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.

Interclonal and Intraclonal Heterogeneity in Patients with Early Myelodysplastic Syndrome (MDS)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1670-1670
Author(s):  
Maya Thangavelu ◽  
Wanlong Ma ◽  
Steven Brodie ◽  
Christopher Mixon ◽  
Wayne Chen ◽  
...  
Keyword(s):  
Allele Frequency ◽  
Mutant Allele ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Refractory Anemia ◽  
Early Event ◽  
Trisomy 8 ◽  
Cytogenetic Abnormalities ◽  
Allele Burden ◽  
Mutant Allele Frequency

Abstract Introduction: Recent data suggest that MDS evolves by accumulating mutations. Early mutations may involve genes that require additional mutations prior to clinical manifestation as MDS. We explored if mutant allele burden and the relative mutation of one gene to another gene could provide information on the interclonal and intraclonal progression of MDS using next generation sequencing (NGS) in patients with early MDS. Methods: NGS data was generated from 96 patients diagnosed with MDS with marrow blast count <5% using a targeted sequencing covering 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 sequencing was 10,000X. Differences in mutant allele frequency between two genes in the same sample were considered significant if they were >10%. A difference of 10% to 20% was considered mild, 20%-30% moderate, and >30% severe. A heat map reflecting these differences in mutant allele frequency was generated. Results: In this group of early MDS patients, 63 patients (66%) had more than one gene mutated and 38 (40%) had a significant (>10%) difference in allele frequency. The median number of genes mutated was 2 (range 1 to 5). Difference in mutant allele frequency was severe in 15 patients (16%), intermediate in 15 patients (16%), and mild in 13 patients (14%). TET2 was the most commonly mutated gene (43 patients, 45%) and was rarely the sole mutation with most cases exhibiting a mutation in a second gene (39 patients, 91%). The mutant allele burden was highest in TET2 in 26 of these 39 patients (67%), reflecting early event in the tumorigenic process. Of the 13 cases with TET2 mutation and allele burden less than the companion gene, 6 had a mutation in SF3B1, 3 had significant cytogenetic abnormalities (monosomy 5, del(7q), and trisomy 8), 2 had a mutation in SRSF2, 1 had a mutation in ZRSR2 and 1 had a mutation in ASXL1, which suggests that these abnormalities might be the initiating event. A second TET mutation (biallelic mutation) was detected in 16 of the 39 patients. SF3B1 was the most common gene having a solitary mutation (10% of all patients), although mutation in SF3B1 was detected in 27 patients (26% of all patients). All solitary SF3B1 mutations were associated with normal karyotypes, except for one patient with del(11q). JAK2 was mutated with SF3B1 in two cases diagnosed as RARS-T (refractory anemia with ring sideroblasts and thrombocytosis). In one case, the JAK2 and SF3B1 mutation allele frequencies were similar, but in the other, the JAK2 mutant allele frequency was 23% higher, suggesting that a myeloproliferative neoplasm was the initiating process. ASXL1 was mutated in 14 cases, 13 of which had additional mutations. DNMT3A gene was mutated in 18 cases, 5 of which were solitary; two of these five showed cytogenetic abnormalities. TP53 was mutated in 13 cases, but except for one case, all had either mutation in another gene or a cytogenetic abnormality. Conclusion: These data suggest that in patients with clinically confirmed early MDS, TET2 mutations are most likely the initiating oncogenic event, but mutations in other genes or cytogenetic abnormalities most likely lead to clinically confirmed MDS. In contrast, patients with SF3B1 mutation can have clinical disease without additional mutations. Our data suggest that SRSF2, ZRSR2, and ASXL1 may initiate mutagenesis in patients with MDS. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Molecular Profiling in Confirming the Diagnosis of Early Myelodysplastic Syndrome

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4639-4639
Author(s):  
Maya Thangavelu ◽  
Ryan Olson ◽  
Li Li ◽  
Wanlong Ma ◽  
Steve Brodie ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Conflicts Of Interest ◽  
Early Stage ◽  
Molecular Profiling ◽  
Control Group ◽  
Molecular Testing ◽  
Cytogenetic Abnormalities ◽  
Molecular Abnormalities ◽  
Blast Count

Abstract Introduction: Diagnosis of myelodysplastic syndrome (MDS) based on bone marrow morphology can be very difficult, when blasts are not increased. The demonstration of cytogenetic abnormalities in these cases can confirm the diagnosis, providing cytopenia is documented. Cytopenia is usually the major reason for initiating work-up for myelodysplasia and , in general, cases with unicytopenia are the most difficult to make the diagnosis. In principle the recent characterization of the molecular abnormalities underlying the biology of MDS should provide objective biomarkers that can be used to confirm the diagnosis of MDS in the absence of cytogenetic abnormalities. Toward this goal, we developed a 14-gene panel to detect molecular abnormalities in patients referred to rule out MDS with blast count <5% without cytogenetic abnormalities, but with documented cytopenia. Method: Cytopenia is defined as having platelets <100,000 /µl, neutrophils <1,800/µl, or hemoglobin <10g/dL. Total nucleic acid was extracted from bone marrow or peripheral blood samples and tested for mutations in any of the following genes: ASXL1, ETV6, EZH2, IDH1, IDH2, NRAS, CBL, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1 and ZRSR2. Direct bidirectional Sanger sequencing, as well as next generation sequencing were used for testing. Samples from 137 patients fulfilling the criteria described above were analyzed. As cytogenetic abnormalities is a marker of MDS, a control group of 14 patients with cytogenetic abnormalities but no increase in blasts were evaluated using the same molecular panel. Results: Fifty three of the 137 patients (39%) had a mutation in one or more genes. Of the 137 patients, three had tricytopenia, 14 had bicytopenia and 120 had unicytopenia. Two of the three with tricytopenia (66%) had mutations and nine of 14 with bicytopenia (64%) had mutations. In contrast 42 of the 120 patients with unicytopenia (35%) had mutation in one or more genes. Thirty of the 53 patients with mutation (57%) had one gene mutated and only 4 (13%) of these patients had bi- or tricytopenia. Of the remaining 23 patients with mutations in two or more genes a higher percentage (30%) of patients had bi- or tricytopenia. Compared to patients without mutations in the tested genes, those with mutation had significantly lower number of neutrophils (P=0.006), but higher percentage of monocytes (P=0.0002) and slightly higher percentage of lymphocytes (P=0.06). Twelve of 14 (86%) patients with cytogenetic abnormalities showed mutation in one or more genes and only three patients of the 14 (21%) had bi- or tricytopenia. Conclusion: Diagnosis of MDS at early stage of disease (blasts <5%) can be significantly enhanced by adding molecular profiling to cytogenetics studies. Molecular profiling using limited number of genes (14) in patients with cytopenia and suspected of having MDS, but no cytogenetic abnormalities, can confirm the diagnosis of MDS in 39% of cases. Compared to MDS with unicytopenia, MDS with bi- or tricytopenia without increase in blasts, is more likely to be confirmed by molecular testing. Disclosures No relevant conflicts of interest to declare.

scholarly journals Differential and Limited Expression of Mutant Alleles in Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2007-2007
Author(s):  
Adam Sperling ◽  
Naim Rashid ◽  
Niccolo Bolli ◽  
David Wedge ◽  
Peter Van Loo ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Exome Sequencing ◽  
Allele Frequency ◽  
Mutant Allele ◽  
Allelic Expression ◽  
Significant Heterogeneity ◽  
Rna Seq ◽  
Mutant Allele Frequency ◽  
Large Numbers ◽  
Significant Difference

Abstract Multiple Myeloma (MM) is a heterogeneous disease but the hallmark genetic changes involve large numbers of genomic rearrangements. Recent studies have focused on attempts to identify individual driver mutations that might provide both prognostic information and unique therapeutic targets. Whole genome and exome sequencing of increasingly large numbers of patient samples have identified a number of commonly mutated genes in MM patients. However, none of these mutations are found in more than one quarter of patients and most are found in less than 10% of samples sequenced. We recently reported a large cohort of MM exome sequences involving 84 samples from 67 patients (Nat Commun. 2014;5:2997). We defined a diverse set of gene mutations with significant heterogeneity across our cohort with a median of 52 (range 21-488) mutations identified per sample. Although computational approaches can be used to prioritize mutations that are expected to alter protein structure and function, it is more challenging to determine which mutations are likely to be clinically meaningful. As a first step towards that understanding, here we report the frequency of expression of mutant alleles in Multiple Myeloma. In this study we report RNA-seq (100 million paired end reads on Illumina HiSeq) data on 14 samples from 10 MM patients for which we have previously performed exome sequencing and correlate allele-specific expression to the DNA mutant allele frequency. We find that a minority, average 27% (range 11-48%), of previously identified DNA mutations are expressed at detectable levels in MM patients. We also compared the allele frequency found in the RNA-seq to that from our exome sequencing to identify genes that demonstrate differential allelic expression and show that this is a common phenomenon in MM patients. We identified 42 such mutations in our analysis supported by at least 10 RNA-seq reads that showed a significant difference as determined by Bayesian hypothesis testing. For instance, the CCND1 mutant allele is expressed at a higher level than would be predicted based on exome-seq frequencies. Another gene showing a similar pattern of increased expression of the mutant allele in one patient was PARP4 (87% in RNA-seq vs 49% in exome-seq). Conversely, the mutant allele frequency of EIF1AX was lower than would be expected suggesting that the mutant allele may be suppressed in our patient (15% in RNA-seq vs 67% in exome-seq). Moreover, among a subset of genes previously identified as recurrently mutated within our patient samples we see that 8/11 (73%) express the mutant allele, providing further evidence that these genes may in fact be important in disease pathogenesis. Therefore, while a large number of mutations have been described in MM, only a small fraction of the mutant alleles have detectable expression and are likely to be biologically relevant. Unbalanced allelic expression of mutant alleles appears to be a relatively common occurrence in MM patients and may help explain why patients with the same identified mutation do not always behave in a similar fashion. This analysis for the first time highlights the important issue that DNA-based reporting of mutations may have significant limitations. It will be important in the future to study expression of mutant alleles in order to understand the biology, generate prognostic models and develop targeted therapies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Classification of Acute Myeloid Leukemia and Myelodysplastic Syndrome Based on Molecular Profiling

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3836-3836
Author(s):  
Sally Agersborg ◽  
Maya Thangavelu ◽  
Wanlong Ma ◽  
Steven Brodie ◽  
Christopher Mixon ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myelodysplastic Syndrome ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
P Value ◽  
Molecular Abnormalities ◽  
Significant Difference ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is currently distinguished from myelodysplastic syndrome (MDS) based on the presence of 20% blasts in bone marrow, an arbitrary cut-off adopted by the WHO classification and replacing the 30% cut-off required by the older FAB (French, American and British) classification. Patients with t(15;17), t(8;21), or inversion 16 cytogenetic abnormalities are classified as having AML irrespective of the percentage of blasts. We explored the possibility that currently defined molecular abnormalities can distinguish AML from MDS without relying on an arbitrary percentage of blasts in the bone marrow. We compared the molecular profiles obtained by next generation sequencing (NGS) from consecutive patients with a clinical diagnosis of AML or MDS by WHO criteria. Methods: NGS data from 251 patients with the diagnosis of AML and 294 patients with the diagnosis of MDS was studied. All samples were analyzed using a panel of 25 genes including FLT3, NPM1 SF3B1, CBL, DNMT3A, ASXL1, BRAF, CEBPA, CSFR3, ETV6, EZH2, IDH1, IDH2, JAK2, c-KIT, KRAS, NRAS, PHF6, PTPN11, RUNX1, SETBP1, TET2, TP53, WT1, and ZRSR2. We compared the frequency of mutations in each gene between AML and MDS patients. Results: Mutations in FLT3 and NPM1 were uniquely and commonly detected in AML (27% and 22%, respectively). In contrast, mutations in SF3B1 gene were uniquely dominant (22%) in MDS and FLT3 and NPM1 mutations were rare (2% and 3%, respectively). SF3B1 mutations were extremely rare in AML (1%). Overall, 102 (41%) of all AML patients had mutations in either FLT3 or NPM1 and 8% of AML patients had mutations in both FLT3 and NPM1. In addition, WT1 gene was mutated in 8% of AML cases, but none of the MDS cases showed WT1 mutation. TET2 gene was commonly mutated in both AML and MDS (25% and 36%, respectively), but the frequency was significantly higher in MDS (P=0.003). IDH1, IDH2, NRAS, and PTPN11 were mutated slightly more often in AML than in MDS, while ASXL1, EZH2, and ZRSR2 were more frequently mutated in MDS than in AML. There was no statistically significant difference in mutation frequency between AML and MDS for the other genes analyzed. Conclusion: Mutations in FLT3, NPM1 and WT1 are molecular abnormalities characteristically detected in patients with AML and can be used as objective criteria for the classification of AML rather than blast count in bone marrow. These mutations are detected in 49% of AML patients. This suggests that approximately half of AML patients can be diagnosed based on the detection of molecular abnormalities, irrespective of bone marrow morphology. The presence of mutation in SF3B1 gene is also a characteristic molecular finding for MDS. Table. AML (No=251) MDS (No=294) P-Value No % No % FLT3 68 27 5 2 0.00001 NPM1 55 22 8 3 0.0001 SF3B1 3 1 66 22 0.00006 CBL 4 2 10 3 NS DNMT3A 51 20 51 17 0.07 ASXL1 44 18 75 26 0.01 BRAF 3 1 1 0 NS CEBPA 38 15 51 17 NS CSFR3 11 4 11 4 NS ETV6 3 1 6 2 NS EZH2 8 3 25 9 0.03 IDH1 20 8 7 2 0.03 IDH2 17 7 7 2 0.04 JAK2 4 2 10 3 NS KIT 2 1 0 0 NS KRAS 11 4 6 2 NS NRAS 34 14 18 6 0.01 PHF6 5 2 2 1 NS PTPN11 26 10 6 2 0.01 RUNX1 31 12 33 11 NS SETBP1 5 2 9 3 NS TET2 64 25 105 36 0.003 TP53 61 24 75 26 NS WT1 19 8 0 0 0.01 ZRSR2 7 3 30 10 0.02 Disclosures No relevant conflicts of interest to declare.

Clonal Leukemic Evolution In Myelodysplastic Syndromes With ASXL1 and EZH2 mutations: A Comparative Analysis Of 58 Paired Samples

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2786-2786
Author(s):  
Hsuan-Jen Shih ◽  
Lee-Yung Shih ◽  
Hung Chang ◽  
Ming-Chung Kuo ◽  
Tung-Huei Lin ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Allele Frequency ◽  
Mutational Analysis ◽  
Conflicts Of Interest ◽  
Gene Mutations ◽  
Additional Gene ◽  
Paired Samples ◽  
Cell Counts ◽  
Significant Difference

Abstract Background and purpose ASXL1 and EZH2 are histone modifiers. Mutations of ASXL1 and EZH2 genes have been described in both myelodyspolastic syndromes (MDS) and acute myeloid leukemia (AML). The role of ASXL1 or EZH2 mutations in the progression from MDS to secondary AML (sAML) is unclear. We aimed to determine the clinical relevance of ASXL1 and EZH2 mutations in patients with MDS and to investigate the role of ASXL1 or EZH2 mutation with its cooperating mutated genes in sAML progression. Methods ASXL1 and EZH2 mutations were analyzed on bone marrow samples from 126 patients with de novo MDS (45 RAEB1, 53 RAEB2, 24 RCMD and 4 RARS). Paired matched MDS/sAML samples were available for ASXL1 mutational analysis in 58 patients and for EZH2 in 54 patients. Mutational analysis of ASXL1 exon 12 was performed by PCR assays followed by direct sequencing, EZH2 mutations were first screened with denaturing high-performance liquid chromatography on amplified PCR fragments covering the whole coding sequencing from exons 2 to 20 of EZH2 gene followed by sequencing of the abnormal profile. Additional 20 known gene mutations in myeloid neoplasms were also examined in patients carrying ASXL1 or EZH2 mutations. Relative allele frequency was determined by pyrosequencing. Results Among the 126 patients, ASXL1 mutations were detected in 18 (14.3%) patients and EZH2 in 11 patients (8.7%), 3 of them had both mutations. Taken together, 20.6% of patients carried mutations of ASXL1 and/or EZH2. ASXL1-mutated patients had male predominance (17 out of 18 patients, P=0.012) and fewer circulating blasts (P=0.007). ASXL1-mutated and -unmutated patients had no difference in hemoglobin levels, white blood cell counts, platelet counts, cytogenetics, WHO subtypes, bone marrow blasts, IPSS-R or risk to sAML. ASXL1 mutations had no impact on overall survival (P=0.765) or time to sAML transformation (P=0.605). No significant difference was observed between EZH2 mutation status and clinicohematologic features or outcomes. Of the 58 paired samples, ASXL1 mutations were detected in 8 cases at diagnosis of MDS, all were also present at the time of sAML progression with no difference in the mutant allele burden (P=0.614), 2 patients acquired ASXL1 mutations. Five had EZH2 mutations at both phases of disease which exhibited a similar allele frequency (P= 0.434), none lost and one acquired EZH2 mutation at sAML phase. Progression to sAML was accompanied by additional gene mutations including RUNX1 (n=4), TET2 (n=3), CEBPα (n=2), PTPN11 (n=2), and one each for FLT3-ITD, CBL, MLL-PTD, DNMT3A, IDH2 and EZH2 in ASXL1-mutated patients; TET2 (n=4), RUNX1 (n=3), N-RAS (n=2) and single cases for DNMT3A, IDH1, and ASXL1 in EZH2-mutated patients. Cooperating mutations were either detected in both MDS and sAML samples or newly appeared in sAML samples except one who lost JAK2V617F mutation while acquired EZH2 and N-RAS mutations during sAML progression. Conclusions Our study on a large cohort of paired MDS and sAML samples demonstrated that ASXL1 or EZH2 mutations remained stable at both phases of disease in most patients. Clonal evolution can occur and cooperation of additional gene mutations is frequently detected in patients harboring ASXL1 or EZH2 mutations in the progression of MDS to sAML. Grant support This work was supported by NHRI-EX102-10003NI and DOH102-TD-C-111-006, Taiwan. Disclosures: No relevant conflicts of interest to declare.

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.

scholarly journals Association of CYP2C19*2 and *17 genetic variants with hypertension in Pakistani population

2021 ◽  
Vol 18 (4) ◽  
pp. 851-855
Author(s):  
Sana Riaz ◽  
Atika Mansoor ◽  
Saima Siddiqi ◽  
Muhammad Usman Tareen ◽  
Sana Rubab ◽  
...  
Keyword(s):  
Allele Frequency ◽  
Mutant Allele ◽  
Allelic Polymorphism ◽  
Optimal Dosage ◽  
Wild Type ◽  
Pakistani Population ◽  
Hypertensive Patients ◽  
Mutant Allele Frequency ◽  
Significant Difference ◽  
Cyp2c19 Gene

Purpose: To investigate the association of *2 and *17 single nucleotide polymorphisms (SNPs) of CYP2C19 gene with hypertension in Pakistani population. Methods: The study was conducted on 527 hypertensive patients and 530 unrelated healthy controls from selected regions of Pakistan. DNA was extracted from leukocytes and all patients and controls were genotyped for two SNPs (rs4244285 and rs12248560) of CYP2C19 gene by allele specific polymerase chain reaction (AS-PCR). Results: Multi-allelic polymorphism in CYP2C19 identified four distinct phenotypes known as ultra-rapid metabolizer (UM), extensive metabolizer (EM), intermediate metabolizer (IM) and poor metabolizer (PM) in hypertensive patients and controls. For CYP2C19*2 polymorphisms, overall wild type and mutant allele frequency were 75 and 25 % in hypertensive patients, and 64.2 and 35.8 % in controls. For CYP2C19*17 polymorphisms, the overall wild type and mutant allele frequency were 66.6 and 33.4 % in hypertensive patients and 75.6 % and 24.4 % in controls. Significant difference in allele frequencies for CYP2C19*2 and *17 was demonstrated between hypertensive and non-hypertensive subjects. Conclusion: To the best of our knowledge, this is the first report on CYP2C19 frequencies in hypertensive Pakistani patients. The finds should help clinicians to determine a suitable optimal dosage of some drugs in order to reduce side effects.

scholarly journals Mutational Characteristics and Changing Pattern from Idiopathic Cytopenia of Undetermined Significance to High-Risk Myelodysplastic Syndrome Stratified By IPSS-R

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3009-3009
Author(s):  
Eun-Ji Choi ◽  
Young-Uk Cho ◽  
Seongsoo Jang ◽  
Chan-jeoung Park ◽  
Han-Seung Park ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Myelodysplastic Syndrome ◽  
Rna Splicing ◽  
Conflicts Of Interest ◽  
Low Risk ◽  
International Prognostic Scoring System ◽  
Intermediate Risk ◽  
Sequencing Data ◽  
Undetermined Significance

Background: Unexplained cytopenia comprises a spectrum of hematological diseases from idiopathic cytopenia of undetermined significance (ICUS) to myelodysplastic syndrome (MDS). Revised International Prognostic Scoring System (IPSS-R) is the standard tool to assess risk in MDS. Here, we investigated the occurrence, characteristics, and changing pattern of mutations in patients with ICUS and MDS stratified by IPSS-R score. Methods: A total of 211 patients were enrolled: 73 with ICUS and 138 with MDS. We analyzed the sequencing data of a targeted gene panel assay covering 141 genes using the MiSeqDx platform (Illumina). The lower limit of variant allele frequency (VAF) was set to 2.0% of mutant allele reads. Bone marrow components were assessed for the revised diagnosis according to the 2016 WHO classification. Lower-risk (LR) MDS was defined as those cases with very low- or low-risk MDS according to the IPSS-R. Higher-risk (HR) MDS was defined as those cases with high- or very high-risk MDS according to the IPSS-R. Results: Patients with ICUS were classified as very low-risk (39.7%), low-risk (54.8%), and intermediate-risk (5.5%) according to the IPSS-R. Patients with MDS were classified as LR (35.5%), intermediate-risk (30.4%), and HR (34.1%). In the ICUS, 28 (38.4%) patients carried at least one mutation in the recurrently mutated genes in MDS (MDS mutation). The most commonly mutated genes were DNMT3A (11.0%), followed by TET2 (9.6%), BCOR (4.1%), and U2AF1, SRSF2, IDH1 and ETV6 (2.7% for each). IPSS-R classification was not associated with mutational VAF and the number of mutations in ICUS. In the 49 LR MDS, 28 (57.1%) patients carried at least one MDS mutation. The most commonly mutated genes were SF3B1 (20.4%), followed by TET2 (12.2%), U2AF1 (10.2%), DNMT3A (10.2%), ASXL1 (10.2%), and BCOR (6.1%). Higher VAF and number of mutations were observed in LR MDS compared to ICUS patients. In the 42 intermediate-risk MDS, 27 (64.3%) patients carried at least one MDS mutation. The most commonly mutated genes were ASXL1 (23.8%), followed by TET2 (21.4%), RUNX1 (16.7%), U2AF1 (14.3%), DNMT3A (14.3%), SF3B1 (9.5%), and SRSF2, BCOR, STAG2 and CBL (7.1% for each). In the 47 HR MDS, 36 (76.6%) patients carried at least one MDS mutation. The most commonly mutated genes were TET2 (25.5%), followed by DNMT3A (14.9%), TP53 (14.9%), RUNX1 (12.8%), U2AF1 (10.6%), ASXL1 (10.6%), and SRSF2 and KRAS (6.4% for each). As the disease progressed, VAF and number of the MDS mutations gradually increased, and mutations involving RNA splicing, histone modification, transcription factor or p53 pathway had a trend for increasing frequency. Specifically, ASXL1, TP53, and RUNX1 mutations were the most striking features in patients with advanced stage of the disease. Cohesin mutations were not detected in ICUS, whereas these mutations were detected at a relatively high frequency in HR MDS. Our data were summarized in Table 1. Conclusions: We demonstrate that on disease progression, MDS mutations are increased in number as well as are expanded in size. Furthermore, a subset of mutations tends to be enriched for intermediate- to HR MDS. The results of this study can aid both diagnostic and prognostic stratification in patients with unexpected cytopenia. In particular, characterization of MDS mutations can be useful in refining bone marrow diagnosis in challenging situations such as distinguishing LR MDS from ICUS. Disclosures No relevant conflicts of interest to declare.

scholarly journals Identification of a Clinical Cutoff Value for Multiplex KRASG12/G13 Mutation Detection in Colorectal Adenocarcinoma Patients Using Digital Droplet PCR, and Comparison with Sanger Sequencing and PNA Clamping Assay

2020 ◽  
Vol 9 (7) ◽  
pp. 2283
Author(s):  
Kyung Ha Lee ◽  
Tae Hee Lee ◽  
Min Kyung Choi ◽  
In Sun Kwon ◽  
Go Eun Bae ◽  
...  
Keyword(s):  
Allele Frequency ◽  
Mutant Allele ◽  
Sanger Sequencing ◽  
Colorectal Adenocarcinoma ◽  
Diagnostic Sensitivity ◽  
Absolute Quantitation ◽  
Tissue Samples ◽  
Mutant Allele Frequency ◽  
Mutational Status

KRAS (Kirsten rat sarcoma 2 viral oncogene homolog) is a major predictive marker for anti-epidermal growth factor receptor treatment, and determination of KRAS mutational status is crucial for successful management of colorectal adenocarcinoma. More standardized and accurate methods for testing KRAS mutation, which is vital for therapeutic decision-making, are required. Digital droplet polymerase chain reaction (ddPCR) is an advanced digital PCR technology developed to provide absolute quantitation of target DNA. In this study, we validated the clinical performance of ddPCR in determination of KRAS mutational status, and compared ddPCR results with those obtained by Sanger sequencing and peptide nucleic acid-clamping. Of 81 colorectal adenocarcinoma tissue samples, three repeated sets of KRASG12/G13 mutation were measured by ddPCR, yielding high consistency (ICC = 0.956). Receiver operating characteristic (ROC) curves were constructed to determine KRASG12/G13 mutational status based on mutant allele frequency generated by ddPCR. Using the best threshold cutoff (mutant allele frequency of 7.9%), ddPCR had superior diagnostic sensitivity (100%) and specificity (100%) relative to the two other techniques. Thus, ddPCR is effective for detecting the KRASG12/G13 mutation in colorectal adenocarcinoma tissue samples. By allowing definition of the optimal cutoff, ddPCR represents a potentially useful diagnostic tool that could improve diagnostic sensitivity and specificity.

Cytogenetic Abnormalities in Primary Myelodysplastic Syndrome Are Highly Predictive of Outcome After Allogeneic Bone Marrow Transplantation

Blood ◽  
1998 ◽  
Vol 92 (6) ◽  
pp. 1910-1917 ◽  
Author(s):  
Thomas J. Nevill ◽  
Henry C. Fung ◽  
John D. Shepherd ◽  
Douglas E. Horsman ◽  
Stephen H. Nantel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Myelodysplastic Syndrome ◽  
Marrow Transplantation ◽  
Allogeneic Bone Marrow Transplantation ◽  
Allogeneic Bone Marrow ◽  
Myelogenous Leukemia ◽  
Allogeneic Bone ◽  
Cytogenetic Abnormalities ◽  
Poor Risk

Abstract Allogeneic bone marrow transplantation (BMT) is the only curative therapy available for patients with myelodysplastic syndrome (MDS). In an attempt to identify prognostic factors influencing outcome, we collected data retrospectively on 60 consecutive adult patients who had undergone BMT at our center for primary MDS or acute myelogenous leukemia evolving from preexisting primary MDS (sAML). Patients were divided into subgroups according to cytogenetic abnormalities based on a recently described International MDS Workshop categorization system. The 7-year actuarial event-free survival (EFS), relapse rate, and nonrelapse mortality (NRM) for all patients were 29% (95% confidence interval [CI], 16% to 43%), 42% (CI, 24% to 67%), and 50% (CI, 37% to 64%), respectively. The EFS for the good-, intermediate-, and poor-risk cytogenetic subgroups were 51% (CI, 30% to 69%), 40% (CI, 16% to 63%), and 6% (CI, 0% to 24%), respectively (P= .003). The corresponding actuarial relapse rates were 19% (CI, 6% to 49%), 12% (CI, 2% to 61%), and 82% (CI, 48% to 99%), respectively (P = .002) with no difference in NRM between the subgroups. Univariate analysis showed cytogenetic category, French-American-British (FAB) subtype, and graft-versus-host disease (GVHD) prophylaxis used to be predictive of relapse and EFS. In multivariate analysis, only the cytogenetic category was predictive of EFS, with the relative risk of treatment failure for the good-, intermediate-, and poor-risk cytogenetic subgroups being 1.0, 1.5, and 3.5, respectively (P = .004). For adults with primary MDS and sAML, even after BMT, poor-risk cytogenetics are predictive of an unfavorable outcome; novel treatment strategies will be required to improve results with allogeneic BMT in this patient population. © 1998 by The American Society of Hematology.

Sign in / Sign up

Export Citation Format

Share Document