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• W1963556688 abstract "MPL mutation testing is recommended in patients with suspected primary myelofibrosis or essential thrombocythemia who lack the JAK2 V617F mutation. MPL mutations can occur at allelic levels below 15%, which may escape detection by commonly used mutation screening methods such as Sanger sequencing. We developed a novel multiplexed allele-specific PCR assay capable of detecting most recurrent MPL exon 10 mutations associated with primary myelofibrosis and essential thrombocythemia (W515L, W515K, W515A, and S505N) down to a sensitivity of 2.5% mutant allele. Test results were reviewed from 15 reference cases and 1380 consecutive specimens referred to our laboratory for testing. Assay performance was compared to Sanger sequencing across a series of 58 specimens with MPL mutations. Positive cases consisted of 45 with W515L, 6 with S505N, 5 with W515K, 1 with W515A, and 1 with both W515L and S505N. Seven cases had mutations below 5% that were undetected by Sanger sequencing. Ten additional cases had mutation levels between 5% and 15% that were not consistently detected by sequencing. All results were easily interpreted in the allele-specific test. This assay offers a sensitive and reliable solution for MPL mutation testing. Sanger sequencing appears insufficiently sensitive for robust MPL mutation detection. Our data also suggest the relative frequency of S505N mutations may be underestimated, highlighting the necessity for inclusion of this mutation in MPL test platforms. MPL mutation testing is recommended in patients with suspected primary myelofibrosis or essential thrombocythemia who lack the JAK2 V617F mutation. MPL mutations can occur at allelic levels below 15%, which may escape detection by commonly used mutation screening methods such as Sanger sequencing. We developed a novel multiplexed allele-specific PCR assay capable of detecting most recurrent MPL exon 10 mutations associated with primary myelofibrosis and essential thrombocythemia (W515L, W515K, W515A, and S505N) down to a sensitivity of 2.5% mutant allele. Test results were reviewed from 15 reference cases and 1380 consecutive specimens referred to our laboratory for testing. Assay performance was compared to Sanger sequencing across a series of 58 specimens with MPL mutations. Positive cases consisted of 45 with W515L, 6 with S505N, 5 with W515K, 1 with W515A, and 1 with both W515L and S505N. Seven cases had mutations below 5% that were undetected by Sanger sequencing. Ten additional cases had mutation levels between 5% and 15% that were not consistently detected by sequencing. All results were easily interpreted in the allele-specific test. This assay offers a sensitive and reliable solution for MPL mutation testing. Sanger sequencing appears insufficiently sensitive for robust MPL mutation detection. Our data also suggest the relative frequency of S505N mutations may be underestimated, highlighting the necessity for inclusion of this mutation in MPL test platforms. The myeloproliferative neoplasms (MPNs) include polycythemia vera, essential thrombocythemia (ET), primary myelofibrosis (PMF), and other less well-characterized chronic myeloproliferative disorders.1Vardiman J.W. Thiele J. Arber D.A. Brunning R.D. Borowitz M.J. Porwit A. Harris N.L. Le Beau M.M. Hellstrom-Lindberg E. Tefferi A. Bloomfield C.D. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes.Blood. 2009; 114: 937-951Crossref PubMed Scopus (3452) Google Scholar Each of these entities is associated with gene mutations that are important diagnostic markers. The most prevalent mutations in ET and PMF are JAK2 V617F (55% and 65% of cases, respectively)2Baxter E.J. Scott L.M. Campbell P.J. East C. Fourouclas N. Swanton S. Vassiliou G.S. Bench A.J. Boyd E.M. Curtin N. Scott M.A. Erber W.N. Green A.R. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders.Lancet. 2005; 365: 1054-1061Abstract Full Text Full Text PDF PubMed Scopus (2300) Google Scholar, 3Tefferi A. Novel mutations and their functional and clinical relevance in myeloproliferative neoplasms: jAK2, MPL, TET2, ASXL1, CBL, IDH and IKZF1.Leukemia. 2010; 24: 1128-1138Crossref PubMed Scopus (432) Google Scholar and MPL (3% and 10% of cases, respectively).3Tefferi A. Novel mutations and their functional and clinical relevance in myeloproliferative neoplasms: jAK2, MPL, TET2, ASXL1, CBL, IDH and IKZF1.Leukemia. 2010; 24: 1128-1138Crossref PubMed Scopus (432) Google Scholar, 4Pardanani A.D. Levine R.L. Lasho T. Pikman Y. Mesa R.A. Wadleigh M. Steensma D.P. Elliott M.A. Wolanskyj A.P. Hogan W.J. McClure R.F. Litzow M.R. Gilliland D.G. Tefferi A. MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients.Blood. 2006; 108: 3472-3476Crossref PubMed Scopus (838) Google Scholar, 5Pikman Y. Lee B.H. Mercher T. McDowell E. Ebert B.L. Gozo M. Cuker A. Wernig G. Moore S. Galinsky I. DeAngelo D.J. Clark J.J. Lee S.J. Golub T.R. Wadleigh M. Gilliland D.G. Levine R.L. MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia.PLoS Med. 2006; 3: e270Crossref PubMed Scopus (1102) Google Scholar Testing for MPL mutations can aid in the diagnosis of these MPNs and is recommended in patients with suspected ET or PMF who lack the JAK2 V617F mutation. In this setting, the presence of an MPL mutation establishes a clonal myeloproliferation and meets a major diagnostic criterion in the most recent revision of the World Health Organization classification of ET and PMF.1Vardiman J.W. Thiele J. Arber D.A. Brunning R.D. Borowitz M.J. Porwit A. Harris N.L. Le Beau M.M. Hellstrom-Lindberg E. Tefferi A. Bloomfield C.D. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes.Blood. 2009; 114: 937-951Crossref PubMed Scopus (3452) Google Scholar, 6Tefferi A. Thiele J. Vardiman J.W. The 2008 World Health Organization classification system for myeloproliferative neoplasms: order out of chaos.Cancer. 2009; 115: 3842-3847Crossref PubMed Scopus (173) Google Scholar The MPL gene (located on chromosome 1p34) includes 12 exons and encodes the thrombopoietin receptor. Five recurrent MPL mutations have been reported in ET and PMF patients to date, all clustering in exon 10 and affecting two amino acids (W515L, W515K, W515A, W515R, and S505N). W515L and W515K represent the vast majority of MPL mutations reported in the literature, whereas the W515A, W515R, and S505N mutations are reported less commonly.4Pardanani A.D. Levine R.L. Lasho T. Pikman Y. Mesa R.A. Wadleigh M. Steensma D.P. Elliott M.A. Wolanskyj A.P. Hogan W.J. McClure R.F. Litzow M.R. Gilliland D.G. Tefferi A. MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients.Blood. 2006; 108: 3472-3476Crossref PubMed Scopus (838) Google Scholar, 7Beer P.A. Campbell P.J. Scott L.M. Bench A.J. Erber W.N. Bareford D. Wilkins B.S. Reilly J.T. Hasselbalch H.C. Bowman R. Wheatley K. Buck G. Harrison C.N. Green A.R. MPL mutations in myeloproliferative disorders: analysis of the PT-1 cohort.Blood. 2008; 112: 141-149Crossref PubMed Scopus (308) Google Scholar, 8Schnittger S. Bacher U. Haferlach C. Beelen D. Bojko P. Burkle D. Dengler R. Distelrath A. Eckart M. Eckert R. Fries S. Knoblich J. Kochling G. Laubenstein H.P. Petrides P. Planker M. Pihusch R. Weide R. Kern W. Haferlach T. Characterization of 35 new cases with four different MPLW515 mutations and essential thrombocytosis or primary myelofibrosis.Haematologica. 2009; 94: 141-144Crossref PubMed Scopus (49) Google Scholar, 9Boyd E.M. Bench A.J. Goday-Fernandez A. Anand S. Vaghela K.J. Beer P. Scott M.A. Bareford D. Green A.R. Huntly B. Erber W.N. Clinical utility of routine MPL exon 10 analysis in the diagnosis of essential thrombocythaemia and primary myelofibrosis.Br J Haematol. 2010; 149: 250-257Crossref PubMed Scopus (79) Google Scholar, 10Millecker L. Lennon P.A. Verstovsek S. Barkoh B. Galbincea J. Hu P. Chen S.S. Jones D. Distinct patterns of cytogenetic and clinical progression in chronic myeloproliferative neoplasms with or without JAK2 or MPL mutations.Cancer Genet Cytogenet. 2010; 197: 1-7Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 11Ding J. Komatsu H. Wakita A. Kato-Uranishi M. Ito M. Satoh A. Tsuboi K. Nitta M. Miyazaki H. Iida S. Ueda R. Familial essential thrombocythemia associated with a dominant-positive activating mutation of the c-MPL gene, which encodes for the receptor for thrombopoietin.Blood. 2004; 103: 4198-4200Crossref PubMed Scopus (273) Google Scholar Notably, the S505N mutation was first identified as an inherited alteration in familial ET,11Ding J. Komatsu H. Wakita A. Kato-Uranishi M. Ito M. Satoh A. Tsuboi K. Nitta M. Miyazaki H. Iida S. Ueda R. Familial essential thrombocythemia associated with a dominant-positive activating mutation of the c-MPL gene, which encodes for the receptor for thrombopoietin.Blood. 2004; 103: 4198-4200Crossref PubMed Scopus (273) Google Scholar, 12Liu K. Martini M. Rocca B. Amos C.I. Teofili L. Giona F. Ding J. Komatsu H. Larocca L.M. Skoda R.C. Evidence for a founder effect of the MPL-S505N mutation in eight Italian pedigrees with hereditary thrombocythemia.Haematologica. 2009; 94: 1368-1374Crossref PubMed Scopus (47) Google Scholar but has also been identified as a somatic mutation in both ET and PMF.7Beer P.A. Campbell P.J. Scott L.M. Bench A.J. Erber W.N. Bareford D. Wilkins B.S. Reilly J.T. Hasselbalch H.C. Bowman R. Wheatley K. Buck G. Harrison C.N. Green A.R. MPL mutations in myeloproliferative disorders: analysis of the PT-1 cohort.Blood. 2008; 112: 141-149Crossref PubMed Scopus (308) Google Scholar The MPL mutations associated with ET and PMF are gain of function and lead to receptor activation in the absence of thrombopoietin binding with constitutional activation of the JAK-STAT signaling.4Pardanani A.D. Levine R.L. Lasho T. Pikman Y. Mesa R.A. Wadleigh M. Steensma D.P. Elliott M.A. Wolanskyj A.P. Hogan W.J. McClure R.F. Litzow M.R. Gilliland D.G. Tefferi A. MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients.Blood. 2006; 108: 3472-3476Crossref PubMed Scopus (838) Google Scholar, 5Pikman Y. Lee B.H. Mercher T. McDowell E. Ebert B.L. Gozo M. Cuker A. Wernig G. Moore S. Galinsky I. DeAngelo D.J. Clark J.J. Lee S.J. Golub T.R. Wadleigh M. Gilliland D.G. Levine R.L. MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia.PLoS Med. 2006; 3: e270Crossref PubMed Scopus (1102) Google Scholar MPL mutations are usually found in MPNs that test negative for the JAK2 V617F mutation, although a small number of patients with both mutations have been reported.4Pardanani A.D. Levine R.L. Lasho T. Pikman Y. Mesa R.A. Wadleigh M. Steensma D.P. Elliott M.A. Wolanskyj A.P. Hogan W.J. McClure R.F. Litzow M.R. Gilliland D.G. Tefferi A. MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients.Blood. 2006; 108: 3472-3476Crossref PubMed Scopus (838) Google Scholar, 13Guglielmelli P. Pancrazzi A. Bergamaschi G. Rosti V. Villani L. Antonioli E. Bosi A. Barosi G. Vannucchi A.M. Anaemia characterises patients with myelofibrosis harbouring Mpl mutation.Br J Haematol. 2007; 137: 244-247Crossref PubMed Scopus (122) Google Scholar It should be noted that although MPL mutations occur in ET and PMF, they have not been reported in polycythemia vera.4Pardanani A.D. Levine R.L. Lasho T. Pikman Y. Mesa R.A. Wadleigh M. Steensma D.P. Elliott M.A. Wolanskyj A.P. Hogan W.J. McClure R.F. Litzow M.R. Gilliland D.G. Tefferi A. MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients.Blood. 2006; 108: 3472-3476Crossref PubMed Scopus (838) Google Scholar The mutant allele burden in specimens harboring MPL mutations is frequently >50%, suggesting that biallelic mutation (or loss of heterozygosity) is somewhat common.7Beer P.A. Campbell P.J. Scott L.M. Bench A.J. Erber W.N. Bareford D. Wilkins B.S. Reilly J.T. Hasselbalch H.C. Bowman R. Wheatley K. Buck G. Harrison C.N. Green A.R. MPL mutations in myeloproliferative disorders: analysis of the PT-1 cohort.Blood. 2008; 112: 141-149Crossref PubMed Scopus (308) Google Scholar, 8Schnittger S. Bacher U. Haferlach C. Beelen D. Bojko P. Burkle D. Dengler R. Distelrath A. Eckart M. Eckert R. Fries S. Knoblich J. Kochling G. Laubenstein H.P. Petrides P. Planker M. Pihusch R. Weide R. Kern W. Haferlach T. Characterization of 35 new cases with four different MPLW515 mutations and essential thrombocytosis or primary myelofibrosis.Haematologica. 2009; 94: 141-144Crossref PubMed Scopus (49) Google Scholar, 10Millecker L. Lennon P.A. Verstovsek S. Barkoh B. Galbincea J. Hu P. Chen S.S. Jones D. Distinct patterns of cytogenetic and clinical progression in chronic myeloproliferative neoplasms with or without JAK2 or MPL mutations.Cancer Genet Cytogenet. 2010; 197: 1-7Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar However, mutations in patients with MPNs have also been reported at lower levels (5% and less).7Beer P.A. Campbell P.J. Scott L.M. Bench A.J. Erber W.N. Bareford D. Wilkins B.S. Reilly J.T. Hasselbalch H.C. Bowman R. Wheatley K. Buck G. Harrison C.N. Green A.R. MPL mutations in myeloproliferative disorders: analysis of the PT-1 cohort.Blood. 2008; 112: 141-149Crossref PubMed Scopus (308) Google Scholar, 8Schnittger S. Bacher U. Haferlach C. Beelen D. Bojko P. Burkle D. Dengler R. Distelrath A. Eckart M. Eckert R. Fries S. Knoblich J. Kochling G. Laubenstein H.P. Petrides P. Planker M. Pihusch R. Weide R. Kern W. Haferlach T. Characterization of 35 new cases with four different MPLW515 mutations and essential thrombocytosis or primary myelofibrosis.Haematologica. 2009; 94: 141-144Crossref PubMed Scopus (49) Google Scholar, 10Millecker L. Lennon P.A. Verstovsek S. Barkoh B. Galbincea J. Hu P. Chen S.S. Jones D. Distinct patterns of cytogenetic and clinical progression in chronic myeloproliferative neoplasms with or without JAK2 or MPL mutations.Cancer Genet Cytogenet. 2010; 197: 1-7Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 14Brisci A. Damin F. Pietra D. Galbiati S. Boggi S. Casetti I. Rumi E. Chiari M. Cazzola M. Ferrari M. Cremonesi L. COLD-PCR and innovative microarray substrates for detecting and genotyping MPL exon 10 W515 substitutions.Clin Chem. 2012; 58: 1692-1702Crossref PubMed Scopus (8) Google Scholar Strategies to detect MPL mutations must therefore have sufficient analytical sensitivity as well as the ability to detect the variety of nucleotide alterations. Unfortunately, mutations below 10% are difficult to detect by commonly used MPL mutation screening methods such as Sanger sequencing. To address this problem, we developed a simple multiplexed allele-specific PCR assay that detects the vast majority of MPL mutations with high sensitivity and that could be easily deployed in our laboratory. We retrospectively reviewed test results from specimens referred to our laboratory for testing and then compared the performance of this assay to Sanger sequencing across a series of 58 MPL mutation–positive specimens. An interlaboratory validation set of 15 specimens (5 positive and 10 negative for MPL mutations) and 1380 consecutive cases referred to our institution for MPL mutation testing were included in this study. Genotyping data and all accompanying case materials were de-identified following clinical testing. No further clinical information other than age and sex was available for most cases. All 15 validation samples were bidirectionally sequenced to verify both positive and negative results. Sanger sequencing was also performed on each clinical specimen reported as positive for MPL mutation by allele-specific PCR. Samples reported as negative for MPL mutations by allele-specific PCR were not further evaluated. When available, medical chart review was performed to determine clinicopathological status of patients with specimens that had discordant results between our assay and the Sanger sequencing assay. This study was performed according to a University of Michigan Institutional Review Board–approved protocol for research use of de-identified specimens. Genomic DNA was extracted from peripheral blood or bone marrow using standard techniques (Qiagen BioRobot EZ1; Qiagen, Valencia, CA) according to the manufacturer’s instructions. For the 15 cases included in the preclinical validation of the assay, genomic DNA from peripheral blood or bone marrow was kindly provided by MD Anderson Cancer Center (Houston, TX). The Catalogue of Somatic Mutations in Cancer (COSMIC) Mutation Database was searched to identify recurrent published mutations (three or more instances) within the MPL coding region in patients with MPNs (ET or PMF) (Sanger Institute, http://cancer.sanger.ac.uk/cosmic/gene/analysis?ln=MPL, last accessed November 10, 2011). A total of 314 mutations were listed. All mutations were nucleotide substitutions affecting either codon 505 or 515. The W515L mutation was most common (210 cases, 67%), followed by W515K (18 cases, 26%), W515A (11 cases, 3.5%), S505N (7 cases, 2%), and two types of W515R mutation (5 cases, 1.5%). Results of this analysis revealed that in patients with ET or PMF, 98% of reported MPL mutations involve a W515L, W515K, W515A, or S505N substitution mutation. Therefore, our goal was to design an assay capable of detecting each of these mutations with high analytical sensitivity and specificity. We therefore designed an allele-specific PCR assay using primers that specifically amplify only from specimens containing the particular mutation. The W515L primer detects the c.1544G>T substitution. The W515K primer detects the c.1543_1544delinsAA mutation found in each of the two types of W515K mutations. The W515A primer detects the c.1543_1544delinsGC mutation found in each of the two types of W515A mutations. The S505N primer detects the c.1514G>A substitution. Nucleotide numbering for each of the mutations listed above is based on GenBank accession number NM_005373.2. Multiplex PCR amplifications were performed in a two-tube format to detect the four most common MPL mutations (W515L, W515K, W515A, and S505N) (Figure 1). Each tube consisted of a 20-μL reaction containing 5 μL (50 ng) of input DNA, 10 μL of 2× AmpliTaq Gold PCR Master Mix (Applied Biosystems, Foster City, CA), and 5 μL of 4× MPL primer mix. The 4× tube A primer mix contained the S505N and the W515L mutation–specific primers, each at 1200 nmol/L. The 4× tube B primer mix contained the W515K and W515A mutation–specific primers, W515K 400 nmol/L and W515A 1200 nmol/L. Both tube A and B primer mixes also contained outer MPL-forward and MPL-reverse primers (each at 600 nmol/L in the 4× mix), which provided amplification of the targeted mutations in each tube and also served to amplify the entire MPL exon 10 as an internal control. Primer sequences are listed in Table 1. A 5% MPL mutation–positive control, an MPL mutation–negative control, and a no template (blank) control (DNA-grade water) were included in each run. The PCR was performed using a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems) with the following cycling conditions: 95°C for 10 minutes, followed by 38 cycles of 94°C for 15 seconds, 64°C for 30 seconds, and 72°C for 1 minute, and a final extension at 72°C for 5 minutes. Following PCR amplification, the products were resolved and analyzed by capillary electrophoresis. For capillary electrophoresis, 1 μL of each PCR amplification product was mixed with 20 μL of Hi-Di Formamide (Applied Biosystems) and 1 μL of GeneScan 500 LIZ Size Standard (Applied Biosystems). Samples were electrokinetically injected at 1.2 kV for 18 seconds on a 3130xl Genetic Analyzer (Applied Biosystems) and run using a 36-cm capillary array and POP-4 polymer at a voltage of 15 kV for 1500 seconds at 60°C under filter set G5. Data were analyzed using Applied Biosystems GeneMapper ID software version 3.2 (Applied Biosystems). All results in this study were obtained using these test conditions.Table 1Primers Used in the Allele-Specific PCR Assay for Detection of MPL Exon 10 MutationsAssayPrimer sequencesTube A MPL-forward5′-FAM-TGGGCCGAAGTCTGACCCTTT-3′ MPL-reverse5′-CAGAGCGAACCAAGAATGCCTGT-3′ W515L-forward5′-FAM-GGCCTGCTGCTGCTGAGATT-3′ S505N-reverse5′-CAGGCCCAGGACGGCGT-3′Tube B MPL-forward5′-NED-TGGGCCGAAGTCTGACCCTTT-3′ MPL-reverse5′-CAGAGCGAACCAAGAATGCCTGT-3′ W515K-forward5′-NED-GCCTGCTGCTGCTGAGGAA-3′ W515A-reverse5′-TAGTGTGCAGGAAACTGCGC-3′ W515A-reverse∗The alternative W515A primer is used in the updated version of the test.5′-GTAGTGTGCAGGAAACTGCGC-3′Primer bases highlighted in bold correspond to the mutation-specific sequence. A mismatch represented in italics was included in the W515L allele-specific primer 3 bp from the 3′ end for increased specificity. The forward primers in both assays are labeled with different fluorophores [FAM (blue) and NED (yellow)] to permit detection by capillary electrophoresis.∗ The alternative W515A primer is used in the updated version of the test. Open table in a new tab Primer bases highlighted in bold correspond to the mutation-specific sequence. A mismatch represented in italics was included in the W515L allele-specific primer 3 bp from the 3′ end for increased specificity. The forward primers in both assays are labeled with different fluorophores [FAM (blue) and NED (yellow)] to permit detection by capillary electrophoresis. The assay was also recently updated and optimized for the AmpliTaq Gold 360 PCR Master Mix (Applied Biosystems) in replacement of the discontinued AmpliTaq Gold PCR Master Mix. Assay conditions were identical except for the following additional substitutions: W515A allele-specific reverse primer sequence, 5′-GTAGTGTGCAGGAAACTGCGC-3′; concentration of W515K primer increased to 600 nmol/L in 4× tube B primer mix; cycling conditions changed to 95°C for 10 minutes, followed by 36 cycles of 95°C for 30 seconds, 61°C for 30 seconds, and 72°C for 1 minute, and a final extension at 72°C for 5 minutes. Performance characteristics of this updated assay were determined to be identical to the original assay, except the analytical limit of detection, which was improved to 0.5% for the W515L and W515K mutations, and 2% for S505N and W515A mutations (data not shown). Capillary fluorescence signal thresholds were established for interpreting positive results. These cutoffs were determined by evaluating results from the sensitivity dilutions (see Assay Analytical Sensitivity), low positive sensitivity controls (approximately 5% mutation), the negative controls, and the negative validation samples. To ensure that only specific peaks representative of the authentic MPL mutant product were interpreted as positive, we considered a peak >500 relative fluorescent units (RFU) in height in any of the mutation bins (S505N, W515L, W515K, or W515A) to be unequivocally positive for an MPL mutation (Figure 2). To eliminate the possible reporting of false-negative results due to poor sample quality, we required the control peak height to be at least 6000 RFU in height in both the tube A and B assays. A 212-bp fragment containing the entire MPL exon 10 sequence was amplified in a 30-μL reaction containing 5 μL (50 ng) of input DNA, 15 μL of 2× Phusion High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA), 7.5 μL of MPL intronic primers [4× mixture with each primer 1200 nmol/L at 4× concentration (forward, 5′-TGGGCCGAAGTCTGACCCTTT-3′ and reverse, 5′-ACAGAGCGAACCAAGAATGCCTGT-3′)], 1.8 μL of 100% dimethyl sulfoxide (New England Biolabs, Ipswich, MA), and 0.7 μL of DNA-grade water. The PCR was performed on a GeneAmp PCR System 9700 thermal cycler with the following amplification conditions: 98°C for 30 seconds, followed by 35 cycles of 99°C for 5 seconds, 65°C for 15 seconds, and 72°C for 15 seconds, and a final extension at 72°C for 2 minutes. An MPL mutation–negative control and no template blank control (DNA-grade water) were included on each run. PCR products were purified by the QIAquick PCR Purification Kit (Qiagen) and bidirectionally sequenced using nested primers (forward, 5′-TGTCTCCTAGCCTGGATCTCC-3′ and reverse,: 5′-TTCGGCTCCACCTGGTCC-3′) and the BigDye v1.1 Terminator Cycle Sequencing Kit (Applied Biosystems) on a 3130xl Genetic Analyzer. The entire coding region of exon 10 was evaluated for mutations using Mutation Surveyor software version 3.10 (SoftGenetics, State College, PA) in comparison to MPL reference sequence NM_005373.2. For each case identified as MPL mutation positive by allele-specific PCR, semiquantitative mutation levels were calculated as the average of the two peak drop values from forward and reverse peaks on sequence chromatograms using Mutation Surveyor software version 3.10. Cases with mutant allele levels >15% and between 5% and 15% were considered positive and equivocal positive, respectively. Cases with undetected mutations were considered negative by sequencing. The test was performed in a two-tube format for PCR amplification (Figure 1). The tube A assay included S505N and W515L mutation–specific primers along with an outer pair of intronic primers (MPL-forward and MPL-reverse) that amplify a 211-bp fragment containing the entire MPL exon 10 sequence. The tube B assay included W515K and W515A mutation–specific primers along with the same outer primers. For both assays, the 211-bp PCR product that is generated by the outer primers served as the amplification control to ensure the adequacy and integrity of the specimen DNA. The forward primers in both assays were fluorescently labeled to permit detection by capillary electrophoresis: blue indicates FAM (tube A assay) and yellow indicates NED (tube B assay). Specimens were interpreted according to the presence or absence of amplification products corresponding to each tested mutation, which were distinguished by amplicon size and assay tube (Figure 2). Specimens negative for the four tested mutations would only display the 211-bp control amplicon in each tube. An interlaboratory validation set consisting of 10 wild-type and 5 mutation-positive cases were tested using the allele-specific PCR assay. All mutations were readily detected (Table 2). Overall correlation was 15 of 15 (100%) compared to the reference result. Each of the 15 cases were also analyzed by Sanger sequencing. Both methods readily identified the mutations in each of the five positive cases. Case 1 was found to harbor two MPL mutations by sequencing (S505N and W515R). This specimen tested positive for only S505N by our allele-specific PCR assay because it was not designed to detect the rare W515R mutation. Analytic specificity of the allele-specific PCR assay was also evaluated in a set of 30 peripheral blood specimens from healthy individuals and 15 bone marrow specimens with the JAK2 V617F mutation. All 45 specimens tested negative for MPL mutations as expected (data not shown). Assay reproducibility was assessed over 15 assay runs using low-positive 5% mutation controls: S505N and W515A (tube A), W515L and W515K (tube B), and a negative control. Expected test results were obtained in all assay runs.Table 2MPL Exon 10 Mutation–Positive Cases Identified in This StudyCase∗Cases 1 to 5 were included in the preclinical validation of the fragment analysis assay.MPL mutation†Include MPL exon 10 mutations identified by allele-specific PCR and/or Sanger sequencing by the primers included in the allele-specific PCR assay.Mutation (cDNA)‡Nucleotide numbering based on NM_005373.2.Level§Level refers to the semiquantitative allelic mutation burden as calculated from the Sanger sequencing data using Mutation Surveyor software version 3.10.Allele-specific PCRDirect sequencing1S505N + W515R1514G>A + 1543T>C55%, 56%PositivePositive2W515A1543_1544delinsGC97%PositivePositive3W515K1543_1544delinsAA76%PositivePositive4W515K1543_1544delinsAA54%PositivePositive5W515L1544G>T49%PositivePositive6W515L1544G>T48%PositivePositive7W515L1544G>T78%PositivePositive8W515L1544G>T37%PositivePositive9W515L1544G>T5%PositiveEquivocal positive¶Low-level (5% to 15%) background peaks on the sequencing electropherogram suggestive of this mutation; however, the level was below what could unequivocally be called positive.10W515L1544G>T10%PositiveEquivocal positive¶Low-level (5% to 15%) background peaks on the sequencing electropherogram suggestive of this mutation; however, the level was below what could unequivocally be called positive.11W515L1544G>T50%PositivePositive12W515L1544G>T13%PositiveEquivocal positive¶Low-level (5% to 15%) background peaks on the sequencing electropherogram suggestive of this mutation; however, the level was below what could unequivocally be called positive.13W515L1544G>T3%PositiveNegative‖Very low-level mutations that were not detected by Sanger sequencing.14W515L1544G>T25%PositivePositive15W515L1544G>T35%PositivePositive16W515L1544G>T9%PositiveEquivocal positive¶Low-level (5% to 15%) background peaks on the sequencing electropherogram suggestive of this mutation; however, the level was below what could unequivocally be called positive.17W515L1544G>T25%PositivePositive18W515L1544G>T51%PositivePositive19W515K1543_1544delinsAA40%PositivePositive20W515L——PositiveNegative‖Very low-level mutations that were not detected by Sanger sequencing.21W515L1544G>T5%PositiveEquivocal positive¶Low-level (5% to 15%) background peaks on the sequencing electropherogram suggestive of this mutation; however, the level was below what could unequivocally be called positive.22W515L1544G>T100%PositivePositive23W515L1544G>T31%PositivePositive24W515L1544G>T38%PositivePositive25W515L1544G>T75%PositivePositive26W515L1544" @default.
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