Low Mutation Rate and No Significant Prognostic Implication of SF3B1, U2AF1 and SRSF2 Genes in Myelodysplastic Syndrome without Harboring Ring Sideroblast

Abstract Abstract 4950 Background: Myelodysplastic syndrome (MDS) is an hematopoietic stem cells (HSCs) disorder, leading to malignant cells that ultimately grow uncontrollably. Somatic mutations of spliceosomal gene such as SF3B1, U2AF1 and SRSF2 has been widely described in MDS and other hematological malignancies. Many reports state that hematopoietic malignancies mostly result from somatic mutations in HSCs in the bone marrow. Some functional studies have been performed and have shown that a high proportion of these cases are associated with somatic mutations in spliceosomal proteins. Also, such kinds of mutation are hallmarks of dominantly acting and growing specific cancer. Many efforts have been made to unravel the mutation considered disease background of MDS. Genotype–phenotype associations have been demonstrated for somatic spliceosomal gene mutation in MDS with ring sideroblasts but there is not much research regarding aberrant splicing pathways in MDS without harboring ring sideroblast. Therefore, this study investigated the prevalence and prognostic implication of the SF3B1, U2AF1 and SRSF2 splice gene mutation in these patients in Korea. Materials and Methods: The study cohort of 92 MDS patients was examined for somatic mutations in SF3B1, U2AF1 and SRSF2 splicing gene using direct sequencing method. The clinical and hematologic data were also recorded. The collected data was analyzed by SPSS for Windows version 18. 0. Pearson's chi-square tests, one way ANOVA analysis, and Student t-test were performed. Survival rates of multiple myeloma patients according to the mutation result of SF3B1, U2AF1 and SRSF2 splicing gene were analyzed using Kaplan-Meier log-rank test. Results: Our 92 MDS patients showed recurrent mutation and polymorphisms. Mutations in K666N, H662Q and K700E of SF3B1; S34T, S34P and Q157P of U2AF1; P95H, P95R and P95L of SRSF2 were found in 8 (8. 7%), 6 (6. 5%), and 11 (11. 9%) patients, respectively. The patients displayed 39198T>T/C polymorphism (88. 0%) in exon 18 of SF3B1, 8345T>T/G polymorphism (7. 6%) in exon 2 of U2AF1 and 5399C/T polymorphism (100%) in exon 1 of SRSF2. In the entire cohort, the number of patients with no polymorphism, one polymorphism, two polymorphisms and three polymorphisms was counted up to 0%, 12%, 80. 4% and 7. 6%, respectively. The T/C polymorphism at position 39198 of SF3B1 exon 18 and the T/G polymorphism at position 8345 of U2AF1 exon 2 were analyzed by allele-specific PCR using normal control. Results in 100 normal controls, polymorphism of SF3B1 exon 18 was taken into account of 82. 0%, the remaining is non polymorphism while U2AF1 exon 2 showed 10. 0% polymorphism and 90. 0% non polymorphism. The patient with either polymorphisms or mutations in both SF3B1, U2AF1 and SRSF2 had no effect on overall survival and disease-free survival. Conclusion: Our results show that mutation rate of SF3B1, U2AF1 and SRSF2 splice gene in Korean MDS patients without harboring ring sideroblast displayed relatively rare and infrequent molecular event. Moreover, alteration of SF3B1, U2AF1 and SRSF2 splice gene was not significantly implicated in the clinical outcomes and prognosis. Disclosures: No relevant conflicts of interest to declare.

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Correlation Analysis of DNA Methylation Level and Clinical Characteristics in JMML Patients

Blood ◽

10.1182/blood-2021-150044 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3699-3699

Author(s):

Yuli Cai ◽

Jingliao Zhang ◽

Meihui Yi ◽

Xiaoming Liu ◽

Xiaoyan Zhang ◽

...

Keyword(s):

Dna Methylation ◽

Correlation Analysis ◽

Gene Mutation ◽

Methylation Level ◽

Somatic Mutations ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasm ◽

Juvenile Myelomonocytic Leukemia ◽

Ptpn11 Gene ◽

Number Of Patients

Abstract Objective: As a rare, aggressive pediatric myeloproliferative disease, juvenile myelomonocytic leukemia (JMML) encompassed both biological features of myelodysplastic syndrome and myeloproliferative neoplasm. Studies have shown that the methylation level in JMML patients is closely related to prognosis, and patients with high methylation level have poor prognosis. This study aimed to find clinical indicators that were associated with different methylation levels and prognosis. Methods: The clinical information of 24 JMML patients with DNA samples admitted to our center from December 2013 to May 2020 was retrospectively analyzed, and the DNA methylation level of their whole genome was detected. Results: The median age of onset was 14.5 months (0.1-153 months) among the 24 cases, including 17 males and 7 females. At diagnosis, the median WBC count was 27.1×10 9/L (6.2-98.1×10 9/L), and the median platelet count was 38×10 9/L (10-277×10 9/L). Chromosome karyotype abnormalities were found in 12.5% (3/24) of patients. Next-generation sequencing results showed that 79.2% (19/24) patients had at least one Ras pathway-related classical gene mutation, and 41.7% (10/24) patients had two or more somatic mutations. Genomic DNA methylation levels were divided into three groups: 10 cases in the hypomethylation group, 4 cases in the moderate methylation group, and 10 cases in the hypermethylation group. There were significant differences in age, platelets, PTPN11 gene mutation and the number of somatic mutations ≥2 in different methylation groups (P<0.05). The age of hypomethylated group was significantly lower than that of hypermethylated group (P<0.05), and the platelets of hypomethylated group was significantly higher than that of hypermethylated group (P<0.05). Patients ≤12m and platelets＞32×10 9/L had lower DNA methylation level (P<0.0001). The number of patients with PTPN11 gene mutation in the hypomethylated group was significantly lower than that in the hypermethylated group (P<0.05), and the number of patients with ≥2 mutations in the low and medium methylated groups was significantly lower than that in the hypermethylated group (P<0.05). Correlation analysis showed that hypermethylation level was significantly correlated with PTPN11 gene mutation and ≥2 somatic mutations (P<0.001). Conclusions: JMML patients with high methylation level in the DNA genome at diagnosis were older and with lower platelet levels, and hypermethylation were significantly correlated with high-risk prognostic factors such as PTPN11 gene mutation and ≥2 somatic mutations. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Decitabine for treatment of children with JMML

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No Recurrent Mutation of SF3B1, U2AF1 and SRSF2 Spliceosomal Gene in Multiple Myeloma but Polymorphisms of These Genes Are Associated with the Prediction of Worse Prognosis

Blood ◽

10.1182/blood.v120.21.3958.3958 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3958-3958

Author(s):

Trang Nguyen Thi Dai ◽

Hye-Ran Kim ◽

Min-Gu Kang ◽

Stephanie J. Won ◽

Hwan-Young Kim ◽

...

Keyword(s):

Multiple Myeloma ◽

Light Chain ◽

Somatic Mutations ◽

Conflicts Of Interest ◽

Bone Lesion ◽

Disease Free Survival ◽

Free Light Chain ◽

Immunoglobulin Level ◽

Exon 2 ◽

Number Of Patients

Abstract Abstract 3958 Background: Recently, a striking example of the effects on acquired somatic mutations in splicing factors such as SF3B1, U2AF1, and SRSF2 has been described. The sequencing of the DNA from abnormal blood cells from patients with several types of leukemia such as AML, CLL, CMML, pre-leukemic syndromes, and MDS, has shown that a high proportion of these cases are associated with somatic mutations in spliceosomal proteins. Also, evidence of cancer-specific alternative splicing and oncogenic somatic mutations in spliceosome subunits has been steadily growing. However, there is not much research regarding aberrant splicing pathways in Multiple myeloma (MM) patients. Therefore, we tried to investigate the presence and prognostic implication of mutation of the SF3B1 and U2AF1 protein in these patients in South Korea. Materials and Methods: We examined a cohort of 87 MM patients and 100 healthy controls for somatic mutations in SF3B1, U2AF1 and SRSF2 by using direct sequencing method. The medical records were reviewed for age, sex, plasma cell percentage, serum M protein, immunoglobulin level, free light chain ratio, calcium, creatinine, hemoglobin, bone lesion, albumin, beta 2 microglobulin, lactate dehydrogenase, treatment outcome, and so on. The collected data was analyzed by SPSS for Windows version 18.0. We performed Pearson's chi-square tests, one way ANOVA analysis, and Student t-test. Survival rates of myeloma patients according to the result of SF3B1, U2AF1 and SRSF2 sequencing were analyzed using Kaplan-Meier log-rank test. Results: Our 87 MM patients showed no mutation including known recurrent ones in SF3B1, U2AF1 and SRSF2 genes. However, the patients displayed 39198T>T/C polymorphism (70.1%) in exon 18 of SF3B1, 8345T>T/G polymorphism (13.8%) in exon 2 of U2AF1 and 5399C/T polymorphism (100%) in exon 1 of SRSF2. In the entire cohort, the number of patients with no polymorphism, one polymorphism, two polymorphisms and three polymorphisms was counted up to 0%, 24.1%, 67.8% and 8.0%, respectively. The T/C polymorphism at position 39198 of SF3B1 exon 18 and the T/G polymorphism at position 8345 of U2AF1 exon 2 were analyzed by allele-specific PCR using normal control. Results in 100 normal controls, polymorphism of SF3B1 exon 18 was taken into account of 82.0%, the remaining is non polymorphism while U2AF1 exon 2 showed 10.0% polymorphism and 90.0% non polymorphism. Sex (p=0.048) and free light chain ratio (p=0.002) showed significant results according to polymorphism status while other clinical characteristics were not associated. The patient with polymorphisms in both SF3B1 and U2AF1 had worse overall survival (P=0.042) and disease-free survival (P<0.01), compared to patients without polymorphism. Conclusion: Our results show no recurrent SF3B1, U2AF1 and SRSF2 mutations in MM patients rather polymorphisms in SF3B1 and U2AF1 gene were significantly implicated in the prediction of poor prognosis. Disclosures: No relevant conflicts of interest to declare.

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No Mutations of SF3B1, U2AF1, and SRSF2 spliceosomal Gene but Polymorphisms Associated with the Worse Prognosis in Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.4190.4190 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4190-4190

Author(s):

Min-Gu Kang ◽

Hye-Ran Kim ◽

Jun Hyung Lee ◽

Thashma Pemmanda Ganapathy ◽

Seung-Hyeon Yang ◽

...

Keyword(s):

Multiple Myeloma ◽

Dna Sequences ◽

Somatic Mutations ◽

Conflicts Of Interest ◽

Disease Free Survival ◽

Cellular Growth ◽

Exon 2 ◽

Number Of Patients ◽

Significant Difference ◽

Worse Prognosis

Abstract Recently, a striking example of the effects of acquired somatic mutations in splicing factors such as SF3B1, U2AF1, and SRSF2 has been described. The DNA sequences from abnormal blood cells of patients with hematologic malignancies has shown that a high proportion of these diseases are associated with somatic mutations of spliceosomal proteins. However, little is known about the aberrant splicing pathways in multiple myeloma (MM) patients. Therefore, this study investigated the presence and prognostic implication of splicing gene aberration in MM patients. Also, we planned functional experiments in cultured normal and Hela cell line to determine the effects of the common spliceosome mutations on cellular growth rate, cell count, and apoptosis. In total, 87 MM patients and 100 healthy controls were examined for somatic mutations in SF3B1, U2AF1, and SRSF2 by direct sequencing. Also, the clinical and laboratory characteristics of MM patients were reviewed. Regarding to the initial findings in recent reports, this study expressed the wild-type and the mutant (Q157P) U2AF1 in Hela cells and examined their proliferation. MM patients did not show mutation in SF3B1, U2AF1,and SRSF2 splicing genes. However, the patients displayed 39198T>C or 39198T>T/C polymorphism (70.1 %) in exon 18 of SF3B1, 8345T>T/G polymorphism (13.8 %) in exon 2 of U2AF1, and 5399C>T or 5399C>C/T polymorphism (100.0 %) in exon 1 of SRSF2 (Fig. 1). In the entire cohort, the number of patients with one polymorphism, two polymorphisms, and three polymorphisms was counted up to 24.1 %, 67.8 %, and 8.0 %, respectively. Meanwhile, in 100 normal controls, polymorphism of SF3B1 exon 18 and U2AF1 exon 2 was counted up to 82.0 % and 10.0 %, respectively. Free light chain (FLC) kappa/lambda (K/L) ratio and chromosome analysis showed significant difference according to polymorphism status (p=0.002) while other clinical characteristics did not. The patient with polymorphisms in both SF3B1 and U2AF1 gene had unfavorable overall survival (p =0.067) and disease-free survival (p=0.001), compared to patients without polymorphism. Our results show no recurrent SF3B1 and U2AF1 mutations in MM patients. Instead, polymorphisms were found in SF3B1 or U2AF1, which were significantly implicated in worse prognosis. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Clonal Hematopoiesis in Patients with Diamond Blackfan Anemia

Blood ◽

10.1182/blood-2020-139652 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 25-26

Author(s):

Michelle Nash ◽

Adrianna Vlachos ◽

Marcin W. Wlodarski ◽

Jeffrey Michael Lipton

Keyword(s):

General Population ◽

Myelodysplastic Syndrome ◽

Somatic Mutation ◽

Somatic Mutations ◽

Conflicts Of Interest ◽

Age Of Onset ◽

Bone Marrow Failure ◽

Large Subunit ◽

Diamond Blackfan Anemia ◽

Clonal Hematopoiesis

Background: Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by anemia, congenital anomalies and a predisposition to cancer. Patients usually present during infancy or early childhood, but can also be diagnosed as adults. In the vast majority of cases DBA is due to a mutation in a gene encoding a small or large subunit-associated ribosomal protein (RP) leading to RP haploinsufficiency. In a study of 702 patients enrolled in the DBA Registry (DBAR), the observed to expected ratio for acute myeloid leukemia (AML) was 28.8 and for myelodysplastic syndrome (MDS), 352.1 (Vlachos et al, Blood, 2018). The average age of onset for MDS in the DBA cohort was 26 years, compared to 60-70 years in the general population. Evolving clonal hematopoiesis (CH) with age has been observed as a precursor to MDS, with CH rarely observed in individuals younger than 40 years of age. Thus we hypothesized that the young age at the development of MDS in DBA would be presaged by evolving CH. Objective: The primary objective was to perform whole exome sequencing (WES) specifically screening for previously reported somatic mutations in 56 genes associated with CH (Jaiswal et al, NEJM, 2014). Design/Method: A total of 69 samples were analyzed from 65 patients, mostly targeting patients older than 18 years (median age 30 years). Multiple samples were run on patients who had available samples in the DBAR Biorepository to determine rate of acquisition of mutations. 468 age- and sex-matched healthy controls were made available from GeneDx who performed the WES for the study. We used a threshold for variant calling of minimum 5% with a minimum of 2 variant reads. Results: Three of the 65 DBA patients (5%) were found to have somatic mutations in STAG1, U2AF1, SF3B1, and DNMT3A at 8, 20, 41, and 70 years, respectively (Table 1). The patient who was 20 years of age had a sample in the DBAR biorepository from when he was age 8 years which was found to have a different somatic mutation (STAG1) than was found at present (U2AF1). This patient did go on to develop MDS at the age of 21 years. In comparison, of the 468 controls, 4 (0.8 %) had a somatic mutation in SF3B1, LUC7L2, DNMT3A, and LUC7L2 at ages 12, 31, 33 and 40 years, respectively. Conclusion: Patients with DBA show more somatic mutations as compared to controls (p<0.05). This early acquisition of mutations may be the driving force for their developing MDS at an earlier age than that of the general population. Further studies with more sensitive methods are warranted to accurately determine the prevalence of somatic CH mutations and their potential association with the development of myelodysplastic syndrome in these patients. Disclosures No relevant conflicts of interest to declare.

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Defining Parameters That Can Guide Public Cord Blood Units (CBUs) in Korea

Blood ◽

10.1182/blood.v126.23.3102.3102 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3102-3102

Author(s):

Junglim Lee ◽

Byung-Su Kim ◽

Soon- Hi Jang ◽

Tai-Gyu KIM

Keyword(s):

Multivariate Analysis ◽

Cord Blood ◽

Umbilical Cord ◽

Conflicts Of Interest ◽

Univariate Analysis ◽

Female Gender ◽

Limiting Factor ◽

Study Cohort ◽

Normal Delivery ◽

Hematopoietic Stem

Abstract Background: It is well known that Total Nucleated Cell (TNC) and CD34 are the greatest limiting factor in the use of umbilical cord blood (UCB) for transplantation. To enhance high UCB quality, it is important that how the factors will affect the UCB. This study is to identify maternal, neonatal, obstetric factors that influence the suitability for banking and transplantation of UCB units collection. Study design and methods: This study examined 6534 UCB (3712 at the Catholic Hematopoietic Stem Cell Bank and 2822 at the Daegu Fatima Hospital Public Umbilical Cord Bank) collected at two banks from October 2003 to June 2015. The variables were collected from retrospective records at the time of donation. The associations between TNC, CD34+ and variables including maternal age (MA), gestational age (GA), fetal body weight (FBW), time from collection to processing (T), collecting volume (CV), preTNC, and delivery type were analyzed by logistic regression. Results: In our study cohort (n=6534, male 2988, female 2991, unkown 555, all Koreans), the median values of TNC, numbers of CD34+, MA, GA, FBW, T, preTNC, and CV were 9.24úI108/unit (range, 3.02-35.64), 2.0úI106/unit(range, 0.04-29.2), 31.0 years(range, 15-46), 277 days (range, 202-382), 3330g (range, 1740-4970), 19 hours (range, 1-54), 11.69úI108/unit (range, 3.41-50.32), and 83.5ml (range, 26.0-218.2) respectively. In univariate analysis, variables that were associated with high TNC (defined as a TNC of > 9.24úI108/unit) included GA (defined as GA > 277 days) [OR 1.29 (95% CI 1.16-1.42 p < 0.001)], FBW (defined as FBW > 3330g) [OR 1.52 (95% CI 1.37-1.68 p < 0.001)])], CV (defined as > 83.5mL) [OR 2.61 (95% CI 2.36-2.88 p < 0.001)], preTNC [OR 25.45 (95% CI 22.34-29.00 p < 0.001)], and T (defined as T> 19 hours) [OR 0.87 (95% CI 0.79-0.96 p < 0.005)]. Variables that were associated with high CD34+ (defined as a number of CD34+ > 2.0úI106/unit) included MA (defined as MA > 31.0 years) [OR 0.90 (95% CI 0.82-0.99 P=0.036)], GA [OR 0.74 (95% CI 0.67-0.82 p < 0.001)], FBW [OR 1.41 (95% CI 1.27-1.56 p<0.001)], preTNC [OR 3.38 95% CI 3.06-3.74 p < 0.001]], and CV [OR 1.41 (95% CI 1.28-1.60 p<0.001)] In multivariate analysis of TNC, preTNC [OR 20.71 (95% CI 17.87-24.00) p < 0.001]] was the best predictor of followed by normal delivery [OR 1.77 (95% CI 1.48-2.11 P<0.001)], FBW [OR 1.35 (95% CI 1.17-1.56 p<0.001)], CV [OR 1.31 (95% CI 1.13-1.53 p<0.001)], and female gender [OR 1.21 (95% CI 1.05-1.39 p=0.01)]. In multivariate analysis of CD34, was preTNC [OR 3.39 (95% CI 3.00-3.83 p < 0.001)] was the best predictor of followed by FBW [OR 1.41 (95% CI 1.25-1.58 P<0.001)], GA [OR 0.59 (95% CI 0.52-0.66 p<0.001)], MA [OR 0.84 (95% CI 0.75-0.94 p=0.003)], and female gender [OR 0.89 (95% CI 0.78-0.98 p=0.02)]. Conclusions: We established referential values of cord blood using large scaled CB units in Korea. In multivariate analysis, maternal/donor characteristics were associated with preTNC, FBW, and gender for both high TNC and CD34+. Our results confirm that is similar values to those reported in previous data. These associations could be used to prioritize donations, collections, optimizing resource utilization and financial modeling in Korean cord blood banks. We are focusing on collection education using the standard operation procedure to facilitate of high cells as well as on more recruits of healthy mothers. Disclosures No relevant conflicts of interest to declare.

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Evidence That T Cells Specific for Non-Hematopoietic Cells Trigger the Development of Acquired Aplastic Anemia

Blood ◽

10.1182/blood.v126.23.1168.1168 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1168-1168

Author(s):

Tatsuya Imi ◽

Hiroyuki Maruyama ◽

Takamasa Katagiri ◽

Yoshitaka Zaimoku ◽

Kana Maruyama ◽

...

Keyword(s):

T Cells ◽

Aplastic Anemia ◽

Progenitor Cells ◽

Response Rate ◽

Conflicts Of Interest ◽

Uniparental Disomy ◽

Target Cells ◽

Principal Mechanism ◽

Hematopoietic Stem ◽

Number Of Patients

Abstract A T-cell attack against hematopoietic stem cells (HSCs) is believed to be the principal mechanism underlying the development of acquired aplastic anemia (AA). The presence of leukocytes that lack HLA class I alleles as a result of the copy number-neutral loss of heterozygosity of the HLA haplotype due to uniparental disomy in the short arm of chromosome 6 (6pLOH) is compelling evidence for the involvement of cytotoxic T lymphocytes (CTLs) in the HSC destruction; this is based on the high response rate to immunosuppressive therapy (IST) in 6pLOH(+) patients (Katagiri, et al. Blood, 2011). However, target cells of the putative CTLs have not yet been characterized because of the small number of patients analyzed via flow cytometry (FCM) in our previous study. FCM can be substituted for SNP arrays by detecting HLA-A allele-lacking leukocytes (HLA-LLs) caused by 6pLOH. To gain insight into the CTL target responsible for the development of AA, we examined a total of 223 patients (213 idiopathic and 10 hepatitis-associated AA; 93 severe and 130 non-severe AA) for the presence of HLA-LLs and determined the lineage combinations of the aberrant leukocyte population. Of the 223 patients, 145 (65.0%) were heterozygous for the HLA-A allele and could be assessed for the presence of HLA-LLs by FCM. Eighteen (25.4%; 10 with severe AA and 8 with non-severe AA) of the 71 pre-treatment patients, and 26 (35.1%; 13 with severe AA and 13 with non-severe AA) of the 74 post-treatment patients were found to be positive for HLA-LLs. The lineage combinations of HLA-LLs in the 44 HLA-LL(+) patients were granulocytes (Gs), monocytes (Ms), B cells (Bs), and T cells (Ts, GMBT) in 13, GMB in 16 and GM in 11 patients. Surprisingly, HLA-LLs were found in Bs alone in three patients, and in one patient, the lineage combination pattern was TB (Figure). The presence of 6pLOH was confirmed via deep sequencing of isolated Bs from one of the three Bs alone patients. These lineage combination patterns were not observed to change for 1-40 months in 22 of 23 patients whose blood samples were available for follow-up analyses. In one patient with the GMB pattern, HLA-LLs decreased from 11.7% before treatment to 0% after 12 months of ATG therapy. The response rate to IST in GMBT patients (3/4, 75%) and in patients with GMB or GM (9/10, 90%) were similarly higher than in patients without HLA-LLs (22/39, 56%) or in patients who were homozygous for the HLA-A allele (23/36, 64%). Two of the three Bs alone patients showed complete responses at the time of sampling three years after ATG therapy and 20 years after CsA therapy, and another patient had a secondary myelodysplastic syndrome two years after response to ATG. The TB alone patient developed AA 20 years earlier, but had not been treated until recently because there was no need for blood transfusions, and is now improving in response to eltrombapag. This study revealed that the targets of putative CTLs in more than half of AA patients are hematopoietic progenitor cells with limited differentiation but long-lasting capacity, and in some patients, they are lymphoid progenitor cells that do not contribute to hematopoiesis. This suggests that CTL attack against non-HSCs including lymphoid precursors could trigger BM failure. Consistent with our previous report, the bystander effects caused by the immune response to non-HSCs such as myelosuppressive cytokines may play a major role in the development of AA. Disclosures No relevant conflicts of interest to declare.

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Identification of GBA Gene Mutations in 19 Pakistani Unrelated Patients of Gaucher Disease

Blood ◽

10.1182/blood-2018-99-120385 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4952-4952 ◽

Cited By ~ 1

Author(s):

Arshi Naz ◽

Qurat Abedin ◽

Shariq Ahmed ◽

Saima Siddiqui ◽

Tahir Shamsi

Keyword(s):

Bone Marrow ◽

Genetic Analysis ◽

Gene Mutation ◽

Gaucher Disease ◽

Conflicts Of Interest ◽

Demographic Data ◽

White Blood Cells ◽

Bone Marrow Morphology ◽

Number Of Patients ◽

Gba Gene

Abstract Introduction: Gaucher disease (GD) is one of the lysosomal storage diseases that is rare and inherited autosomal recessively. There is insufficiency of glucocerebrosidase enzyme that leads to the build up of un-degraded substrates in white blood cells causing anemia, hepatosplenomegaly and skeletal disease. This enzyme deficiency is linked with the defect of its gene (GBA) that codes for this enzyme. Initial diagnosis is made by the estimation of glucocerebrosidase level in blood and confirmed by genetic analysis of GBA gene. To identify the mutations of GBA gene in Pakistani patients with GD from different regions of Pakistan. Sampling & methodology: The sample and demographic data was collected in National Institute of Blood Disease and Bone marrow Transplantation after approval of IRB and written informed consent of patients. We collected total 19 blood samples, out of which 5 had Gaucher's disease, 10 samples were parents of the index cases and 4 were control. The methodology consisted of DNA extraction and quantification from peripheral blood. Genetic analysis of coding regions of GBA gene was done via gene amplification, gel electrophoresis and sequencing. Result: Mutation was found in two out of five families that makes the prevalence of GBA gene mutation 40%. These were diagnosed on reduced enzyme levels and found to have L444P (c.1448T>C) mutation in homozygous form in 10th exon of GBA gene. The parents of that patient carried the same mutation in one allele. Rest of the patients who were diagnosed on bone marrow morphology showed no mutation in GBA gene. Conclusion: Our results illustrate that GBA gene mutation was found in those patients who were diagnosed by the estimation of β-glucosidase enzyme levels rather than on bone marrow morphology. In our population, the mutation L444P was found, which is the most frequent gene mutation found in the world. Since this study is conducted in a small number of patients therefore it is recommended that large cohorts of patients should be evaluated in future for genetic mutations among Gaucher's patients in Pakistan Key words: Gaucher disease, storage disorder, GBA gene Disclosures No relevant conflicts of interest to declare.

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Genetic Mutation in Cytopenic Patients: Distinctive Genomic Profile between Preclinical Vs. Clinical Myelodysplastic Syndrome

Blood ◽

10.1182/blood-2019-128037 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5429-5429

Author(s):

Kritanan Songserm ◽

Amornchai Suksusut ◽

Sunisa Kongkiatkamon ◽

Kitsada Wudhikarn ◽

Chinnachote Teerapakpinyo ◽

...

Keyword(s):

Bone Marrow ◽

High Risk ◽

Myelodysplastic Syndrome ◽

Somatic Mutations ◽

Conflicts Of Interest ◽

Gene Mutations ◽

Genetic Alterations ◽

Genetic Mutation ◽

Low Risk ◽

Genomic Profile

Genetic mutation in cytopenic patients: Distinctive genomic profile between preclinical vs. clinical myelodysplastic syndrome. Introduction Myelodysplastic syndromes (MDS) are heterogeneous groups of clonal hematopoietic disorders. The current diagnosis of MDS is based on morphologic assessments of dysplasia which are subjected to inter-observer variability and cytogenetic abnormalities which are frequently absent. Somatic mutations in myeloid-related genes have been identified in MDS. However, they are also found in idiopathic cytopenia of unknown significance (ICUS) that shows no significant dysplasia. Therefore, we aimed to explore the clinical implications of genetic mutations in ICUS and compared with MDS. The secondary objective was to find association between degree of dysplasia and somatic mutations. Materials and Methods The patients with peripheral cytopenia ≥1 lineage (ANC < 1,800/mm3, hemoglobin < 10 gm/dL, platelet < 100x109/mL) without explainable causes were enrolled. Bone marrow aspirates were evaluated independently by 2 hematologists. Of note, dysplasia are defined by WHO 2008 classification (eg. Erythroid lineage: ring sideroblasts, megaloblastoid change; granulocytic lineage: hypogranularity, pseudopelger-huet anomaly; megakaryocytic lineage: hypolobate, micro-megakaryocyte). The significant dysplasia cut off was 10% in single lineage or more. If there was a discrepancy, the third hematologist would help to reach the final consensus. We extracted DNA from bone marrow and performed next generation sequencing (NGS) that targeted 143 myeloid-related genes. Results Forty-eight patients were enrolled in this study. The median age at diagnosis was 70 years (71-96). Results of bone marrow examinations were categorized by morphology into 3 groups; non-significant dysplasia (dysplasia < 10%) 27%, low risk MDS (IPSS-R ≤3.5) 42% and high-risk MDS/sAML (IPSS-R >3.5/Blast≥20% in BM or peripheral blood) 31%. Most of cases (77%) carried normal cytogenetics while other genetic alterations were complex chromosome (6%), -Y (6%), del(5q) (4%), trisomy 8 (2%), del(20q) (2%), i(17q) (2%). Thirty from 48 cases (62%) harbored more than 1 somatic mutation. Twenty-eight gene mutations were identified. Mutations were detected 1.6 mutation per 1 patient in average. Most frequent somatic mutations were ASXL1:10/80 (12%), TET2:9/80 (11%), MFDS11: 6/80 (7%), TP53:6/80 (7%), and RUNX1:5/80 (6.25%). The proportions of cases with somatic mutations were not different across the groups (no dysplasia 50%, non-significant dysplasia 80% and significant dysplasia 62%). According to mutation types in each group, mutations in epigenetic pathways were the most frequent mutations across all patient subgroups (ICUS 64.7%, low-risk MDS 51.8 %, and high-risk MDS 52.5%). Mutations in transcription factor were predominated in MDS (18.5% and 25.0% in low-risk and high-risk MDS, respectively) compared to ICUS (11.7%). Individual average frequency of gene mutations was significantly different between disease subtype (high risk MDS 2.7 gene/person, low risk MDS 1.1 gene/person, ICUS 1.3 gene/person (P=.038). Higher variant allele frequency (VAF) of mutated genes was significantly observed in high risk MDS (38.3%) compared to low risk MDS (30.8%) and preclinical MDS (29.0%) (P=.03). Conclusion In conclusion, molecular profiling was significantly different between preclinical MDS and MDS groups in terms of types of somatic mutations and VAF. This unique contrast could be used to distinguish between preclinical MDS and clinically significant MDS. In contrast, degree of marrow dysplasia was not associated with number of gene mutations in this study. Prediction for clinical consequent of somatic mutations in CCUS requires long term follow up. Disclosures No relevant conflicts of interest to declare.

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Life History of Normal Human Lymphocytes Revealed By Somatic Mutations

Blood ◽

10.1182/blood-2019-128188 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1045-1045 ◽

Cited By ~ 1

Author(s):

Heather E Machado ◽

Nina Friesgaard Øbro ◽

Emily Mitchell ◽

Megan Davies ◽

Anthony R Green ◽

...

Keyword(s):

T Cells ◽

Life History ◽

Somatic Mutations ◽

Lymphocyte Subsets ◽

Conflicts Of Interest ◽

Human Lymphocytes ◽

Hematopoietic Stem ◽

Mutation Burden ◽

History Of ◽

Mutational Processes

Introduction: Mature blood cells harbor a mixture of mutations inherited from ancestral hematopoietic stem cells (HSCs) and mutations accumulated after maturation. The landscape of these somatic mutations in normal blood is poorly mapped, with questions as simple as "how many mutations does a memory T cell accumulate throughout life?" remaining unanswered. This gap in our knowledge is particularly relevant for hematopoietic malignancy- while we know that lymphomas derive from lymphocytes of particular stages of differentiation, we do not know if the patterns we see are reflected in their normal counterparts. Results: In order to characterize normal somatic mutation in lymphocytes, we performed single-cell expansion and whole genome sequencing of over 600 T and B lymphocytes and 200 HSC and progenitor cells across 5 individuals (ages 0-85). All lymphocyte subsets show increased mutation burden with respect to HSCs across all classes of variants (Figure 1). Some of this increase can explained by lymphocyte-specific mutational processes, such as the activity of RAG, accounting for at least 20% of observed structural variants. We also find a striking variation in mutation burden within and between lymphocyte subsets. Microenvironment specific mutational processes dominate the observed differences. Examples of this include germinal center ("non-canonical AID") mutations in memory B cells and UV-like mutations in memory T cells (putatively skin resident cells). Naive B and T cells show a lack of variation in discrete mutational patterns relative to their memory counterparts, and have mutational profiles and mutation burdens more similar to that of HSCs. We also observe differences in the mutational patterns between B and T cells that are indicative of the increased divergence of T lymphocytes from the HSC pool. In general, the mutation burden we observe in normal lymphocytes approach those seen in lymphoma. Conclusions: Our work highlights the substantial genetic diversity in normal lymphocytes, with some cells accumulating thousands of mutations on top of those inherited from the HSC compartment. These mutations can be used to describe the life history of each individual lymphocyte including their exposure to specific microenvironments. Our findings shed light on the biology of these cells and will help differentiate between normal and disease processes. Figure 1 Disclosures No relevant conflicts of interest to declare.

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Extreme Thrombocytosis Under Azacitidine in Patients with Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v120.21.4961.4961 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4961-4961

Author(s):

Charikleia Kelaidi ◽

Dimitrios Kokkinidis ◽

Maria Protopappa ◽

Georgios Papaioannou ◽

Ioannis Batsis ◽

...

Keyword(s):

Platelet Count ◽

Myelodysplastic Syndrome ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasm ◽

Response To Treatment ◽

Jak2 Mutation ◽

Hematopoietic Stem ◽

Platelet Counts ◽

Hemorrhagic Event ◽

Count Response

Abstract Abstract 4961 Background: Platelet increase under azacitidine in patients with myelodysplastic syndrome (MDS) has been acknowledged as an early predictive factor of response to treatment. However, extreme thrombocytosis under azacitidine has not been reported. Methods: We studied consecutive patients with MDS or MDS/myeloproliferative neoplasm (MDS/MPN) who had platelet counts near or over 1, 000 G/L under azacitidine. Results: Four patients, sex ratio 1:1, with median age of 65 years, had extreme thrombocytosis under azacitidine. Baseline characteristics were: WHO classification RAEB-2/CMML-1/CMML-2 in 2/1/1 patients, median platelet count 248 G/L (<400 G/L in all), normal karyotype/+8, −9/−7 in 2/1/1 patients, IPSS low/int-2/high in 1/2/1 patients. None had reticulinic fibrosis or ring sideroblasts>15% at baseline. A median number of 8 cycles of azacitidine was administered. Individual platelet counts reached 2, 960 G/L, 800 G/L, 1, 188 G/L and 2, 740 G/L. Thrombocytosis occured early after treatment onset or resumption (Figure 1). Histologic findings under treatment were: Increased cellularity (N=4), micromegakaryocytes and other signs of megakaryocytic dysplasia (N=4), reticulinic fibrosis grade I and II in 1 and 2 patients, respectively. JAK2 V617F mutation was detected in 1 patient (with maximum platelet count of 2, 900 G/L) and was undetectable in the remaining patients. None had a thrombotic or hemorrhagic event. Two patients had a concomitant increase of WBC count. Response to azacitidine was CR, PR and stable disease in 1/1/2 patients. Three patients received hydroxyurea (HU) in addition to azacitidine and one patient underwent hematopoietic stem cell transplantation (HCT). AML transformation occurred in 1 patient 25 months after azacitidine onset. Median overall survival after azacitidine onset was 25 months. Conclusion: Extreme thrombocytosis of the range of essential thrombocytosis, with megakaryocytic dysplasia and hyperplasia, was noted under azacitidine in 4 patients with MDS-MDS/MPN and normal baseline platelet count. Hypothetically, azacitidine may induce the expression of critical genes of megakaryopoiesis or platelet release in patients with rare mutations. Notably, JAK2 mutation was detected in only one patient. Alternatively, demethylation could unmask an underlying unclassified MDS/MPN similar to RARS-T. Disclosures: No relevant conflicts of interest to declare.

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