. In target sequencing, 20 somatic mutations were detected in 19 patients (11.3%). BCOR (n = 9) and PIGA (n = 4) were recurrently mutated. The mutational frequency of DNMT3A and ASXL1 was very low (0.6%) in our cohort and was clearly different from that of an adult cohort. The majority of somatic mutations carried low variant allele frequency. In case of BCOR mutations, the variant allele frequency tended to be low, suggesting subclonal composition. In case of U2AF1 mutations, the variant allele frequency tended to be high, which suggests that the U2AF1 -mutated clone was dominant in the bone marrow. The difference in the frequency of somatic mutations in AA, RCC, and RCMD was not statistically significant (p = 0.49). However, with regard to the mutated genes, two patients with RCMD carried U2AF1 plus SETBP1 and TP53 mutations, respectively, which are well-known predictors of poor prognosis in adult MDS. The patient who carried U2AF1 plus SETBP1 developed MDS later and underwent bone marrow transplantation. Of the 19 patients with mutated genes, 15 patients were treated with immunosuppressive therapy (IST). The response rate to IST at 6 months was 60% in the patients with somatic mutations, which was equivalent to those without mutations. Conclusions In our cohort of children who were clinically diagnosed with AA/RCC/RCMD, the frequency of cryptic IBMFS was considered low. Furthermore, the frequency of detectable somatic mutations in childhood AA was low compared with that in adult AA. No novel mutational target was identified with WES. Idiopathic bone marrow failure syndromes in children were characterized by a paucity of gene mutations irrespective of the histopathological classification. Mutations in adult MDS-related genes suggest molecular pathogenesis is different between RCMD and AA/RCC. In conclusion, our study clarified the yet unrevealed genetic background of idiopathic bone marrow failure syndromes in children. Disclosures No relevant conflicts of interest to declare.

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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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Whole Exome Sequencing Shows a Paucity Of Somatic Gene Mutations In Pediatric Idiopathic Bone Marrow Failure Syndrome

Blood ◽

10.1182/blood.v122.21.3708.3708 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3708-3708

Author(s):

Atsushi Narita ◽

Hideki Muramatsu ◽

Kenichi Yoshida ◽

Yusuke Okuno ◽

Asahito Hama ◽

...

Keyword(s):

Bone Marrow ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Somatic Mutations ◽

Bone Marrow Failure ◽

Gene Mutations ◽

Bone Marrow Failure Syndrome ◽

Cell Lineages ◽

Bone Marrow Failure Syndromes ◽

Whole Exome

Abstract Introduction Pancytopenia accompanied by a severe decrease in bone marrow (BM) cellularity in children can be due to a broad variety of underlying disorders. Appropriate classification of bone marrow failure syndrome in children is challenging, particularly in relation to the morphological distinction between aplastic anemia (AA), refractory cytopenia of childhood (RCC), and refractory cytopenia with multilineage dysplasia (RCMD). The goal of this study was to characterize the molecular pathogenesis of these conditions by identifying the full spectrum of gene mutations in 29 patients with these disorders through the use of exome sequencing. Patient and Methods Diagnosis of AA, RCC, or RCMD was made on basis of the 2008 World Health Organization (WHO) classification criteria. AA patients exhibited no morphologically dysplastic changes in any of their hematopoietic cell lineages, while RCC patients had<10% dysplastic changes in two or more cell lineages or >10% in one cell lineage. Patients classified as RCMD exhibited >10% of the dysplastic changes in two or more cell lineages. Blood and BM samples were obtained from 29 children (16 boys and 13 girls) with AA (n = 8), RCC (n = 11), or RCMD (n = 10). The median age at diagnosis was 11 years (range, 2–15 years). Exome capture from paired DNA (non-T cells/CD3+ lymphocyte) was performed using SureSelect® Human All Exon V3 (Agilent Technologies, Santa Clara, CA) covering 50 Mb of the coding exons, followed by massive parallel sequencing using HiSeq 2000 (Illumina, San Diego, CA) according to the manufacturer’s protocol. Candidate somatic mutations were detected through our pipeline for whole exome sequencing (genomon: http://genomon.hgc.jp/exome/index.html). All candidate somatic nucleotide changes were validated by Sanger sequencing. Results Exome sequencing pipeline identified a total of 193 non-synonymous somatic mutations or indels candidates among the 29 patients (range, 2–15 per patient). After validation by Sanger sequencing, one nonsense, 11 missense, and two frame-shift mutations were confirmed as non-silent somatic mutations. The average numbers of mutations per sample were not significantly different when comparing morphological diagnostic groups (0.50 in AA, 0.36 in RCC, 0.60 in RCMD). Of these validated genes, BCOR (n = 2) and CSK (n = 2) mutations were recurrent genetic events. BCOR is a frequent mutational target in myelodysplastic syndrome, whereas CSK somatic mutations were not reported in human cancers. BCOR mutations were found both in AA (c.472delA:p.S158fs; patient 13) and in RCMD (c.G3856T:p.E1286X; patient 39). Both patients with CSK mutations were classified as RCC (c.G994A:p.D332N; patient 23 and 27). When comparing the clinical outcomes of patients with somatic mutations (n = 7) versus those without somatic mutations (n = 22), response rate to immunosuppressive therapy at 6 months (50% vs. 50%), 5-year clonal evolution rate (95% confidential interval) [0% (0% - 0%) vs. 6% (0% - 26%)], and the 5-year overall survival rate (95% confidential interval) [100% (100% - 100%) vs. 95% (70% - 99%)] were not significantly different. Conclusion Whole exome sequencing analysis was used for gene mutational profiling of patients with idiopathic bone marrow failure syndromes; i.e., AA, RCC, and RCMD. Although BCOR and CSK somatic mutations were recurrently identified, idiopathic bone marrow failure syndromes in children are characterized by a paucity of gene mutations, irrespective of morphological diagnosis. These findings suggest that morphological diagnosis based on WHO classification system does not discriminate the mutational profile and pathogenesis of bone marrow failure in children. Disclosures: No relevant conflicts of interest to declare.

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Telomere Length in Inherited Bone Marrow Failure Syndromes

Blood ◽

10.1182/blood.v124.21.1609.1609 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1609-1609

Author(s):

Blanche P. Alter ◽

Neelam Giri ◽

Sharon A Savage ◽

Philip S. Rosenberg

Keyword(s):

Bone Marrow ◽

T Cells ◽

Bone Marrow Failure ◽

Canonical Variate ◽

Dna Repeats ◽

Z Score ◽

Essentially Normal ◽

Bone Marrow Failure Syndromes ◽

Z Scores ◽

The Mean

Abstract Background: Telomeres are short DNA repeats and a protein complex at chromosome ends that are essential for genome integrity. Telomeres are very short in patients with dyskeratosis congenita (DC) due to germline mutations in telomere biology genes. Telomere lengths (TL) were reported to be “short” in a few small studies of patients with other inherited bone marrow failure syndromes (IBMFS), specifically Fanconi anemia (FA), Diamond-Blackfan anemia (DBA), and Shwachman-Diamond syndrome (SDS), although their relation to the very short TL seen in DC was not clear, and various analytic methods were used. Objectives: 1) To compare TL in a cohort of patients with FA, DBA, and SDS, with TL in DC. 2) To determine whether any individuals with non-DC IBMFS had TL as short as in DC. Methods: Blood was obtained from 100 patients with DC, 30 FA, 34 DBA, and 14 SDS enrolled in the NCI IBMFS Cohort Study. TL was measured by flow cytometry with fluorescence in situ hybridization (flow-FISH) in 6 leukocyte subsets: granulocytes, total lymphocytes, CD45RA-positive/CD20-negative naïve T cells, CD45RA-negative memory T cells, CD20-positive B cells, and CD57-positive NK/NKT cells. “Very short” TL was below the 1st percentile for age (defined by 400 normal controls). The diagnosis of DC required very short TL in 3 of 4 lymphocyte lineages including total lymphocytes. Z-scores were used to adjust for age; a Z-score of -2.3 equals the 1st percentile. Canonical Variate Analysis (CVA) including all 6 Z-score measurements was used for group comparisons and outlier identification. Results: Two patients with FA and 1 each with DBA and SDS had TL slightly below the 1st percentile in 3 or more lymphocyte lineages. In univariate analysis, the mean of the DC TL Z-scores was approximately -4, versus approximately -0.5 in FA, DBA, and SDS. Most individual DC Z-score measurements were well below -2.3, and all were below zero (the mean for the control data). Seventy percent of the FA, DBA and SDS lymphocyte TL Z-scores clustered in the bottom half of the normal range (i.e. -2.3 to 0, p <0.001), compared with the expected 50% if the non-DC patients had essentially normal TLs. In addition, short lymphocyte TL correlated with severity of marrow failure in DC; there was a non-significant trend in FA and no trend in DBA or SDS. Further refinement of interpretation utilized the CVA analysis. The first canonical variate captured 89.7% of the total variance, and separated DC widely from the other three IBMFS. The second canonical variate captured 10% of the variance, and separated DBA, FA, and SDS. In the CVA analysis, only 2 patients with FA and 1 each with DBA and SDS had results closer to the mean for DC patients than to the mean for their own disorder. Conclusions: Only 5% of individuals with FA, DBA, or SDS had TL in the DC range, and thus non-DC IBMFS in general do not have “very short” TL. However, 70% of non-DC IBMFS patients did have average TLs below the average for normal individuals. Thus disorders of DNA repair or ribosome biogenesis may result in TL changes, but the deficit in TL is not nearly as extreme as in DC itself. Disclosures No relevant conflicts of interest to declare.

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Very Short Telomeres Are Characteristic of Dyskeratosis Congenita and Not Other Inherited Bone Marrow Failure Syndromes

Blood ◽

10.1182/blood.v112.11.1044.1044 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1044-1044

Author(s):

Blanche P. Alter ◽

Gabriela Baerlocher ◽

Neelam Giri ◽

Peter M. Lansdorp ◽

Sharon A Savage

Keyword(s):

Bone Marrow ◽

T Cells ◽

B Cells ◽

Telomere Length ◽

Bone Marrow Failure ◽

Dyskeratosis Congenita ◽

Z Score ◽

Bone Marrow Failure Syndromes ◽

Z Scores ◽

The Mean

Abstract Telomeres protect the ends of chromosomes, shorten with age, and are very short in dyskeratosis congenita (DC), an inherited bone marrow failure syndrome (IBMFS) associated with mutations in telomere biology genes. “Short telomeres” were reported in Fanconi Anemia (FA), Diamond-Blackfan Anemia (DBA) and Shwachman-Diamond Syndrome (SDS) using telomere restriction fragment length or Q-FISH assays of total leukocyte or mononuclear cell DNA. These reports focused on group averages, not results from individual patients. Our objective was to determine which categories of IBMFS patients have very short telomeres, and in which leukocyte subsets, using a more sensitive and specific assay. Telomere length was measured in granulocytes, lymphocytes, naïve T-cells, memory T-cells, B-cells, and NK cells using automated multicolor flow fluorescence in situ hybridization (FISH). We previously showed that very short telomeres (<1st percentile for age) in lymphocytes, naïve T-cells, and B-cells were sensitive and specific for the diagnosis of DC (Alter et al, Blood110:1439, 2007). Granulocytes were not specific, since in that study neutropenic patients without DC often had very short granulocyte telomeres. The current study included 53 DC patients and 87 relatives, 19 non-transplanted FA patients and 12 relatives, 21 DBA patients and 11 relatives, and 7 SDS patients and 7 relatives. There were 400 normal controls, ages 0 to 100; very short telomeres were defined as <1st percentile of normal for age. Z-scores were calculated to adjust for age; 0 is normal and -2 or below is significantly short (i.e. <2 standard deviations below the mean). Lymphocyte telomeres were <1st percentile (very short) in 96% of DC patients, 7% of DC relatives, 16% of FA patients, 5% of DBA patients, 14% of SDS patients, and none of the non-DC relatives. Granulocyte telomeres were very short in 94% of DC patients, 16% of DC relatives, 44% of FA patients, 19% of DBA patients, 14% of SDS patients, 8% of FA relatives, and none of the non-DC relatives. All three hallmark leukocyte subsets (lymphocytes, naïve T-cells, and B-cells) were very short in 81% of DC patients, 5% of DC relatives, 5% of DBA patients, and none of the FA or SDS patients or their relatives. Although 4 out of 47 (9%) IBMFS other than DC had very short telomeres in lymphocytes, only one, a patient with DBA (without mutations in known DC genes) had very short telomeres in all 3 of the hallmark lineages. Four of 87 DC relatives also had very short telomeres in all 3 lineages; they may be silent carriers in families in which DC genes have not yet been identified. The mean Z-score for lymphocyte telomeres in DC was -4.7; it was above -2 in all other categories (-0.3 in FA, -0.9 in DBA, -1 in SDS). The mean Z-score for granulocytes was -4.2 in DC, -1.9 in FA, -1.2 in DBA, and -1.5 in SDS. Thus, based on the Z-scores, the average telomere length was very short in DC lymphocytes and granulocytes; normal in FA lymphocytes and borderline in FA granulocytes; and normal in DBA and SDS lymphocytes and granulocytes. Flow-FISH provides analysis of multiple cell types in individual patients, and identification of specific individuals with very short telomeres, as well as the age-adjusted mean telomere length in a diagnostic subset. No FA or SDS patients, and only one with DBA, met the DC diagnostic criteria of very short telomeres in three or more lymphocyte subsets. The telomere length deficit in DC appears to be more severe and more frequent than in the other inherited bone marrow failure syndromes.

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Assessment of SBDS Expression Patterns in Human Marrow Hematopoietic and Stromal Cells by Immunohistochemistry and Its Use as a Diagnostic Screen for Schwachman-Diamond Syndrome

Blood ◽

10.1182/blood.v112.11.41.41 ◽

2008 ◽

Vol 112 (11) ◽

pp. 41-41 ◽

Cited By ~ 1

Author(s):

Trisha Wong ◽

Monica Calicchio ◽

Mark D. Fleming ◽

Akiko Shimamura ◽

Marian Hester Harris

Keyword(s):

Bone Marrow ◽

Gene Conversion ◽

Expression Pattern ◽

Plasma Cells ◽

Bone Marrow Failure ◽

Expression Patterns ◽

Missense Mutations ◽

Bone Marrow Failure Syndromes ◽

The Mean ◽

Normal Controls

Abstract Objective. Shwachman Diamond Syndrome (SDS) is an autosomal recessive syndrome characterized clinically by exocrine pancreatic insufficiency, bone marrow dysfunction, skeletal abnormalities and a predisposition to leukemia. Molecularly, it is characterized by mutations in the Shwachman-Bodian-Diamond-Syndrome (SBDS) gene encoded on chromosome 7. The majority of mutant alleles (74%) associated with SDS are the result of gene conversion events with an adjacent SBDS pseudogene, leading to truncation, splice site, and frameshift mutations and a marked decrease in SBDS expression. The remaining pathogenic alleles include frameshift and, rarely, missense changes1. In this study, we examined the SBDS expression pattern in bone marrows from patients with SDS compared to normal controls or patients with other bone marrow failure syndromes using a new immunohistochemical (IHC) assay. Methods. Archived bone marrow biopsy tissue sections from patients with SDS (21 specimens from 9 patients), idiopathic aplastic anemia (AA, 12 patients), Fanconi Anemia (FA, 4 patients), Diamond-Blackfan Anemia (DBA, 9 patients), Severe Congenital Neutropenia (SCN, 3 patients) and 13 normal controls were stained with an antibody directed against the C-terminus of SBDS. Four cell lineages (myeloid precursors, megakaryocytes, plasma cells and osteoblasts) were scored in a blinded fashion by two pathologists on a scale from 0 (negative) to 3 (strong). The scores from each cell lineage within each diagnostic group were averaged to yield an overall IHC score. Results. We first characterized the SDS expression pattern in normal controls. Promyelocytes, early myelocytes, megakaryocytes, plasma cells, and osteoblasts all showed high levels of SBDS protein. In the myeloid lineage, SBDS expression decreased to undetectable levels in mature granulocytes, consistent with prior reports that SBDS is not necessary for terminal neutrophil differentiation. SBDS staining was not detectable in erythroid precursors or lymphocytes. We next compared SBDS IHC staining in these normal controls to that seen in SDS patients and patients with other bone marrow failure syndromes. The mean overall IHC score for SDS patients was 0.6 whereas it was 2.5 in normal controls, 2.23 in AA, 2.4 in DBA, 2.67 in FA and 2.38 in SCN. Four samples from two SDS patients with rare missense mutations in SBDS had moderate staining, consistent with previous studies that showed intermediate levels of SBDS expression in patients with missense mutations. When the analysis was restricted to patients harboring biallelic gene conversion SBDS mutations, the mean SBDS IHC score dropped to 0.14. Conclusion. SBDS protein is highly expressed in early myeloid progenitors, megakaryocytes, and osteoblasts. This novel SBDS IHC assay may provide a rapid screen for the most common SBDS mutations.

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

Blood ◽

10.1182/blood.v122.21.522.522 ◽

2013 ◽

Vol 122 (21) ◽

pp. 522-522

Author(s):

Swapna Thota ◽

Sarah McMahon ◽

Bartlomiej Przychodzen ◽

Thomas LaFramboise ◽

Hideki Makishima ◽

...

Keyword(s):

Bone Marrow ◽

Family History ◽

Research Funding ◽

Somatic Mutations ◽

Germ Line ◽

Bone Marrow Failure ◽

Telomere Maintenance ◽

Myeloid Neoplasms ◽

History Of ◽

The Mean

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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Randomized, Single-Blind, Multicenter Phase II Study of Two Doses of Imetelstat in Relapsed or Refractory Myelofibrosis

Journal of Clinical Oncology ◽

10.1200/jco.20.02864 ◽

2021 ◽

pp. JCO.20.02864

Author(s):

John Mascarenhas ◽

Rami S. Komrokji ◽

Francesca Palandri ◽

Bruno Martino ◽

Dietger Niederwieser ◽

...

Keyword(s):

Bone Marrow ◽

Allele Frequency ◽

Phase Ii ◽

Phase Ii Study ◽

Variant Allele ◽

Variant Allele Frequency ◽

Bone Marrow Fibrosis ◽

End Points ◽

Symptom Response ◽

Marrow Fibrosis

PURPOSE Patients with myelofibrosis who are relapsed or refractory (R/R) to Janus-associated kinase inhibitors (JAKis) have poor clinical outcomes including dismal overall survival (OS) that ranges between 13 and 16 months. Imetelstat, a telomerase inhibitor, was evaluated in patients with intermediate-2 or high-risk myelofibrosis R/R to JAKi in a phase II multicenter study (ClinicalTrials.gov identifier: NCT02426086 ). PATIENTS AND METHODS Patients were randomly assigned to receive either imetelstat 9.4 mg/kg or 4.7 mg/kg intravenous once every 3 weeks. Spleen response (≥ 35% spleen volume reduction) and symptom response (≥ 50% reduction in total symptom score) rates at week 24 were coprimary end points. Secondary end points included OS and safety. RESULTS Study enrollment was closed early, and patients treated with 4.7 mg/kg were permitted to continue treatment with 9.4 mg/kg. At week 24, spleen and symptom response rates were 10.2% and 32.2% in the 9.4-mg/kg arm and 0% and 6.3% in the 4.7-mg/kg arm. Treatment with imetelstat 9.4 mg/kg led to a median OS of 29.9 months and bone marrow fibrosis improvement in 40.5% and variant allele frequency reduction of driver mutations in 42.1% of evaluable patients. Fibrosis improvement and variant allele frequency reduction correlated with OS. Target inhibition was demonstrated by reduction of telomerase activity and human telomerase reverse transcriptase level and correlated with spleen response, symptom response, and OS. Most common adverse events on both arms were grade 3 or 4 reversible cytopenias. CONCLUSION In this phase II study of two imetelstat doses, 9.4 mg/kg once every 3 weeks demonstrated clinical benefits in symptom response rate, with an acceptable safety profile for this poor-risk JAKi R/R population. Biomarker and bone marrow fibrosis assessments suggested selective effects on the malignant clone. A confirmatory phase III study is currently underway.

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