Clonal hematopoiesis in acquired aplastic anemia

AbstractClonal hematopoiesis (CH) in aplastic anemia (AA) has been closely linked to the evolution of late clonal disorders, including paroxysmal nocturnal hemoglobinuria and myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML), which are common complications after successful immunosuppressive therapy (IST). With the advent of high-throughput sequencing of recent years, the molecular aspect of CH in AA has been clarified by comprehensive detection of somatic mutations that drive clonal evolution. Genetic abnormalities are found in ∼50% of patients with AA and, except for PIGA mutations and copy-neutral loss-of-heterozygosity, or uniparental disomy (UPD) in 6p (6pUPD), are most frequently represented by mutations involving genes commonly mutated in myeloid malignancies, including DNMT3A, ASXL1, and BCOR/BCORL1. Mutations exhibit distinct chronological profiles and clinical impacts. BCOR/BCORL1 and PIGA mutations tend to disappear or show stable clone size and predict a better response to IST and a significantly better clinical outcome compared with mutations in DNMT3A, ASXL1, and other genes, which are likely to increase their clone size, are associated with a faster progression to MDS/AML, and predict an unfavorable survival. High frequency of 6pUPD and overrepresentation of PIGA and BCOR/BCORL1 mutations are unique to AA, suggesting the role of autoimmunity in clonal selection. By contrast, DNMT3A and ASXL1 mutations, also commonly seen in CH in the general population, indicate a close link to CH in the aged bone marrow, in terms of the mechanism for selection. Detection and close monitoring of somatic mutations/evolution may help with prediction and diagnosis of clonal evolution of MDS/AML and better management of patients with AA.

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Clonality in context: hematopoietic clones in their marrow environment

Blood ◽

10.1182/blood-2017-07-794362 ◽

2017 ◽

Vol 130 (22) ◽

pp. 2363-2372 ◽

Cited By ~ 40

Author(s):

James N. Cooper ◽

Neal S. Young

Keyword(s):

Direct Relationship ◽

Somatic Mutations ◽

Clonal Selection ◽

Bone Marrow Failure ◽

Clonal Evolution ◽

Clinical Results ◽

Clonal Hematopoiesis ◽

Relationship Of ◽

Apparently Healthy

Abstract Clonal hematopoiesis occurs normally, especially with aging, and in the setting of disease, not only in myeloid cancers but in bone marrow failure as well. In cancer, malignant clones are characterized by recurrent somatic mutations in specific sets of genes, but the direct relationship of such mutations to leukemogenesis, when they occur in cells of an apparently healthy older individual or after recovery from immune aplastic anemia, is uncertain. Here we emphasize a view of clonal evolution that stresses natural selection over deterministic ontogeny, and we stress the selective role of the environment of the marrow and organism. Clonal hematopoieses after chemotherapy, in marrow failure, and with aging serve as models. We caution against the overinterpretation of clinical results of genomic testing in the absence of a better understanding of clonal selection and evolution.

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A brief, but comprehensive, guide to clonal evolution in aplastic anemia

Hematology ◽

10.1182/asheducation-2018.1.457 ◽

2018 ◽

Vol 2018 (1) ◽

pp. 457-466 ◽

Cited By ~ 8

Author(s):

Daria V. Babushok

Keyword(s):

Aplastic Anemia ◽

Somatic Mutations ◽

Clonal Evolution ◽

Clinical Care ◽

Hla Class I ◽

The Elderly ◽

Hematologic Malignancies ◽

Class I ◽

Clonal Hematopoiesis ◽

Immune Mediated

Abstract Acquired aplastic anemia (AA) is an immune-mediated bone marrow aplasia that is strongly associated with clonal hematopoiesis upon marrow recovery. More than 70% of AA patients develop somatic mutations in their hematopoietic cells. In contrast to other conditions linked to clonal hematopoiesis, such as myelodysplastic syndrome (MDS) or clonal hematopoiesis of indeterminate potential in the elderly, the top alterations in AA are closely related to its immune pathogenesis. Nearly 40% of AA patients carry somatic mutations in the PIGA gene manifested as clonal populations of cells with the paroxysmal nocturnal hemoglobinuria phenotype, and 17% of AA patients have loss of HLA class I alleles. It is estimated that between 20% and 35% of AA patients have somatic mutations associated with hematologic malignancies, most characteristically in the ASXL1, BCOR, and BCORL1 genes. Risk factors for evolution to MDS in AA include the duration of disease, acquisition of high-risk somatic mutations, and age at AA onset. Emerging data suggest that several HLA class I alleles not only predispose to the development of AA but may also predispose to clonal evolution in AA patients. Long-term prospective studies are needed to determine the true prognostic implications of clonal hematopoiesis in AA. This article provides a brief, but comprehensive, review of our current understanding of clonal evolution in AA and concludes with 3 cases that illustrate a practical approach for integrating results of next-generation molecular studies into the clinical care of AA patients in 2018.

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Myeloid Tumor-Related Somatic Mutations in 293 Children and Adults with Acquired Aplastic Anemia in Chinese

Blood ◽

10.1182/blood-2019-129813 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5024-5024

Author(s):

Hailong He ◽

Hua Yin ◽

Xiaoqing Liu ◽

Lang Cheng ◽

Guangsheng He ◽

...

Keyword(s):

Aplastic Anemia ◽

Somatic Mutations ◽

Response Rate ◽

Conflicts Of Interest ◽

Clonal Evolution ◽

Telomere Maintenance ◽

Adult Group ◽

Myeloid Malignancies ◽

Clonal Hematopoiesis ◽

Age Related

ABSTRACT Objectives and Methods: It was postulated that the subset of aplastic anemia ( AA ) with somatic mutations typically seen in myeloid malignancies were at risk of clonal evolution[1,2]. But the response rate to immunosuppressive therapy (IST) was different or not between patients with or without mutations, remain unclear[1,2]. Age of patients with AA is a strong predictor of outcome for IST, and the presence of somatic mutations also rise appreciably in frequency with age[3]. Here we checked myeloid tumor-related somatic mutations from 293 children and adult patients who have acquired AA. We excluded Fanconi anemia, mutations of telomere maintenance, and a family history of BM failure (BMF) or cancer. We looked for somatic mutations by identifying previously characterized single-nucleotide variants in 25 genes that are recurrently mutated in myeloid malignancies in the peripheral blood cells by second-generation gene sequencing. And clinical significance of mutations in different ages was also annalyzed. Results : Somatic mutations in myeloid tumor-related genes are present in 6.48% of the patients. The the variants occurred in ten genes: ASXL1 (n = 2 ), KRAS (n = 1), PIGA (n = 2 ), TP53 (n = 2 ), BCOR (n = 2 ), TET2 (n = 5 ), SF3B1 (n = 1), DNMT3A (n = 2 ), SH2B3 (n = 1), MPL (n = 1) (Figure 1). The majority of the variants occurred in epigenetic genes: TET2, DNMT3A, ASXL1. Among patients of children and teen agers' group, adult group, the clonal mutations were observed in 2.82%, 9.93% (P= 0.013) (Figure 2). The presence of a somatic mutation was not associated with gender ( male or female, 3.87% vs 9.42%, P=0.054), severity ( non severe AA or severe AA, 7.87% vs 4.35%, P=0.233). Patients with somatic mutations had a simillar response rate, compared with patients without mutations in children and teen agers' group, or adult group. Somatic mutations in AA patients were not associated with high risk of transformation to MDS (P > 0.5 ). Conclusions: Age-related clonal hematopoiesis rise along age in patients with AA in chinese, but somatic mutations in AA patients were associated with efectiveness of IST, and risk of clonal evolution. References 1 Kulasekararaj AG, Jiang J, Smith AE, et al. Somatic mutations identify a subgroup of aplastic anemia patients who progress to myelodysplastic syndrome. Blood. 2014; 124(17):2698-2704). 2 Yoshizato T, Dumitriu B, Hosokawa K et al. Somatic Mutations and Clonal Hematopoiesis in Aplastic Anemia. N Engl J Med. 2015 Jul 2; 373(1):35-47. 3 Jaiswal S, Fontanillas P, Flannick J, et al. Age-Related Clonal Hematopoiesis Associated with Adverse Outcomes. N Engl J Med 2014; 371:2488-98 Disclosures No relevant conflicts of interest to declare.

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Paroxysmal Nocturnal Hemoglobinuria Clones Are Infrequent in Hepatitis-Associated Aplastic Anemia at the Time of Diagnosis

Blood ◽

10.1182/blood-2019-125620 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5016-5016

Author(s):

Wenrui Yang ◽

Xin Zhao ◽

Guangxin Peng ◽

Li Zhang ◽

Liping Jing ◽

...

Keyword(s):

Aplastic Anemia ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Clonal Evolution ◽

Extrinsic Factors ◽

Clone Size ◽

Hematopoietic Stem ◽

Over Time ◽

Pnh Clone

Aplastic anemia (AA) is an immune-mediated bone marrow failure, resulting in reduced number of hematopoietic stem and progenitor cells and pancytopenia. The presence of paroxysmal nocturnal hemoglobinuria (PNH) clone in AA usually suggests an immunopathogenesis in patients. However, when and how PNH clone emerge in AA is still unclear. Hepatitis associated aplastic anemia (HAAA) is a special variant of AA with a clear disease course and relatively explicit immune pathogenesis, thus serves as a good model to explore the emergence and expansion of PNH clone. To evaluate the frequency and clonal evolution of PNH clones in AA, we retrospectively analyzed the clinical data of 90 HAAA patients that were consecutively diagnosed between August 2006 and March 2018 in Blood Diseases Hospital, and we included 403 idiopathic AA (IAA) patients as control. PNH clones were detected in 8 HAAA patients (8.9%,8/90) at the time of diagnosis, compared to 18.1% (73/403) in IAA. Eight HAAA patients had PNH clone in granulocytes with a median clone size of 3.90% (1.09-12.33%), and 3 patients had PNH clone in erythrocytes (median 4.29%, range 2.99-10.8%). Only one HAAA patients (1/8, 12.5%) had a PNH clone larger than 10%, while 24 out of 73 IAA patients (32.9%) had larger PNH clones. Taken together, we observed a less frequent PNH clone with smaller clone size in HAAA patients, compared to that in IAAs. We next attempted to find out factors that associated with PNH clones. We first split patients with HAAA into two groups based on the length of disease history (≥3 mo and < 3mo). There were more patients carried PNH clone in HAAA with longer history (21.4%, 3/14) than patients with shorter history (6.6%, 5/76), in line with higher incidence of PNH clone in IAA patients who had longer disease history. Then we compared the PNH clone incidence between HAAA patients with higher absolute neutrophil counts (ANC, ≥0.2*109/L) and lower ANC (< 0.2*109/L). Interestingly, very few VSAA patients developed PNH clone (5%, 3/60), while 16.7% (5/30) of non-VSAA patients had PNH clone at diagnosis. We monitored the evolution of PNH clones after immunosuppressive therapy, and found increased incidence of PNH clone over time. The overall frequency of PNH clone in HAAA was 20.8% (15/72), which was comparable to that in IAA (27.8%, 112/403). Two thirds of those new PNH clones occurred in non-responders in HAAA. In conclusion, PNH clones are infrequent in HAAA compared to IAA at the time of diagnosis, but the overall frequency over time are comparable between the two groups of patients. In SAA/VSAA patients who are under the activated abnormal immunity, longer clinical course and relatively adequate residual hematopoietic cells serve as two important extrinsic factors for HSCs with PIGA-mutation to escape from immune attack and to expand. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v126.23.300.300 ◽

2015 ◽

Vol 126 (23) ◽

pp. 300-300 ◽

Cited By ~ 1

Author(s):

Bhumika J. Patel ◽

Bartlomiej Przychodzen ◽

Michael J. Clemente ◽

Cassandra M. Hirsch ◽

Tomas Radivoyevitch ◽

...

Keyword(s):

Aplastic Anemia ◽

Board Of Directors ◽

Deep Sequencing ◽

Somatic Mutations ◽

Clonal Evolution ◽

Clonal Expansion ◽

Quantitative Detection ◽

Sequencing Analysis ◽

Advisory Committees ◽

Before And After

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

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HLA Class I Allele-Lacking Hematopoietic Stem/Progenitor Cells Support Long-Term Clonal Hematopoiesis without Oncogenic Driver Mutations in Acquired Aplastic Anemia

Blood ◽

10.1182/blood.v128.22.3894.3894 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3894-3894

Author(s):

Tatsuya Imi ◽

Takamasa Katagiri ◽

Kazuyoshi Hosomichi ◽

Noriharu Nakagawa ◽

Yoshitaka Zaimoku ◽

...

Keyword(s):

Aplastic Anemia ◽

Progenitor Cells ◽

Somatic Mutations ◽

Hla Class I ◽

Driver Mutations ◽

Hematopoietic Stem ◽

Target Sequencing ◽

Clonal Hematopoiesis ◽

Oncogenic Driver

Abstract [Background] Clonal hematopoiesis is currently known to be common in patients with acquired aplastic anemia (AA). One of the most common abnormalities underlying clonal hematopoiesis in AA patients is copy-number neutral loss of heterozygosity (LOH) in the short of 6 chromosome (6pLOH) caused by acquired uniparental disomy. Hematopoietic stem/progenitor cells (HSPCs) having undergone 6pLOH are thought to evade attack by cytotoxic T lymphocytes (CTLs) specific to auto-antigens by lacking particular HLA-A alleles. These HSPCs then produce HLA class I allele-lacking [HLA(-)] leukocytes to support hematopoiesis in patients with AA patients in remission. Our recent study showed that HLA(-) granulocytes are detected in about 24% of newly-diagnosed AA patients, and the aberrant granulocytes often account for more than 95% of the total granulocytes and persist for many years. The sustainability of 6pLOH(+) HSPC clones suggests that these HSPCs may suffer from secondary somatic mutations that confer a proliferative advantage on them over normal HSPCs. Alternatively, 6pLOH(+) HSPCs may persist and continue to support hematopoiesis according to their inherent sustainability, just like the PIGA mutant HSPCs we previously described (Katagiri et al. Stem Cells, 2013). To test these hypotheses, we determined the sequences of genes associated with the clonal expansion of HSPCs in HLA(-) granulocytes. [Patients and Methods] Eleven AA patients whose percentages of HLA(-) granulocytes ranged 6.4%-99.8% (median 94.2%) of the total granulocyte population were chosen for this study. The patients (male/female, 5/6 and age 27-79 [median 53] years) had been diagnosed with severe (n=5) or non-severe (n=6) AA 2-25 [median 12.5] years earlier, and 7 and 4 patients achieved complete response and partial response, respectively after treatments with cyclosporine (CsA) alone (n=4), CsA+antithymocyte globulin (ATG, n=3), CsA+anabolic steroids (AS, n=2), AS+romiplostim (n=1), and AS alone (n=1). The lineage combinations of HLA(-) cells were granulocyte, monocytes, B cells and T cells (GMBT) in 6, GMB in 4 and GM in 1. HLA(-) and normal [HLA(+)] granulocytes were sorted from the blood leukocytes of the 11 patients and the DNA of each cell population as well as that of buccal mucosa cells was subjected to target sequencing of 61 myelodysplastic syndrome (MDS)-related genes with MiSeq. DNA samples from 5 patients including 4 patients whose HLA(-) cell percentages were greater than 95% were further analyzed by whole-exome sequencing (WES) using HiSeq. The percentage of 6pLOH(+) cells in the total granulocytes or sorted HLA(-) granulocytes were estimated using digital droplet PCR or deep sequencing of HLA alleles. [Results] Target sequencing of 8 of the 11 patients revealed somatic mutations in the HLA(-) granulocytes of 3 patients. HLA(-) granulocytes-specific mutations were found in DNMT3A, PRR5L, SMC3A, and LRCH1 (Table). The variant allele frequencies (VAF) of these mutations were far lower (5.1%-20%) than those of HLA(-) granulocytes that accounted for 95% of sorted cells. WES revealed 22 non-synonymous and 9 synonymous mutations in the HLA(-) granulocytes from 4 of the 5 patientsthat included 3 new patients and 2 patients whose samples were negative for mutations revealed by the target sequencing. The VAF of these mutations ranged from 20.7-52.5% (median 44.1%, Table). Very-high VAFs of several mutant genes suggested that these mutations occurred simultaneously with or soon after the occurrence of 6pLOH. A patient who achieved remission after romiplostim therapy without ATG showed various gene mutations that were thought to have occurred after 6pLOH. Despite of their highly biased hematopoiesis supported by single or few clones, recurrent or MDS-related oncogenic mutations were not detected in any of the 11 patients. Of note, the percentages of 6pLOH(+) cells in the sorted HLA(-) granulocytes were ≤75% (36.7%, 46%, 74%, and 75%) in 4 patients, indicating the presence of granulocytes lacking HLA-A alleles through mechanisms other than 6pLOH. [Conclusions] HLA(-) HSPCs caused by 6pLOH or other unknown mechanisms support long-term hematopoiesis without the development of oncogenic driver mutations that are associated with clonal hematopoiesis of MDS; as such, clonal hematopoiesis by 6pLOH(+) HSPCs may not portend a poor prognosis. Disclosures Nakao: Alexion Pharmaceuticals: Honoraria, Research Funding.

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Comprehensive analysis of mutations and clonal evolution patterns in a cohort of patients with cytogenetically normal acute myeloid leukemia

Molecular Cytogenetics ◽

10.1186/s13039-021-00561-2 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Yuslina Mat Yusoff ◽

Fadly Ahid ◽

Zahidah Abu Seman ◽

Julia Abdullah ◽

Nor Rizan Kamaluddin ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Somatic Mutations ◽

Clonal Selection ◽

Clonal Evolution ◽

Selective Advantage ◽

Remission Induction ◽

Leukemic Clone ◽

Disease Stages ◽

Acute Myeloid

Abstract Background Relapsed acute myeloid leukemia (AML) is associated with the acquisition of additional somatic mutations which are thought to drive phenotypic adaptability, clonal selection and evolution of leukemic clones during treatment. We performed high throughput exome sequencing of matched presentation and relapsed samples from 6 cytogenetically normal AML (CN-AML) patients treated with standard remission induction chemotherapy in order to contribute with the investigation of the mutational landscape of CN-AML and clonal evolution during AML treatment. Result A total of 24 and 32 somatic variants were identified in presentation and relapse samples respectively with an average of 4.0 variants per patient at presentation and 5.3 variants per patient at relapse, with SNVs being more frequent than indels at both disease stages. All patients have somatic variants in at least one gene that is frequently mutated in AML at both disease presentation and relapse, with most of these variants are classic AML and recurrent hotspot mutations including NPM1 p.W288fs, FLT3-ITD, NRAS p.G12D and IDH2 p.R140Q. In addition, we found two distinct clonal evolution patterns of relapse: (1) a leukemic clone at disease presentation acquires additional mutations and evolves into the relapse clone after the chemotherapy; (2) a leukemic clone at disease presentation persists at relapse without the addition of novel somatic mutations. Conclusions The findings of this study suggest that the relapse-initiating clones may pre-exist prior to therapy, which harbor or acquire mutations that confer selective advantage during chemotherapy, resulting in clonal expansion and eventually leading to relapse.

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Clinical Relevance of Clonal Hematopoiesis in the Oldest-Old Population: Analysis of the "Health and Anemia" Study

Blood ◽

10.1182/blood-2018-99-114717 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 750-750

Author(s):

Marianna Rossi ◽

Manja Meggendorfer ◽

Matteo Zampini ◽

Mauro Tettamanti ◽

Emma Riva ◽

...

Keyword(s):

Somatic Mutations ◽

Clonal Evolution ◽

Oldest Old ◽

Vascular Diseases ◽

Employment Equity ◽

Clonal Hematopoiesis ◽

Increased Risk ◽

Equity Ownership ◽

The Relationship

Abstract Background. Age-dependent clonal expansion of somatic mutations in the hematopoietic system is associated with an increased risk of hematological cancers (including myelodysplastic syndromes, MDS) and other illnesses (coronary heart disease and stroke). However, the presence of clonal hematopoiesis per se in a given individual has only limited predictive power. We hypothesized that the study of oldest-old population can define more specifically the relationship between mutations in the hematopoietic system and risk for MDS, inflammation and vascular diseases. Methods. We analyzed 1004 oldest-old subjects (median age 84.2y, range 80-105) included in the "Health and Anemia" population-based study [Haematologica 2010;95:1849]. Using peripheral blood DNA, we looked for somatic mutations in 47 genes recurrently mutated in hematologic cancers. Results. Clonal mutations were observed in 32.8% of individuals (range 1-5). The majority of variants occurred in 3 genes: DNMT3A (36.4%), TET2 (24.3%) and ASXL1 (6.5%). Mutations in splicing genes, PPM1D and TP53 were found in 7.4%, 5% and 2% of cases, respectively. The mutation frequencies increased with age, up to 50% in individuals aged over 90 years (P=.011). Clonal hematopoiesis was associated with a lower 5-y probability of survival (P=.03), and prognosis was even poorer in patients carrying ≥2 mutations (P=.002) We first focused on the relationship between clonal hematopoiesis and MDS phenotype. Carrying a somatic mutation with a variant allele frequency (VAF) ≥.10, carrying ≥2 mutations, spliceosome gene mutations and co-mutation patterns involving TET2, DNMT3A had a positive predictive value for MDS (from .85 to 1.0). The most frequent early phenotypic changes in patients who developed MDS included an increasing red blood cell distribution width (RDW) and mean corpuscular volume (MCV). Preliminary analyses suggested that the combination of mutations and non-mutational factors (RDW, MCV, after excluding iron/vitamin depletion and thalassemia) may improve the capability to capture individual risk of developing MDS with respect to molecular data alone (P=.01) We studied clonal evolution in 72 patients with multiple samples available over a period of 5y. Clonal hematopoiesis was found at baseline in 22 cases: 2 individuals acquired additional mutations during follow-up, and 5 displayed significant increase in VAF. In 9 subjects without clonal hematopoiesis, mutations were acquired during follow-up. RDW and MCV changes, induction of unexplained cytopenia and overt MDS phenotype were significantly restricted to subjects displaying clonal evolution. We hypothesized that in oldest-old populations MDS could be underdiagnosed (many patients are not considered for bone marrow aspiration because of age). Cytopenia was a common finding in our cohort (20%) the underlying cause remaining unexplained in 27% of cases. In patients with unexplained anemia, carrying a somatic mutation had a positive predictive value for persistent, progressive, multilineage cytopenia (findings consistent with a MDS phenotype) and shorter survival (from .8 to .94). On this basis, 8% of all cytopenias might be undiagnosed MDS. Finally, we investigated the association between clonal hematopoiesis with inflammatory and vascular diseases. Mutations in DNMT3A, TET2, and ASXL1 were each individually associated with risk of coronary heart disease and death, and preliminary analyses suggest that clonal hematopoiesis is also associated with increased risk of rheumatological diseases (P from .03 to.009). We identified mutations in macrophages isolated from synovial fluid of 4/17 patients with rheumatoid arthritis and from atherosclerotic plaques of 3/25 patients with carotid stenosis. Functional studies of macrophages (expression of specific chemokine and cytokine gene patterns) are ongoing. All these findings are under validation in an independent cohort of 800 subjects enrolled in the "Monzino 80-plus" study [Alzheimers Dement 2015;11:258]]. Conclusion. Clonal hematopoiesis was associated with reduced survival in an oldest-old population. Specific mutational profiles define different risks of developing MDS and inflammatory/vascular diseases. Non mutational factors, such as early changes in red blood cell indices, may improve the capability to identify patients at increased risk of developing myeloid cancers. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Bolli:Celgene: Honoraria. Vassiliou:KYMAB: Consultancy, Equity Ownership; Celgene: Research Funding. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Aplastic Anemia Associated with PNH-Clone - a Single Centre Experience

Blood ◽

10.1182/blood.v128.22.5080.5080 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5080-5080

Author(s):

Elena Shilova ◽

Tatiana Glazanova ◽

Zhanna Chubukina ◽

Lubov Stelmashenko ◽

Sergey Gritsaev ◽

...

Keyword(s):

Aplastic Anemia ◽

Combined Therapy ◽

Clonal Evolution ◽

Pregnant Patient ◽

Observation Group ◽

Intravascular Hemolysis ◽

Clone Size ◽

Dynamic Observation ◽

Long Term Observation ◽

Pnh Clone

Abstract Introduction: Pathogenetic relationship of aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) was registered a long time ago. Approximately in 50-60% of patients with AA is detected PNH-clone, and during long-term observation the transformation of AA into classic PNH is likely. At the time of diagnosis PNH-clone sizes range from small proportion of PNH-cells, requiring only monitoring, up to combination of clinically manifested AA and PNH requring combined therapy. Nevertheless, the peculiar characteristics of the disease in cases of different AA-PNH-combinations still remain unclear. Aim: to evaluate the relationship between size and dynamics of PNH-clone with clinical features of AA/PNH. Materials and methods: We have evaluated blood samples of 81 patients with AA (whole group) for the presence of PNH-clone using flow cytometry according to ICCS standards. 23 patients aged 23 to 54 years (median 46 years) were under constant observation for 3 years or more, control examination was performed once every 6 months (dynamic observation group). Of them 17 had severe AA (SAA) and 6 - nonsevere AA (NAA). Twelve patients received standard immunosupressive therapy (IST) for the treatment of AA, other patients were in remission and did not require IST. Results: In the whole group of patients (n=81) 47 patients (58%) were AA/PNH-positive (PNHpos) with clone size ranging from 0.01% to 97.9%. PNH-clone less than 1% was identified in 10% of subjects; PNH-clone more than 10% was observed in 50% of subjects. In dynamic observation group 18 patients were AA/PNHpos with initial clone size from 0.1% to 95.9%, and 5 patients - AA/PNH negative (AA/PNHneg). In AA/PNHneg patients pathological clone still has not been detected in repeated studies for the observed period. Among patients with AA/PNHpos stable clone was observed in 11 subjects (61%), it increased to 10% and more in 5 subjects (28%) and decreased in 2 patients (11%) with small PNH-clone. Clinical and laboratory signs of chronic intravascular hemolysis were usually observed in patients with pathological clone over 25%. PNH-clone less than 10% was not accompanied by significant deviations in clinico-laboratory indices. Most of patients in whom PNH-clone size increased, had remission of AA. Four patients had clonal evolution to manifest classical PNH, of them in 2 patients with SAA аplasia of hematopoiesis persisted. In one patient, with complete remission of SAA lasting more than 3 years and minor PNH-clone, was observed transformation into myeloid leukemia leading to death. Ekulizumab was prescribed to 4 patients: 2 with clinically manifest hemolysis and clone size > 90% (1 - in conjunction with IST for AA), and 2 with clone size 24% and 70% and LDH > 1.5 ULN due to unplanned pregnancies (both on Cyclosporin therapy). All of them achieved the target level of LDH <1.5 ULN in the first 2 weeks of therapy. Of 2 transfusion-dependent patients, one still does not require transfusion support after the Ekulizumab initiation, and in other patient transfusion dependency decreased, despite refractory TAA. In one patient with PNH-clone 70% Ekulizumab therapy was initiated in III trimester of pregnancy, delivery was without complications, full-term baby, 8-9 points Apgar. In the 2nd pregnant patient with PNH-clone 24% Ekulizumab was prescribed because of pre-eclampsia and LDH>4 ULN at 21-22 weeks with antenatal fetal death, and it allowed for accelerating cure of pathological symptoms. Conclusion: PNH-clone was detected in 58% of patients with AA. Our data confirm the need for monitoring of PNH clone in patients with AA and for timely adequate therapy, including inhibitors of intravascular hemolysis, according to specific indications in each case. Disclosures Fominykh: Novartis Pharma: Honoraria; BMS: Honoraria.

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Clinical implications of somatic mutations in aplastic anemia and myelodysplastic syndrome in genomic age

Hematology ◽

10.1182/asheducation-2017.1.66 ◽

2017 ◽

Vol 2017 (1) ◽

pp. 66-72 ◽

Cited By ~ 5

Author(s):

Jaroslaw P. Maciejewski ◽

Suresh K. Balasubramanian

Keyword(s):

Stem Cell ◽

Myelodysplastic Syndrome ◽

Aplastic Anemia ◽

Cell Biology ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Somatic Mutations ◽

Bone Marrow Failure ◽

Clonal Evolution ◽

Clonal Diversity ◽

Clinical Aspects

AbstractRecent technological advances in genomics have led to the discovery of new somatic mutations and have brought deeper insights into clonal diversity. This discovery has changed not only the understanding of disease mechanisms but also the diagnostics and clinical management of bone marrow failure. The clinical applications of genomics include enhancement of current prognostic schemas, prediction of sensitivity or refractoriness to treatments, and conceptualization and selective application of targeted therapies. However, beyond these traditional clinical aspects, complex hierarchical clonal architecture has been uncovered and linked to the current concepts of leukemogenesis and stem cell biology. Detection of clonal mutations, otherwise typical of myelodysplastic syndrome, in the course of aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria has led to new pathogenic concepts in these conditions and created a new link between AA and its clonal complications, such as post-AA and paroxysmal nocturnal hemoglobinuria. Distinctions among founder vs subclonal mutations, types of clonal evolution (linear or branching), and biological features of individual mutations (sweeping, persistent, or vanishing) will allow for better predictions of the biologic impact they impart in individual cases. As clonal markers, mutations can be used for monitoring clonal dynamics of the stem cell compartment during physiologic aging, disease processes, and leukemic evolution.

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