Higher Mutation Rate in Patients with Aplastic Anemia Using Peripheral Blood cfDNA As Compared with Bone Marrow Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3902-3902
Author(s):  
Adam Albitar ◽  
Danielle Townsley ◽  
Wanlong Ma ◽  
Ivan De Dios ◽  
Vincent Funari ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Allele Frequency ◽  
Peripheral Blood ◽  
Research Funding ◽  
Mutant Allele ◽  
San Diego ◽  
Somatic Mutations ◽  
Plasma Samples ◽  
Significant Difference

Abstract Background:We have reported that peripheral blood cell-free DNA (cfDNA) is reliable for detecting bone marrow molecular abnormalities in patients with hematologic neoplasms. However, not clear is whether cfDNA is sufficient to detect mutations present at low variant allele frequency (VAF). Since patients with aplastic anemia (AA usually have relatively small clones in blood and bone marrow (BM), we compared mutations detected in BM cells with those detected in peripheral blood cfDNA from patientswith this disease. Methods: A total of 120 paired BM aspirate and PB plasma samples were tested by the commercially available TruSight Myeloid Sequencing Panel (Illumina; San Diego, CA). We extracted DNA from bone marrow aspirate using the QIAamp DNA Mini Kit. We used NucliSenS EasyMAG automated platform for extracting total nucleic acid from PB plasma collected in EDTA. All paired BM and plasma samples were tested by the commercially available TruSight Myeloid Sequencing Panel (Illumina; San Diego, CA), which covers hot spot mutations in 54 genes. The average depth of sequencing was 10,000X. Results: One hundred twenty paired BM and cfDNA samples from 96 patients with aplastic anemia were tested. Of the 96 patients, 33 (34%; equivalent to 48 samples, 40%) had one or more mutations. We identified 54 different somatic mutations in these patients, of which 45 were unique. There was no significant difference (P=0.71, Sign test) in allele frequency between cfDNA and BM. The median mutant allele frequency was 10.9% in cfDNA and 12.6% in BM cells, and 40 of the 54 mutations had allele frequency ≤20% in BM cells, while 45 samples had allele frequency ≤20 in cfDNA. Six of the 33 patients with somatic mutations (18%) showed mutations in plasma cfDNA but not in BM. In contrast, 2 patients (6%) showed mutations in BM cells and not in cfDNA. One of these two patients had a mutation in ASXL1 gene detected in BM cells but not in cfDNA and a subsequent sample showed the same ASXL1mutation in BM cells and not in cfDNA, and a second clone with a different ASXL1 mutation detected in both BM cells and cfDNA. Overall concordance between BM cells and cfDNA in the 120 samples was 92% and there was no statistically significant difference between the two sample types (P=0.6). Summary and Conclusions: Seven samples (from 7 patients) of the 120 tested samples showed mutations in cfDNA and not in BM cells while 3 samples (from 2 patients) showed mutations in BM and not in cfDNA. VAF of mutations in cfDNA were similar to those in BM cells. Therefore, peripheral blood cfDNA should be tested in addition to BM cells for detecting mutations in patients with AA. Peripheral blood cfDNA can be used as a reliable means for monitoring patients with AA. cfDNA testing can be used as an alternative testing to bone marrow even when mutant allele frequency in bone marrow is <20%. cfDNA may be an especially valuable source of mutation detection in marrow failure, in which marrow aspirates may not contain sufficient cells for accurate mutation analysis. Disclosures Albitar: Neogenomics Laboratories: Employment. Townsley:Novartis: Research Funding. Ma:Neogenomics Laboratories: Employment. De Dios:Neogenomics Laboratories: Employment. Funari:Neogenomics Laboratories: Employment. Young:GSK/Novartis: Research Funding. Albitar:Neogenomics Laboratories: Employment, Equity Ownership.

Fertility Recovery Following Allogeneic Bone Marrow Transplantation in Aplastic Anemia; A Study of 157 Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4783-4783
Author(s):  
Feras Alfraih ◽  
Shad Ahmed ◽  
Dennis Dong Hwan Kim ◽  
Walid Rasheed ◽  
Ghuzayel Aldawsari ◽  
...  
Keyword(s):  
Risk Factors ◽  
Bone Marrow ◽  
Risk Factor ◽  
Aplastic Anemia ◽  
Median Duration ◽  
Research Funding ◽  
Independent Risk Factor ◽  
Age Group ◽  
Significant Difference ◽  
Fertility Recovery

Abstract Introduction : Infertility is a major late effect of hematopoietic stem cell transplants (HSCT). In aplastic anemia (AA) patients, although the fertility recovery rate is relatively higher than other diseases but the exact incidence and risk factors are not very well studied. In this study, we attempted to evaluate incidence and the impact of patientÕs characteristics and transplantation procedures on fertility recovery following allogeneic HSCT for adolescent and adults patients with AA. Methods : A total of 157 patients who were at least 14 years old with AA receiving HSCT between year 1987 and 2014 at our center were reviewed. Patients who survived at least 2 years following HSCT and either married or in relationship were included in the analysis and evaluated for fertility following HSCT. 87 patients were eligible for the study. Questionnaire survey and long-term charts were used for data collection. With a response rate and or available information of 63% patients, 55 patients were identified and stratified into fertility recovery (FR+) versus non-fertility recovery (FR-) group. Fertility recovery was defined by a pregnancy of the patient or his partner. Results: Median age for all patients is 23 years (range, 14 -50), 44% (n=24) between 14-20 years old, 51% (n=28) between age 20-40 years and 5% (n=3) > 40 years. 51% (n=28) were females. Matched related donor was used for majority of patients 96% (n=53). GVHD prophylaxis was CSA/MTX for 93% (n=51,). Conditioning regimen was Cyclophosphamide/Flu in 25 (45%), Cyclophosphamide /ATG in 18 patients (35%) and others in 12 patients (20%). Bone marrow was the source of stem cells for 52 patients (94%). A median follow-up of 8 years for survivors (range, 0.3 -23) showed 45 patients (82%) had FR+ while 10 patients (18%) were FR-. Median duration of fertility recovery (from delivery to BMT) was 6 years (range, 0.8-19) with significant difference based on age groups, 4 years for patients 20-40 years (n=29, 53%) versus 8 years for those < 20 years (n=24, 44%), (p=0.002), (Figure 1). None of the patients >40 years old (n=2, 4%) had fertility recovery. Comparison based on gender showed no significant difference. Males had a median duration of fertility recovery of 5.9 years, (range 0.6-14.9) versus 6.2 years, (range, 0.8-15.2) (p=0.31) females. The overall median number of pregnancies was 2 (range, 1-6). For males, it was 2 (range, 1-6) while 1.5 (range, 1-5) for females (p=0.26). Deliveries occurred in natural ways in (95%) while C-section for (5%). All deliveries were without fetal abnormalities. Univariate analysis of risk factors for fertility recovery showed age group (p=0.03) and chronic GVHD (p=0.05) are important factors. Neither gender of patients or type of preparative regimens used for HSCT (Cyclo/ATG vs Cyclo/Flu) was a risk factor. In multivariate analysis, age group was the only confirmed an independent risk factor for fertility recovery (p=0.02) [HR= 2.02, CI=1.012-3.64). Conclusion: The present study suggested that the incidence of fertility recovery following HSCT for patients with aplastic anemia is high with no significant differences between males and females. Patients between the ages of 20-40 years at the time of HSCT have significantly shorter recovery period. Age was the only independent risk factor for fertility recovery while there was no impact of whether ATG or Fludarabine was used in addition to Cyclophosphamide as preparative regimen. Figure 1. Figure 1. Disclosures Kim: Bristol-Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding.

Decoding Bone Marrow Fibrosis in Myelodysplastic Syndrome

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1985-1985
Author(s):  
Megan Melody ◽  
Najla H Al Ali ◽  
Ling Zhang ◽  
Hanadi Ramadan ◽  
Eric Padron ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
Somatic Mutations ◽  
Bone Marrow Aspirate ◽  
Bone Marrow Fibrosis ◽  
Advisory Committees ◽  
Significant Difference ◽  
Baseline Characteristics ◽  
Grade 3 ◽  
Severe Fibrosis

Abstract Background Bone marrow fibrosis (BMF) is observed in 10-20% of patients diagnosed with MDS. A study conducted at the University of Pavia, Italy evaluated 180 cases of MDS with varying grades of BMF and found that the presence of grade 2-3 BMF in MDS (graded by European classification) was associated with worse outcomes, especially in patients with lower risk MDS, however there remains little data pertaining to the treatment and outcomes in the MDS-F patient population. The primary objective of this study is to examine a large MDS patient population to further determine the relationship between BMF and survival outcomes in the context of new risk stratification models. Methods All bone marrow aspirate and biopsy reports, obtained at the time of diagnosis or prior to any therapy, in the Moffitt Cancer Center MDS database were reviewed and the degree of BMF was determined using the European classification system. Patients with less than 5% myeloblasts and grade 2 or 3 BMF were identified and these bone marrow aspirate and biopsies were reviewed by two hematopathologists (LZ and JMB) to confirm the grade of fibrosis reported. Patients were then divided into two groups: grade 0-2 BMF and severe BMF (grade 3). These two groups were compared to evaluate differences in clinical characteristics, response to treatment and survival. NexGen sequencing was available for 251 patients and distribution of acquired somatic mutations were compared between grade 0-2 BMF and grade 3 BMF. Results There were 2357 cases included in this analysis of which 2237 (95%) were determined to have grade 0-2 BMF and 120 (5 %) to have grade 3 BMF. Table-1 summarizes baseline characteristics. There was no statistical difference in age at diagnosis, gender, or race. A greater percentage of patients with severe BMF (39%) met the criteria for poor/very poor IPSS-R score category than patients with grade 0-2 BMF (29%), p = 0.011. Complex karyotype was observed more frequently among patients with severe fibrosis (23% versus 15%, p = 0.031). Patients with severe BMF also had a higher incidence of elevated LDH (51%) than patients with grade 0-2 BMF (33%), P < 0.005. The median overall survival (OS) was 38.7 months (mo) for grade 0-2 BMF and 23.1mo for grade 3 BMF, p < 0.005. When examined by revised IPSS (R-IPSS), severe BMF only impacted OS among the lower risk group. The median OS in patients with very low/low R-IPSS risk was 47mo in patients with severe BMF compared to 77 for those with grade 0-2 BMF, p = 0.015. In multivariable analysis, adjusting for age and R-IPSS, severe BMF was independently associated with inferior OS (HR 1.7, P 0.01). The rate of AML transformation was 28% in both the severe and grade 0-2 BMF groups, p = 0.98. Eighty patients with severe BMF were treated with hypomethylating agents (HMA). The overall response, by IWG 2006 criteria, of HI or better was 30% among patients with severe BMF compared to 32% of patients with grade 0-2 BMF, p 0.4. Among patients with severe BMF treated with lenalidomide (n=30), 25% of patients had HI response compared to 16% in patients with grade 0-2 BMF, p 0.9. The median OS for the 100 patients with severe BMF who did not undergo allogenic SCT was 23mo compared to 30mo for the 19 patients with severe BMF who did undergo allogenic SCT, p 0.29 Among somatic gene mutations, SF3B1 mutation was observed in 14% of grade 0-2 and 0% of grade 3 fibrosis. However, SRSF2 was present in 35.7% of patients with grade 3 fibrosis and only 9.7% of patients with grade 0-2 BMF, p .009. There was no statistically significant difference in the rate of occurrence of TP53 and RUNX1 mutations between the grade 0-2 BMF and the grade 3 BMF groups. In addition, there was no significant difference in the rate of occurrence of JAK2 mutation across the two groups. (table-2) Conclusions In our MDS cohort, only the presence of severe BMF (grade 3) was associated with worse outcome with reduced overall survival namely among patients with very low/low R-IPSS disease, whereas BMF grade did not impact response to HMA or lenalidomide treatment. SRSF2 gene mutation occurred with greater frequency among patients with severe fibrosis. Table 1 Baseline characteristics Table 1. Baseline characteristics Table 2 Acquired Somatic Mutations Table 2. Acquired Somatic Mutations Disclosures Sweet: Incyte Corporation: Research Funding; Ariad: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Pfizer: Speakers Bureau; Karyopharm: Honoraria, Research Funding. Bennett:Celgne: Membership on an entity's Board of Directors or advisory committees. Komrokji:Incyte: Consultancy; Novartis: Consultancy, Speakers Bureau; Boehringer-Ingelheim: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Characterization of Myelodysplastic Syndromes (MDS) with T-Cell Large Granular Lymphocyte Proliferations (LGL)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 113-113 ◽  
Author(s):  
Christa Roe ◽  
Najla Alali ◽  
Eric Padron ◽  
Pearlie K Burnette ◽  
Kendra L. Sweet ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Peripheral Blood ◽  
Scoring System ◽  
Lower Risk ◽  
Research Funding ◽  
Somatic Mutations ◽  
Gene Mutations ◽  
Significant Difference ◽  
Somatic Gene

Abstract Introduction: MDS include a spectrum of hematopoietic stem cell malignancies characterized by bone marrow failure and dysplastic morphology. LGL is a clonal proliferation of cytotoxic T cells, which manifest as neutropenia, anemia, and thrombocytopenia and is associated with autoimmune disorders. LGL in association with MDS has been previously reported. However, clinicopathological features, prognostic, and predictive factors in those patients diagnosed with both LGL and MDS is not well studied. Methods: We identified patients at Moffitt Cancer Center (MCC) diagnosed with MDS who were previously tested for the presence of LGL clonal populations by peripheral blood flow cytometry at time of first visit. An LGL population was defined by the standard flow cytometry immunophenotype and clonality confirmed by T-cell receptor gamma and beta gene rearrangement.. Next Generation sequencing data was available for 151 patients. Recurrent somatic gene mutations were compared between patients with an LGL clone and those without. Results: Of the 675 patients with MDS tested for LGL in the database, 206 (30.5%) had an LGL clonal population. The mean LGL absolute cell count in the peripheral blood was 335/µL. Table-1 summarizes the baseline characteristics of the two groups. There was no difference in response to azacitidine therapy. Among 50 patients with LGL clone who received azacitidine with available data on response, the rate of hematological improvement or better (HI+) was 38%. The (HI+) was 28% among 105 patients evaluable for response without LGL clone. P .14 The median overall survival (OS) was for patients with no LGL clone was 65 months (mo) compared to 46 mo (p .024). The median OS for lower risk MDS patients (low/int-1 by International Prognostic Scoring System [IPSS]) was 68 mo versus 97 mo for those with or without LGL proliferation, respectively (P .005). In higher risk MDS, there was no difference in median OS between those with or without LGL expansion, respectively (20 mo versus 16 mo, p .7). The median OS for patients with very low/ low Revised-Internatinal Prognostic Scoring System (R-IPSS) was 96 mo if LGL proliferation was detected compared to 128 mo if it was not, (p value .016). For intermediate R-IPSS the median OS was 65 mo and 41 mo with or without LGL proliferation (p .16). Finally, for high/very high R-IPSS the median OS was 18 and 16 mo with or without LGL proliferation, (p .84) In cox regression analysis the presence of an LGL clone was independently prognostic for OS after adjusting for age and R-IPSS, Hazard ratio 1.3, p = .05. Somatic gene mutation data were available for 151 patients; there was no statistically significant difference in the distribution of any mutation except IDH-2 (Table-2). The most common somatic mutations observed among patients with LGL clone were SF3B1 19%, TET-2 16%, U2AF1 13%, IDH-2 13%, RUNX-1 13%, and ASXL-1 10%. In patients without an LGL clone the most common somatic mutations were TET-2 26%, ASXL-1 20%, DNMT3A 16%, TP53 13%, SF3B1 12%. Conclusion: An LGL clone is demonstrable in approximately 30% of patients with MDS in association with advancing age. The presence of LGL proliferation was associated with worse OS in lower risk MDS pts. Although the spectrum of somatic gene mutations were similar, the presence of IDH-2 mutation and absence of DNMT3A or TP53 gene mutationscharacterized LGL+ cases. Table 1. Table 1. Table 2. Table 2. Disclosures Roe: Celgene: Speakers Bureau; Alexion: Speakers Bureau; Seattle Genetics: Speakers Bureau. Sweet:Pfizer: Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Incyte Corporation: Research Funding; Karyopharm: Honoraria, Research Funding. Sokol:Seattle Genetics: Consultancy; Spectrum: Consultancy. Komrokji:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy.

Role of S-Phase Fraction in Differentiating Aplastic Anemia from Hypoplastic Myelodysplastic Syndrome

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4118-4118
Author(s):  
Anil Tripathi ◽  
Payal Tripathi ◽  
Ashutosh Kumar ◽  
Rizwan Ahmad ◽  
Anil Balapure ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Aplastic Anemia ◽  
Peripheral Blood ◽  
S Phase ◽  
Phase Fraction ◽  
Control Subjects ◽  
Significant Difference ◽  
The Mean ◽  
Made In

Abstract Among the patients with bone marrow hypoplasia, differentiating aplastic anemia (AA) from hypoplastic myelodysplastic syndrome (HMDS) can be a difficult and challenging task because of the considerable clinical, cytological and histological similarities between these two disorders. The distinction between AA and HMDS is important because the clinical course and management of these two entities differ. There is a higher risk of progression to acute leukemia in patients with HMDS compared with AA. Various attempts have been made in the past to differentiate these entities. Different patterns of proliferation of bone marrow cells in AA and HMDS have been reported in past using proliferating cell nuclear antigens (PCNA). S-Phase Fraction (SPF) reflects the cellular proliferation and has been proven to be a useful diagnostic and prognostic marker in various hematological malignancies and solid tumors. In the present study, we examined whether flow cytometric analysis of SPF could be used as a tool to differentiate AA from HMDS. The study group comprised of 25 consecutive patients with AA, 18 patients with HMDS diagnosed on the basis of peripheral blood and bone marrow findings along with 30 age and sex matched healthy controls. The mean age of AA patients and HMDS patients was 27.1 ± 12.7 years (range 13–65 years with median age of 23 years) and 38.8 ± 20.6 years (range 15–75 years with median age of 32.5 years) respectively. The most common clinical presentation in patients with AA and HMDS was anemia. Other manifestations were bleeding and pyrexia. No etiological association could be made in any of these cases. Peripheral blood leucocytes were stained with propidium iodide and analyzed for SPF through flow cytometry using Modfit-LT V 3.0 software. The mean SPF value in the patients with AA and HMDS was 0.49 ± 0.33% and 0.79 ± 0.28% respectively. The mean SPF value in control subjects was 0.67 ± 0.22%. The SPF value in patients with AA was significantly lower than that of control (p=0.01) whereas there was no significant difference in SPF values in patients with HMDS and control subjects. The SPF value was statistically significant higher in HMDS patients as compared to AA (p=0.003). During follow-up, 3 patients (12%) with AA have revealed the evidence of dysplasia on repeat bone marrow examination. These patients had high SPF values as compared to the median SPF value in AA patients. We conclude that SPF value may be an important parameter in patients with AA to predict their propensity to evolve into HMDS. SPF value may also be useful in the early diagnosis of HMDS before morphologically evidence of dysplasia is apparent.

The Role of Molecular Profiling of Bone Marrow Samples in Confirming the Diagnosis of Myelodysplastic Syndrome in Patients Presenting with Cytopenia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1667-1667
Author(s):  
Maya Thangavelu ◽  
Wanlong Ma ◽  
Steven Brodie ◽  
Christopher Mixon ◽  
Wayne Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Allele Frequency ◽  
Gene Mutation ◽  
Mutant Allele ◽  
Conflicts Of Interest ◽  
Molecular Profiling ◽  
Cytogenetic Abnormalities ◽  
Mutant Allele Frequency ◽  
Significant Difference

Abstract Introduction: Diagnosis of myelodysplastic syndrome (MDS) can be very difficult when blast count in bone marrow is <5%. The demonstration of a mutation in one or more of the MDS-related genes is usually considered an objective confirmation of MDS. However, recent reports suggest that normal individuals may have circulating clonal hematopoietic cells carrying MDS-related mutations. We studied the relevance the mutated allele frequency and number of mutated genes in confirming the diagnosis of MDS in patients with cytopenia as determined using bone marrow samples. Methodology: We analyzed Next Generation Sequencing (NGS) data from of 294 consecutive bone marrow samples referred to rule out MDS and were reported to be positive for mutation in one or more MDS-related genes. All samples were tested for mutations in the following genes: TET2, SF3B1, ASXL1, DNMT3A, SRSF2, RUNX1, NRAS, ZRSR2, EZH2, ETV6, TP53, CBL, NPM1, JAK2, U2AF1, IDH1, KRAS, IDH2, FLT3, PTPN11, SETBP1, and BCOR. The average depth of NGS testing in this targeted sequencing was approximately 10,000X. Results: Of the 294 MDS samples with mutations, 103 (35%) had blasts <5%. Of the 103 samples, 36 (35%) showed mutations in one gene; the remaining (65%) had mutations in more than one gene. The frequency of the mutant allele was <20% in only 11 of 103 cases (11%). The remaining 92 patients had either mutations in two genes or in one gene, but the mutant allele frequency was >20%. Four of the 11 patients (36%) with one gene mutation and <20% allele frequency had cytogenetic abnormalities confirming the diagnosis of MDS [der(1;7)(q10;p10), del(5q), trisomy 8. and del(11)(q23)]. Of the remaining 7 patients with allele frequency <20%, 3 had mutations in DNMT3A, 1 in U2AF1 gene, 1 in TET2 gene, 1 in TP53 and 1 in SF3B1 gene. Of these 7 cases, only two cases had an allele frequency <10%, one in TP53 gene and one in SF3B1 gene. Of the 92 cases with mutations in two genes or in one gene with allele frequency >20%, 26 patients (28%) had cytogenetic abnormalities confirming the diagnosis of MDS. In fact in this group of 26 patients with cytogenetic abnormalities, only one patient had mutations at <20% in all mutated genes (TET2, DNMT3A and TP53), but also had del(17p). Of the remaining patients 65 cases without cytogenetic abnormalities, with more than one gene mutation, at least one gene had mutant allele at >20%. There was no statistically significant difference in the degree of cytopenia between patients with <20% one mutation and no cytogenetic abnormalities (N=7) and the 96 cases with mutations in two genes or in one gene with allele frequency >20%. There was no significant difference in the degree of cytopenia between the 36 patients with one gene mutation and 67 patients with more than one gene mutation. Conclusion: This data suggests that bone marrow samples from patients with peripheral cytopenia should be tested by cytogenetic and molecular profiling using NGS and the analysis of MDS-related genes. Our data suggests that when proper criteria are used, molecular profiling of bone marrow in the proper clinical presentation can help in confirming the diagnosis of MDS. Our data suggests that the presence of mutations in more than one gene and the detection of mutant allele frequency >20% may comprise reliable criteria for the diagnosis of MDS. The presence of mutation in 20% of DNA usually reflects mutation in 40% of the bone marrow cells. Patients with mutant allele frequency between 10% and 20% in the bone marrow and cytopenia most likely have MDS, but further studies are needed. Mutant allele frequency in bone marrow of <10% is extremely rare when testing is performed in patients presenting with cytopenia. Disclosures No relevant conflicts of interest to declare.

scholarly journals Myeloid Neoplasm Gene Somatic Mutations in Patients with Severe Aplastic Anemia Treated with Eltrombopag and Standard Immunosuppression

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 727-727 ◽  
Author(s):  
Danielle M. Townsley ◽  
James N. Cooper ◽  
Thomas Winkler ◽  
Phillip Scheinberg ◽  
Olga Rios ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Research Funding ◽  
Somatic Mutations ◽  
Clonal Evolution ◽  
Response Rates ◽  
Complete Response ◽  
Hematologic Response ◽  
Treatment Naïve ◽  
Cytogenetic Evolution

Abstract The major complication of severe aplastic anemia is clonal evolution, defined as any new cytogenetic abnormality or progression to MDS/AML, which occurs in about 15% of SAA patients, usually many months to years after the diagnosis. Eltrombopag, a thrombopoietin receptor agonist, appears capable of stimulating hematopoietic stem cell proliferation in patients with bone marrow failure. Addition of eltrombopag to standard immunosuppressive treatment (IST) with horse antithymocyte globulin (hATG) and cyclosporine (CsA) markedly increases hematologic response rates in treatment-naive SAA, with overall response rates up to 90% and complete response rates approaching 60% (Townsley DM et al, ASH 2015, clinicaltrials.gov NCT01623167). In comparison, IST alone achieves 60% overall response rates, of which 10% are complete (Scheinberg Blood 2012). Somatic mutations in myeloid cancer candidate genes are present in one-third of patients after IST alone (Yoshizato T et al, NEJM 2015). Specific subsets of mutations were associated with clinical outcomes: a group including ASXL1 and DNMT3A with a poor response to IST, inferior survival, and clonal evolution, while BCOR and PIGA were associated with good response and favorable outcomes. Monosomy 7, the most prevalent cytogenetic abnormality defining clonal evolution, can develop in the absence of gene mutations, underscoring the non-determinative and complex role mutations play in clonal evolution (Dumitriu B et al, Blood 2015). Of patients with disease refractory to IST who were subsequently treated with single-agent eltrombopag, 19% (8/43) developed cytogenetic abnormalities, usually within the first year of treatment, but only rarely with morphologic changes consistent with MDS/AML (Desmond R et al, Blood 2014). The frequency of somatic mutations following treatment with eltrombopag added to IST in treatment-naïve SAA patients is unknown. We used amplicon-based next-generation sequencing to assess mutations in 54 candidate genes recurrently mutated in myeloid neoplasms. Bone marrow cells of 90 subjects who had been treated with IST/eltrombopag were obtained at 6 months following treatment initiation, or at the time of clonal evolution. At least one detectable mutation was identified in 21 (23%) subjects. All 21 patients had exhibited a hematologic response to treatment by 6 months; of those patients with somatic mutations, 14 of 19 (74%) had a complete hematologic response. In comparison, of the 69 patients who lacked mutations, 20 (29%) had a complete response. Nine different genes were mutated in total, with the most frequent genes being ASXL1 and BCOR. BCOR was associated with more robust responses (6 of 7 had a complete response) and younger age (range 12 - 49 years; Table). One subject with a longstanding history of JAK2-positive essential thrombocytosis and myelofibrosis at baseline had two additional mutations detectable following treatment, TET2 and ASXL1. With a median follow up of 21 months, clonal cytogenetic evolution occurred in 7/90 (8%) subjects. Three of seven patients also had a mutation in a myeloid cancer gene (two with DNMT3A and one with ASXL1/RUNX1); in the other four, no somatic mutation was detected, either at the 6-month time point or at time of cytogenetic evolution. Four patients had monosomy or partial deletion of chromosome 7: one patient had complex (t(3;3)(q21;q26), -7), one patient had deletion 13q that later disappeared, and one patient had trisomy 6 and trisomy 15 in 2 metaphases. The patient with complex cytogenetics did not have somatic mutations detected at evolution, and she later died due to relapsed AML following transplant. The rates of somatic mutations in myeloid cancer genes and of cytogenetic evolution in patients treated with IST/eltrombopag do not appear to be higher than we and others have reported in aplastic anemia treated with standard IST, without eltrombopag (Kulasekararaj AG et al, Blood 2014). The distribution of genes mutated and the allelic frequency of these mutations also were similar to patients treated with standard IST. These results suggest that the benefits of a higher response rate and quality of response associated with the addition of eltrombopag to IST for the initial treatment of SAA are not associated with a higher risk of clonal progression. Disclosures Townsley: GSK/Novartis: Research Funding. Cooper:GSK/Novartis: Research Funding. Winkler:GSK/Novartis: Research Funding. Scheinberg:Novartis: Consultancy, Speakers Bureau. Rios:GSK/Novartis: Research Funding. Weinstein:GSK/Novartis: Research Funding. Desierto:GSK/Novartis: Research Funding. Fernandez Ibanez:GSK/Novartis: Research Funding. Dunbar:GSK/Novartis: Research Funding. Ma:Neogenomics Laboratories: Employment. Albitar:Neogenomics Laboratories: Employment, Equity Ownership. Young:GSK/Novartis: Research Funding.

scholarly journals The Mutation Profile of Myelodysplastic Syndrome Associated with Auto-Immune Rheumatological Disorders

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3081-3081
Author(s):  
Lih En Hong ◽  
Deepak Singhal ◽  
Amilia Wee ◽  
Rakchha Chhetri ◽  
Mihir D Wechalekar ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Repair ◽  
Allele Frequency ◽  
Research Funding ◽  
Germline Mutations ◽  
Cytotoxic Agents ◽  
Royal Adelaide Hospital ◽  
Repair Pathway ◽  
Mutation Profile ◽  
Significant Difference

Abstract Introduction: A subset of patients with MDS and related myeloid disorders present with concomitant autoimmune rheumatological diseases (AIRD); however the prevalence ranges from 10-48% based on limited literature. Further, use of immunosuppressive agents in AIRD patients could confound the secondary diagnosis of MDS and in some cases cause it (therapy-related myeloid neoplasm; t-MN). The prevalence of cytopenia in AIRD patients is unknown and the genetic characteristics of MDS patients with concomitant AIRD have not been described. Hence, we interrogated two large multi-institutional databases -Royal Adelaide Hospital Rheumatology Database (RAH-RD) and South-Australian MDS (SA-MDS) registry in this study. Methods: Demographic, clinical, laboratory and treatment data of 2663 AIRD and 1157 MDS patients were analysed. In AIRD patients (autoimmune inflammatory arthritis, spondyloarthritis, vasculitis and connective tissue diseases), cytopenia (persisting >6 months) were defined as follows: hemoglobin <100g/dL, absolute neutrophil <1800/mm3 and platelet <100,000/mm3. Targeted massively parallel sequencing of a custom panel of 43 myeloid neoplasms associated genes and 20 Fanconi (FA) DNA repair pathway genes (all coding regions) was performed on diagnosis bone marrow samples (n=237). An in-house well established filtering pipeline was used for identification of somatic mutations. Matched germline material was available for 62/194 (32%) patients. Only variants with Genome Aggregation Database minor allele frequency of ≤0.01% and variant allele frequency of ≥35% were selected for further analysis of germline variants. Results: During follow up of 2663 AIRD patients, 36 (1.3%) patients satisfied the criteria for at least one cytopenia. Anemia (19/36, 53%) was most common followed by neutropenia (8/36, 22%), thrombocytopenia (4/36, 11%) and bi-cytopenia (5/36, 14%). Twenty-two patients had bone marrow examination which was non-diagnostic in 16 patients, while 7/2663 (0.3%) patients were diagnosed with MDS. Importantly, 5 patients with MDS and 11 patients with cytopenia did not receive any cytotoxic agents. In the MDS database, 69(5.4%) were diagnosed with AIRD, with rheumatoid arthritis (n=20, 29%) being the most common AIRD. Among these 69 patients, 24 (34.8%) had low risk MDS and 15 (21.7%) had higher risk MDS. The remaining 30 patients had t-MN (n=19, 27.5%), MDS/MPN (n=8, 11.6%) and AML (n=3, 4.3%). Overall, in a combined population of 2663 RAH-RD and 1157 SA-MDS, 76(2%) had concomitant MDS and AIRD. Genetic profile of patients with MDS and AIRD: The cytogenetic and mutational profile of MDS patients with (n=20) or without (n=217) AIRD were compared. No significant difference was seen in the cytogenetic profile (normal, complex or monosomal karyotype, chr. 5 or 7 abnormalities) between the two groups but in mutational analysis, 56 mutations were seen in 20 MDS patients with AIRD (Fig1). In these patients, mutations in epigenetic pathways were most common (23/56, 41%) followed by transcription pathway (10/56, 18%). Splicing mutations were seen in 5 patients, with SRSF2 mutations being more common than SF3B1. Mutations in TP53 were present in 4 (24%) patients; 3/4 patients developed MDS following therapy for AIRD (t-MDS). IDH1 mutations were found in significantly higher frequency in MDS patients with AIRD compared to MDS without AIRD (30% vs 3%, p=0.04). There was no significant difference in the frequency of other mutations or overall mutation frequency between the two groups. Interestingly, 2 (10%) patients with MDS and AIRD also had rare, deleterious germline mutations in FA pathway genes (BRCA2 V2601M and L2512F) which could suggest either genetic predisposition to both these conditions or compromised DNA repair capability increasing susceptibility to t-MN. Conclusions: In a large multi-institutional cohort of autoimmune rheumatological disorders, 1.3% patients developed persistent cytopenia with 0.3% diagnosed with MDS. This is significantly higher than incidence of MDS in the general population (30-50/100,000). Similarly, 5% MDS patients had AIRD. The mutation profile of MDS patients with AIRD shows higher frequency of IDH1 and SRSF2 mutations. A small proportion of cases also had deleterious rare germline mutations in the DNA repair pathway. Our findings warrant further study and have potential implications for selection of immunosupressive agents for AIRD. Disclosures Hiwase: Novartis: Research Funding; Celgene: Research Funding.

scholarly journals Clinical observations of bone marrow transfusion for promoting bone marrow reconstruction after chemotherapy for AIDS-related lymphoma

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yixuan Liu ◽  
Suhong Xie ◽  
Lei Li ◽  
Yanhui Si ◽  
Weiwei Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Immune System ◽  
T Lymphocytes ◽  
Peripheral Blood ◽  
Autologous Bone Marrow ◽  
Control Group ◽  
Peripheral Vein ◽  
Significant Difference ◽  
Autologous Bone ◽  
Aids Related Lymphoma

Abstract Background This study investigates the effect of autologous bone marrow transfusion (BMT) on the reconstruction of both bone marrow and the immune system in patients with AIDS-related lymphoma (ARL). Methods A total of 32 patients with ARL participated in this study. Among them, 16 participants were treated with conventional surgery and chemotherapy (control group) and the remaining 16 patients were treated with chemotherapy followed by autologous bone marrow transfusion via a mesenteric vein (8 patients, ABM-MVI group) or a peripheral vein (8 patients, ABM-PI group). Subsequently, peripheral blood and lymphocyte data subsets were detected and documented in all patients. Results Before chemotherapy, no significant difference in indicators was observed between three groups of ARL patients. Unexpectedly, 2 weeks after the end of 6 courses of chemotherapy, the ABM-MVI group, and the ABM-PI group yielded an increased level of CD8+T lymphocytes, white blood cells (WBC), and platelet (PLT) in peripheral blood in comparison to the control group. Notably, the number of CD4+T lymphocytes in the ABM-PI group was significantly higher than that in the other two groups. Additionally, no significant difference in haemoglobin levels was observed before and after chemotherapy in both the ABM-MVI and ABM-PI groups, while haemoglobin levels in the control group decreased significantly following chemotherapy. Conclusions Autologous bone marrow transfusion after chemotherapy can promote the reconstruction of both bone marrow and the immune system. There was no significant difference in bone marrow recovery and reconstruction between the mesenteric vein transfusion group and the peripheral vein transfusion group.

scholarly journals The Necessary for Long-Term Follow-up of Mutated Genes and Clonal Evolutions in Multiple Myeloma Combined with cfDNA Assay

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5515-5515
Author(s):  
Yuko Mishima ◽  
Yuji Mishima ◽  
Masahiro Yokoyama ◽  
Noriko Nishimura ◽  
Yoshiharu Kusano ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Genomic Dna ◽  
Research Funding ◽  
Somatic Mutations ◽  
Clinical Course ◽  
Bone Marrow Aspiration ◽  
Long Term Follow Up

Introduction)Somatic mutations in multiple myeloma (MM) are strongly related to the clinical outcome and clonal evolution over the clinical course, and are a major problem. From a clinical viewpoint, although numerous novel drugs have been utilized, achieving long-lasting and complete remission remains difficult. Recent studies have elucidated the mutated genes using next-generation sequencing, and have examined how clonal change can be acquired in myeloma. In this study, we traced the transition of the somatic mutations of bone marrow tumor cells in patients with MM over a long-term follow-up. Furthermore, we compared the somatic mutations found in serum cell-free DNA (cfDNA) and mutated genes obtained from bone marrow myeloma cells. Material and Methods)Patients diagnosed with multiple myeloma who provided written informed consent to participate in the study were enrolled. Patients were treated by immuno-chemotherapy with or without radiation between 2000 and 2017 at our institute. Bone marrow aspiration and biopsy were performed at the time of diagnosis and upon disease progression. Around the time of bone marrow aspiration, serum was obtained from a peripheral blood sample for cfDNA analysis. Myeloma cells were separated from bone marrow samples with MicroBeads of CD138 antibody and genomic DNA was extracted. The peripheral blood samples derived from myeloma patients. The cfDNA was extracted from the serum using a Maxwell RSC cfDNA Plasma kit. Using genomic DNA derived from cfDNA and bone marrow, multiplex polymerase chain reaction (PCR) was performed, and a sequence library was then constructed with an Ion Custom Amplicon panel. The panel for the sequence library was designed using an Ion AmpliSeq DesignerTM. 126 targeted genes were selected. The genomes were sequenced using the Ion ProtonTM System. This protocol was approved by the institutional review board and the Genomic Review Board of the Japanese Foundation for Cancer Research. Result)We followed 7 patients' long term-clinical course and the transition of mutations (8.5 year average). The expression of myeloma driver genes, such as RAS, BRAF, and MYC, were not critical. We did, however, detect a relationship between an increase in the dominant mutated gene, such as TP53, DIS3, FAM46C, KDM6B, and EGR1 and poor prognosis in patients with myeloma. Next, we calculated the cfDNA concentrations from 34 cases. The cfDNA concentrations were significantly higher than 10 control cases (average 62.0 ng/mL (0-200 ng/mL) and 8.18 ng/mL (4.3-14.1 ng/mL), P=0.0046). The 2.5 year-progression free survival (PFS) during the first treatment of MM were tend to be poorer in the group with cfDNA>50 ng/mL (72.9%) than the group with cfDNA<50 ng/mL(25.9%), however there are no statistical significance (P = 0.15).We caluculated concordance rate of derived mutations from bone marrow MM cells and cfDNA in 7 cases. The somatic mutations found in serum cell-free DNA (cfDNA) and bone marrow MM cells were determined the correlation coefficients. However, there are few difference expression pattern in each source. In cfDNA assay, CREEP, EGR1, HDAC4, HDAC6, and JMJD1C were highly expressed as 57.1% (4/7) - 85.7% (6/7), and these results were almost the same as those for bone marrow MM cells. On the other hand, KDM1A (85.7%), PI3KCD (71.4%), and KDM3B (57.1%) were highly detected in cfDNA, although those were not frequently expressed in bone marrow. Discussion)Our data demonstrate the importance of the long-term follow-up of somatic mutations during the clinical course of myeloma. Serum cfDNA is a useful alternative source for detecting somatic mutations in MM patients during long-term follow-up. Disclosures Mishima: Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Yokoyama:Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Nishimura:Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy; Celgene K.K.: Honoraria. Hatake:Celgene K.K.: Research Funding; Janssen Pharmaceutical K.K.: Research Funding; Takeda Pharmaceutical Co.,Ltd.: Honoraria. Terui:Bristol-Myers Squibb K.K.: Research Funding; Bristol-Myers Squibb, Celgene, Janssen, Takeda, MSD, Eisai, Ono, and Chugai-Roche Pharmaceuticals Co.,Ltd.: Honoraria.

scholarly journals Genome-Wide Association Study Identifies an Immune-Related Etiology for Severe Aplastic Anemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1224-1224
Author(s):  
Sharon A. Savage ◽  
Mathias Viard ◽  
Weiyin Zhou ◽  
Meredith Yeager ◽  
Shengchao Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Association Study ◽  
Research Funding ◽  
Genome Wide Association Study ◽  
Genome Wide Association ◽  
Class I ◽  
Severe Aplastic Anemia ◽  
Genome Wide ◽  
Validation Set

Introduction. Acquired severe aplastic anemia (SAA) is a life-threatening disorder characterized by severe progressive pancytopenia and hypocellular bone marrow. The etiology of acquired SAA is not understood but believed to be related to abnormal immune responses to environmental exposures. We conducted a genome-wide association study (GWAS) to identify common germline variants associated with SAA. Methods. We identified 895 patients with SAA who underwent related or unrelated hematopoietic cell transplant (HCT) with clinical data and pre-HCT blood samples available in the Center for International Blood and Marrow Transplant Research (CIBMTR) database and biorepository. Pre-HCT DNA was extracted from blood of patients with SAA and genome-wide genotyping was conducted using the Illumina OmniExpress array. We excluded 93 inherited bone marrow failure cases. The SAA cases were grouped cases into discovery and validation sets based on time of batch sample receipt. Analyses were limited to patients of European ancestry based on principal component analyses to minimize the potential effect of population stratification. Controls were genomically matched and selected from previously scanned cancer-free subjects at the Cancer Genomics Research Laboratory, Division of Cancer Epidemiology and Genetics, NCI. The final analysis included 534 acquired SAA cases (359 in the discovery set and 175 in the validation set), and 2,455 controls (1,396 in the discovery set, and 1,059 in the validation set). Results. Patients with SAA in this study received HCT between 1989-2015 at a median age of 21 years, 56% were male, and the median time between SAA diagnosis and HCT was 11 months. Strong genome-wide association signals were identified across the human leukocyte antigen (HLA) genes encoded at the major histocompatibility complex (MHC) on chromosome 6p21 (Figure 1). The top SNP was located in the P4 binding pocket of the HLA class II gene HLA-DPB1(rs1042151A>G, p.Met105Val, pooled-odds ratio [OR]=1.75, 95% confidence interval [CI]=1.50-2.03, p=1.94x10-13). The expression of HLA-DP in CD19+ cells from 175 healthy donors was significantly different by rs1042151 A>G genotype (p=2.04x10-6) (Figure 2). A second SNP near HLA-B, rs28367832G>A, also reached genome-wide significance (pooled-OR=1.49, 95% CI=1.22-1.78, p=7.27x10-9). Copy-number variant analysis and next generation sequencing also identified somatic, clonal copy-neutral loss-of-heterozygosity affecting class I HLA genes in 8.6% of the SAA cases and none of the controls. Conclusion. This SAA GWAS identified strong association signals between common germline genetic variants in HLA class I and II genes and SAA. The main SNP is associated with changes in HLA-DP expression suggesting a key role for this locus in SAA etiology. This study adds further evidence to the connection between SAA and immune dysregulation. Disclosures Cerhan: Janssen: Membership on an entity's Board of Directors or advisory committees; NanoString: Research Funding; Celgene: Research Funding. Lee:Incyte: Research Funding; Syndax: Research Funding; Amgen: Research Funding; Novartis: Research Funding; Takeda: Research Funding; Kadmon: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; AstraZeneca: Research Funding.

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