scholarly journals Direct comparison of quantitative digital PCR and 2-hydroxyglutarate enantiomeric ratio for IDH mutant allele frequency assessment in myeloid malignancy

Leukemia ◽  
2015 ◽  
Vol 29 (12) ◽  
pp. 2421-2423 ◽  
Author(s):  
D H Wiseman ◽  
E A Struys ◽  
D P Wilks ◽  
C I Clark ◽  
M W Dennis ◽  
...  
Keyword(s):  
Allele Frequency ◽  
Mutant Allele ◽  
Digital Pcr ◽  
Myeloid Malignancy ◽  
Enantiomeric Ratio ◽  
Mutant Allele Frequency

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.

scholarly journals Real-Time PCR Genotyping Assay for GM2 Gangliosidosis Variant 0 in Toy Poodles and the Mutant Allele Frequency in Japan

2014 ◽  
Vol 76 (2) ◽  
pp. 295-299 ◽  
Author(s):  
Mohammad Mahbubur RAHMAN ◽  
Akira YABUKI ◽  
Moeko KOHYAMA ◽  
Sawane MITANI ◽  
Keijiro MIZUKAMI ◽  
...  
Keyword(s):  
Real Time ◽  
Allele Frequency ◽  
Real Time Pcr ◽  
Mutant Allele ◽  
Gm2 Gangliosidosis ◽  
Genotyping Assay ◽  
Mutant Allele Frequency ◽  
Pcr Genotyping

Determination of the mutant allele frequency in patients with neurofibromatosis type 2 and somatic mosaicism by means of deep sequencing

2015 ◽  
Vol 54 (8) ◽  
pp. 482-488 ◽  
Author(s):  
Melanie Spyra ◽  
Benjamin Otto ◽  
Gerhard Schön ◽  
Hildegard Kehrer-Sawatzki ◽  
Victor-Felix Mautner
Keyword(s):  
Allele Frequency ◽  
Deep Sequencing ◽  
Mutant Allele ◽  
Somatic Mosaicism ◽  
Neurofibromatosis Type ◽  
Neurofibromatosis Type 2 ◽  
Mutant Allele Frequency

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.

Alteration of mutant allele frequency in visceral metastases of melanoma.

2018 ◽  
Vol 36 (15_suppl) ◽  
pp. e21528-e21528 ◽  
Author(s):  
Viktória Doma ◽  
Orsolya Papp ◽  
Erzsébet Rásó ◽  
Tamás Barbai ◽  
Lilla Reiniger ◽  
...  
Keyword(s):  
Allele Frequency ◽  
Mutant Allele ◽  
Mutant Allele Frequency ◽  
Visceral Metastases

High-Throughput Sequencing For The Identification Of NOTCH1 mutations In Early Stage Chronic Lymphocytic Leukemia: Biological and Clinical Implications

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1622-1622
Author(s):  
Marta Lionetti ◽  
Sonia Fabris ◽  
Giovanna Cutrona ◽  
Luca Agnelli ◽  
Carmela Ciardullo ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Allele Frequency ◽  
Deep Sequencing ◽  
Mutant Allele ◽  
Sanger Sequencing ◽  
Early Stage ◽  
Lymphocytic Leukemia ◽  
Mutant Allele Frequency ◽  
Mutational Activation ◽  
Notch1 Mutations

Abstract NOTCH1 mutations have recently emerged as new genetic lesions significantly correlated with survival in chronic lymphocytic leukemia (CLL). NOTCH1 c.7541_7542delCT is by far the most frequently observed NOTCH1 mutation in the disease. To estimate the prevalence and clonal evolution of NOTCH1 c.7541_7542delCT mutation, and prospectively investigate its clinical significance in early stage CLL and clinical monoclonal B cell lymphocytosis (cMBL), we analyzed by next generation sequencing (NGS) 384 cases at diagnosis enrolled in the GISL O-CLL1 multicenter trial. The patient cohort included 100 cMBL and 284 Binet stage A CLL cases, 48 of whom were also longitudinally investigated at progression or during follow-up (32 and 16, respectively) in absence of treatment. Deep sequencing of the NOTCH1 mutation hotspot was performed by Roche 454 pyrosequencing on the Genome Sequencer Junior instrument. NOTCH1 mutation was validated by an extremely sensitive PCR-based approach and Sanger sequencing. The association between NOTCH1c.7541_7542delCT and clinical, molecular and biological variables, as well as its impact on progression free survival (PFS), were tested. Deep sequencing analysis of NOTCH1 mutation hotspot in our cohort (median depth of coverage 1510x, ranging from 605 to 2842) revealed a mutant allele frequency ranging from 0.02% to 75% of total reads in 145 cases. The occurrence of the mutation was subsequently assessed by an extremely sensitive ARMS (amplification refractory mutation system)-PCR, which allowed to confirm the presence of delCT in the 49 cases with frequency of mutated sequencing reads greater than 0.7%, specifically in 11% of cMBL (11/100) and 13.4% of CLL patients (38/284). Furthermore, mutated samples were subjected to DNA Sanger sequencing: in line with the expected sensitivity of the method, the mutation was identified only in samples with higher mutation loads according to NGS (mutant allele frequency ≥ 7%, n=25). Our data revealed that often NOTCH1 mutational activation affected a neoplastic sub-clone, especially in cMBL patients. NOTCH1 mutated patients utilized unmutated IGHV genes more frequently, and had higher expression of CD38 and ZAP-70 (P=3.2e-11, P=2.6e-08, P=3.4e-05, respectively). Trisomy 12 was more frequent in this patient group (P=5.4e-04), whereas 13q14 deletion was less represented than in the NOTCH1 wild-type patients (P=2.8e-03). NOTCH1 mutation was associated with the occurrence of stereotyped HCDR3 (P=5.6e-03); in addition, compared with other major BCR subsets, CLL subset #10 was significantly enriched in NOTCH1 mutations (P=0.032). The prevalence of the analyzed dinucleotide deletion was not significantly different between cMBL and CLL patients, even if only Rai 0 cases (28/197 cases, 14.2% mutation frequency) were considered. The percentages of variant sequencing reads in NOTCH1-mutated cases were slightly higher in CLL (median 19.6%) than in cMBL (median 4.2%), a finding confirmed by a regression analysis that highlighted the association of the CLL presentation with higher percentages of NOTCH1 delCT reads (P=0.033). NOTCH1-mutated cases, both at sub-clonal and clonal levels, displayed a significant reduction in median PFS (P=0.0018), although NOTCH1 mutation prognostic value, in multivariate analysis, was not independent if 11q and/or 17p deletion, IGHV mutational status, and cMBL or CLL status were considered. Finally, sequential analyses in a representative fraction of cases of our dataset indicated that (i) NOTCH1 mutation did not occur during the course of the disease and that (ii) the mutational load in positive cases was stable over time. These findings highlight the importance of using high sensitive methods for an accurate detection of NOTCH1 mutation in cMBL/early stage CLL. This is required for a better prognostic stratification and also to obtain useful information for potential therapeutic approaches, since sub-clonal mutations in untreated CLL can possibly anticipate the dominant genetic composition of the relapsing tumor. Disclosures: No relevant conflicts of interest to declare.

The Majority of RAS Pathway Mutations Detected in Relapsed B-Cell Precursor Acute Lymphoblastic Leukemia Are Present in Major or Minor Subclones at Diagnosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2397-2397 ◽  
Author(s):  
Jiangyan Yu ◽  
Esmé Waanders ◽  
Simon V. van Reijmersdal ◽  
Edwin Sonneveld ◽  
Peter M Hoogerbrugge ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Allele Frequency ◽  
Deep Sequencing ◽  
Mutant Allele ◽  
Sanger Sequencing ◽  
Lymphoblastic Leukemia ◽  
Read Depth ◽  
Cell Precursor ◽  
Ras Pathway ◽  
Mutant Allele Frequency

Abstract B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is one of the most common cancers in children. The relapse incidence varies between 15% and 25%, dependent on the treatment protocol. It has been demonstrated that the presence of mutations in the RAS pathway genes, which regulate signal transduction upon binding of ligand to a variety of membrane receptors, are frequently associated with high-risk ALL and may be relapse drivers (Zhang et al., Blood 2011 118:3080-7; Irving et al., ASH 2013). Relapses in ALL are thought to result from the outgrowth of therapy-resistant residual leukemia cells and recent studies have shown a complex, dynamic architecture of clonal diversity in ALL (Anderson et al., Nature 2011 469:356-61; Notta et al. Nature 2011 496:362-7). In this study, we have investigated the clonal origin of RAS pathway mutations in relapsed BCP-ALL. We screened 146 relapse samples from children with relapsed BCP-ALL for mutations in five RAS pathway genes using IonTorrent sequencing with a read-depth of approximately 150x. A total of 30% of relapse samples carried RAS pathway mutations, including mutations in KRAS (n=22), NRAS (n=13), PTPN11 (n=8), and FLT3 (n=2). No RAS mutations were found in 103 patients, whereas one patient had both a PTPN11 and a KRAS mutation, and another patient had two KRAS mutations. For 28 mutations we collected matched diagnosis samples and determined mutation presence using Sanger sequencing. We found that 12 of the RAS mutations were also present at diagnosis (43%) and 16 were initially not detected at diagnosis (57%). In order to gain more insight into the clonal evolution of relapse development, we performed amplicon based ultra-deep sequencing on the diagnosis samples, with an average read-depth of 50,000x for each mutation. The ultra-deep sequencing allows for sensitive and accurate detection of relapse-prone clones at diagnosis. A total of 22 mutations were identified at diagnosis samples (79%), of which 10 mutations had a low mutant allele frequency (average 3.8%), and were initially missed by Sanger sequencing (Fig.1). The 12 mutations determined using Sanger sequencing were detected at an average mutant allele frequency of 30.8%. We were unable to detect 6 mutations in the matched diagnosis samples, indicating that these mutations were newly acquired in the relapse clone at a time point after relapse. Our results indicate that cells harboring RAS pathway mutations are recurrently present in subclones at diagnosis. These cells may survive initial therapy and subsequently emerge at relapse. Patients with (minor clones with) RAS pathway mutations identified by ultra-deep sequencing, may benefit from treatment with MEK inhibitors added to the frontline therapy strategy. Figure 1. Backtracking of RAS pathway mutations by ultra-deep sequencing. A total of 22 mutations (out of 28) were detected in matched diagnosis samples. The 12 mutations (solid grey) determined by Sanger sequencing were detected at high mutant allele frequency ranging from 22% to 42%. Ten mutations (diagonally striped) were missed by Sanger sequencing but detected by ultra-deep sequencing. These mutations showed a low mutant allele frequency varying from 0.3% to 8%. Figure 1. Backtracking of RAS pathway mutations by ultra-deep sequencing. A total of 22 mutations (out of 28) were detected in matched diagnosis samples. The 12 mutations (solid grey) determined by Sanger sequencing were detected at high mutant allele frequency ranging from 22% to 42%. Ten mutations (diagonally striped) were missed by Sanger sequencing but detected by ultra-deep sequencing. These mutations showed a low mutant allele frequency varying from 0.3% to 8%. 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.

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