Clonal Hematopoiesis in Older Women with Extremely Skewed, Non-Random X-Inactivation Is Associated with Previous Cardiovascular and Cerebrovascular Events

Age-related clonal hematopoiesis (CH) is a common condition that is associated with an increased risk of hematologic malignancies (HM) and cardiovascular disease (CVD). The majority of candidate driver mutations occur in epigenetic regulatory genes such as ASXL1, DNMT3A, and TET2 genes. However, a significant proportion of older people harbor clonal hematopoiesis without candidate driver mutations. In older women, clonal hematopoiesis can also be detected by the human androgen receptor A gene (HUMARA) assay regardless of the presence or absence of candidate driver mutation(s). The HUMARA assay evaluates non-random X-inactivation (NRX-I) as a marker for clonal hematopoiesis. The purpose of this study is to evaluate the association between NRX-I and cardiovascular risk factors and other health correlates. We screened for NRX-I in 904 women ages 65 and older participating in an ongoing, prospective cohort study in Oregon (Women Engaged in Advancing Health Research [WEAR] study). This approach was used to enhance the investigation of changes in CH that occur over time that may prove to be impactful for health outcomes. We examined the HUMARA results and any association with previous health history using a cross-sectional study design. Analysis of variance and logistic regression analyses were used to examine the relationship between HUMARA assay results and baseline CVD risk, controlling for age and race. HUMARA assay results were quantified and 3 groups were established: random X-inactivation, NRX-I, and extremely skewed NRX-I. No significant differences were detected between groups with respect to age, race, education, reproductive health indices or body mass index. With respect to cardiovascular risk factors, women with extreme NRX-I were more likely to be lifetime non-smokers compared to women without NRX-I and those with NRX-I with less skewing (P: 0.049), though no difference was seen in the proportion of current smokers (P: 0.213) and the total pack years smoked by those with a smoking history (P: 0.846).The proportion of subjects reporting statin or other cholesterol lowering medication did significantly differ between groups, with 24.3% women without NRX-I reporting statin use, versus 30.4% in women with NRX-I, and 36.7% in women with NRX-I extreme skewing (P: 0.005). Post-hoc pairwise tests showed significant differences between women with no NRX-I and women with NRX-I extreme skewing (P: 0.004). Cardiovascular event history differed significantly between the three groups of interest. 5.6% of women without NRX-I reported a history of transient ischemic attack (TIA), cerebral vascular accident (CVA), or myocardial infarction (MI) versus 7.0% in the NRX-I group and 11.0% in the NRX-I with extreme skewing group (P: 0.05). Post-hoc pairwise tests showed significant differences between women with no NRX-I and women with NRX-I extreme skewing (P: 0.043). The adjusted odds of TIA, CVA, or MI more than doubled (OR: 2.15, 95%CI: 1.14-3.42) among women with extremely skewed HUMARA results compared to women with NRX-I, controlling for age, high cholesterol, smoking history, hypertension, and type II diabetes. Cholesterol was also found to be independently associated with TIA, CVA, and MI. Women with hypercholesterolemia were 1.84 times as likely to report a history of TIA, CVA, or MI compared to women without hypercholesterolemia (95% CI: 1.06, 3.20). In summary, prediction of major adverse cardiac events is based on the presence of traditional risk factors including high blood pressure, high cholesterol, uncontrolled diabetes, smoking, and family history. Yet there is significant residual risk; many will still die from CVD without these risk factors. NRX-I, in addition to enriching for mutations known to confer CVD and HM risk; may be a marker for additional and unique health risks. An association between NRX-I, high cholesterol, and history of TIA, CVA, and MI, supports the current biological hypothesis that clonal hematopoiesis, perhaps irrespective of the cause or underlying driver mutation, is a driver for CVD. Disclosures Druker: Celgene: Consultancy; Fred Hutchinson Cancer Research Center: Research Funding; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; McGraw Hill: Patents & Royalties; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Research Funding; Monojul: Consultancy; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Research Funding; Henry Stewart Talks: Patents & Royalties; Millipore: Patents & Royalties; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding. Dao:Incyte: Consultancy.

Clonal Hematopoiesis in AML Patients in Hematological CR Is Present in Many Patients with Intermediate Risk AML and Is Associated with a High Prevalence of DNMT3A gene Mutations

Background De novo acute myeloid leukemia (AML) is a malignant disorder of hematopoietic stem and progenitor cells usually characterized by a rapid clinical onset. Despite this clinical manifestation, recent evidence suggests that premalignant stem cells might be present in patients with AML, which can persist after chemotherapy in some patients and induce clonal hematopoiesis. Little is known about the prevalence of clonal persistence and about the molecular basis. In order to study the prevalence of this phenomenon and to better understand the underlying molecular mechanisms, we investigated AML patients in remission for clonal persistence of cells after chemotherapy. Patients and methods: All patients included in this analysis were treated within a prospective treatment protocol of the Study Alliance Leukemia (SAL). The primary study cohort consisted of 61 female patients with intermediate risk cytogenetics achieving hematologic complete remission (CR), whose DNA material was available at CR. Clonality analysis was based on X-chromosome inactivation testing using the HUMARA assay. DNA from diagnostic samples of patients presenting with evidence for X-chromosome skewing in CR was analyzed using amplicon based resequencing on a MiSeq next generation sequencing (NGS)-system for DNMT3A, ASXL1, ASXL2, TET1, TET2, EZH1, EZH2, IDH1 and IDH2. Results: Of the 61 patients included, 52 were heterozygous for the STR in the human androgen receptor gene. In CR, 22 of these 52 patients (42%) showed evidence for a skewed X-chromosome representation, indicating persistence of clonal hematopoiesis in remission. The NGS-based analysis of genes involved in epigenetic regulation revealed mutations in 13/22 (59%) of the patients. DNMT3A was most frequently mutated (11/13 patients), either alone or in combination with other alterations (TET2, EZH2). Interestingly, two patients showed somatic alterations in the TET1 gene. In remission, clonal persistence of these alterations was detected in all 13 patients with mutations at diagnosis at levels between 0.8 and 50% as documented using ultradeep-NGS. To get an idea on the prevalence of clonal persistence in other cytogenetic groups, we analyzed 22 low risk (i.e. CBF-leukemias) as well as 18 poor risk (-7, complex karyotype) patients using the HUMARA assay. Here we observed similar results, with 13/19 informative patients showing clonal persistence in low-risk group (68%) compared to 7/14 patients (50%) in the poor risk population. Since all these analyses were confined to female patients and potentially limited by the sensitivity of the HUMARA method, we went on to look for persistence of clonal molecular markers using more sensitive ultra-deep NGS. Because DNMT3A exon 23 was the common alteration in this initial analysis, we screened a cohort of 48 patients with mutations in NPM1 and comutations in DNMT3A. In this separate cohort, persistence of the DNMT3A mutations at CR or during follow-up (FU) was detected in 42 patients (87.5%) at levels between 0.5 and 50% (median 11.1%). No difference was seen between male and female patients, the median age was 51 years, persistence was seen even in young patients at 26 years of age. During FU, the DNMT3A VAF level rose further in all patients analyzed, arguing for a clonal advantage of the mutant cells. All patients with relapse and available material showed high levels of DNMT3A at time of relapse. However, correlation of DNMT3A mutant allele levels at CR1 with the incidence of relapse showed no significant impact of the VAF for the development of relapse. Conclusions: Our data indicate that clonal persistence of premalignant cells carrying clonal alterations in epigenetic regulator genes is a common phenomenon in patients in continuous CR. DNMT3A is the most common lesion persisting, the majority of patients with this mutations retain it at CR and during FU. These data indicate that de novo AML develops from preleukemic stem and progenitor cells in many patients. Preliminary data indicate that this persistence per se is not associated with inferior outcome. Disclosures Schuster: AgenDix GmbH: Employment. Thiede:AgenDix GmbH: Equity Ownership, Research Funding; Illumina: Research Support, Research Support Other.

An Active Isodicentric X Chromosome in a Case of Refractory Anaemia with Ring Sideroblasts Associated with Marked Thrombocytosis

Refractory anaemia with ring sideroblasts and marked thrombocytosis (RARS-T) is a provisional entity in the World Health Organization (WHO) classification. It displays features characteristic of both myelodysplastic syndrome and myeloproliferative neoplasia plus ring sideroblasts ≥15% and marked thrombocytosis. Most patients with RARS-T show a normal karyotype. We report a 76-year-old woman diagnosed with RARS-T (76% of ring sideroblasts) withJAK2(V617F) mutation and a load of 30–40%. Classical and molecular cytogenetic (FISH) studies of a bone marrow sample revealed the presence of isodicentric X chromosome [(idic(X)(q13)]. Moreover, HUMARA assay showed the idic(X)(q13) as the active X chromosome. This finding was correlated with the cytochemical finding of ring sideroblasts. To our knowledge, this is the first reported case of an active isodicentric X in a woman with RARS-T.

Clonality analysis of giant cell lesions of the jaws

Despite the importance of clonality to understand the pathogenesis and progression of tumors, it has not been investigated yet in giant cell lesions of the jaws. The aim of this study was to analyze the clonality of peripheral giant cell lesions (PGCL) and central giant cell lesions (CGCL) of the jaws. Six samples of PGCL and 5 samples of CGCL were analyzed in this study using the polymorphic human androgen receptor locus (HUMARA) assay. Three out of the 5 samples of the CGCL and 3 out of 6 samples of PGCL exhibited a monoclonal pattern. Our findings demonstrate that some giant cell lesions of the jaws are clonal, which indicate that these lesions may have a common genetic mechanism of development. Further studies are necessary to better elucidate the molecular mechanisms involved in the pathogenesis of such lesions.

Clonality in Granulocytes Detected by an Improved HUMARA Assay: A Good Prognostic Marker in Bone Marrow Failure Patients Exhibiting Chromosomal Abnormalities of Indefinite Significance.

Some patients with aplastic anemia (AA) and approximately 40% of patients with refractory anemia (RA) of myelodysplastic syndrome exhibit karyotypic abnormalities in bone marrow dividing cells. Although some of the patients undergo evolution to acute myeloid leukemia (AML), others follow a clinical course similar to AA patients without chromosomal abnormalities. Except for several abnormalities such as −7 and 5q-, the clinical significance of such chromosomal abnormalities in bone marrow failure patients remains unclear. We recently developed a reliable HUMARA assay capable of detecting a clonal population in granulocytes which constitutes 30% or more of total granulocytes (Blood. 2003;102:1211–1216). Studying correlation between chromosomal abnormalities and the presence of clonality may help in understanding the pathogenetic role of chromosomal abnormalities in AA and RA. We thus analyzed 50 acquired AA and 28 RA female patients who were heterozygous for the HUMARA gene. Chromosomal abnormalities such as add(5)(q13), 9q–9q+ and del(7)(q14q22) were found in 8% of AA and 21% of RA patients. Clonality was detected in 38% of AA patients and 39% of RA patients. Incidence of chromosomal abnormalities in patients with clonality (27%) was higher than that in patients without clonality (4%, p<0.01). In two AA patients who respectively exhibited add(5)(q13) in 10% and +8 in 38% dividing cells, clonality was not detected and these abnormal clones became undetectable at the time of subsequent bone marrow examination. Clonality was detected in the other 2 AA patients respectively exhibiting 9q–9q+ in 40% and del(7)(q14q22) in 25% dividing cells, and in all 5 RA patients respectively exhibiting +8 in 10%, del(5)(q13q31), dup(1)(q32q12) in 90%, del(5)(q13), add(11)(q23), inv(9) in 65% and X,-X in 100% of dividing cells. None of the 50 AA patients including 2 patients with clonality and chromosomal abnormalities underwent evolution to AML during 2-year follow up while one of 28 RA patients who exhibited del(5)(q13q31) progressed to AML. The proportion of clonal granulocytes in total granulocytes estimated by the HUMARA assay remained unchanged in most patients with clonality except for the transformed one. These data indicate that the chromosomal abnormality in bone marrow dividing cells is not necessarily associated with presence of clonal granulocyte population in peripheral blood and that detection of clonality in granulcytes in bone marrow failure patients with chromosomal abnormalities of indefinite significance is useful in predicting prognosis of these patients.

Identification of a Subset of Aplastic Anemia Patients Highly Responsive to Danazol.

Treatment of patients refractory to immunosuppressive therapy is a major problem in the management of aplastic anemia (AA). Some patients who are ineligible for allogeneic stem cell transplantation have been successfully treated with anabolic steroids although little is known about the common characteristics of these patients. In order to characterize high responders to danazol, we retrospectively studied 44 AA (13 male and 31 female) patients who were treated with 300 mg of danazol daily for at least 3 months. All patients had been previously treated with ATG+CsA or CsA alone without appreciable effects before danazol therapy. Peripheral blood of these patients was examined for the presence of PNH-type cells and clonality in granulocytes using sensitive flow cytometry (Blood100: 3897, 2002) and the improved HUMARA assay (Blood102: 1211, 2003). 24 of 44 (55%) patients attained PR or CR according to the response criteria proposed by Camitta. All 3 lineage cells increased in the responders. The rate of response in female patients (21/31, 68%) was significantly higher than that (3/13, 23%) in male patients (P=0.0058). There was no difference in the response rate between older (&gt;50 years) patients (75%) and younger (&lt;50 years) patients (60%) in female patients, indicating that the effect of danazol is not affected by a female sex hormone. Increased PNH-type cells were detectable in 2 (8.3%) of 24 responders and in 6 (40%) of 15 non-responders. The HUMARA assay revealed presence of clonal granulocyte population in 13 (76%) of 17 responders and 2 (33%) of 6 non-responders, respectively. For a female patient without increased PNH type cells who has not responded to immunosuppressive therapy, the rate of response to danazol was 64%. Probability of 3-year survival in responders to danazol was 100% while that in non-responders to the therapy was 90%. Univariate analysis revealed that negativity for increased PNH-type cell and being female were significantly associated with good response to danazol. These findings indicate that danazol is a useful drug in the treatment of a subset of female AA patients characterized by refractoriness to conventional immunosuppressive therapy and absence of increased PNH-type cells. Given the high frequency of clonal hematopoiesis in responders to danazol, this anabolic steroid may stimulate expansion of a small number of stem cells that survived non-immune marrow insult.