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MSIplus for Integrated Colorectal Cancer Molecular Testing by Next-Generation Sequencing

2015; Elsevier BV; Volume: 17; Issue: 6 Linguagem: Inglês

10.1016/j.jmoldx.2015.05.008

ISSN

1943-7811

Autores

Jennifer A. Hempelmann, Sheena M. Scroggins, Colin C. Pritchard, Stephen J. Salipante,

Tópico(s)

Cancer Genomics and Diagnostics

Resumo

Molecular analysis of colon cancers currently requires multiphasic testing that uses various assays with different performance characteristics, adding cost and time to patient care. We have developed a single, next-generation sequencing assay to simultaneously evaluate colorectal cancers for mutations in relevant cancer genes (KRAS, NRAS, and BRAF) and for tumor microsatellite instability (MSI). In a sample set of 61 cases, the assay demonstrated overall sensitivity of 100% and specificity of 100% for identifying cancer-associated mutations, with a practical limit of detection at 2% mutant allele fraction. MSIplus was 97% sensitive (34 of 35 MSI-positive cases) and 100% specific (42 of 42 MSI-negative cases) for ascertaining MSI phenotype in a cohort of 78 tumor specimens. These performance characteristics were slightly better than for conventional multiplex PCR MSI testing (97% sensitivity and 95% specificity), which is based on comparison of microsatellite loci amplified from tumor and matched normal material, applied to the same specimen cohort. Because the assay uses an amplicon sequencing approach, it is rapid and appropriate for specimens with limited available material or fragmented DNA. This integrated testing strategy offers several advantages over existing methods, including a lack of need for matched normal material, sensitive and unbiased detection of variants in target genes, and an automated analysis pipeline enabling principled and reproducible identification of cancer-associated mutations and MSI status simultaneously. Molecular analysis of colon cancers currently requires multiphasic testing that uses various assays with different performance characteristics, adding cost and time to patient care. We have developed a single, next-generation sequencing assay to simultaneously evaluate colorectal cancers for mutations in relevant cancer genes (KRAS, NRAS, and BRAF) and for tumor microsatellite instability (MSI). In a sample set of 61 cases, the assay demonstrated overall sensitivity of 100% and specificity of 100% for identifying cancer-associated mutations, with a practical limit of detection at 2% mutant allele fraction. MSIplus was 97% sensitive (34 of 35 MSI-positive cases) and 100% specific (42 of 42 MSI-negative cases) for ascertaining MSI phenotype in a cohort of 78 tumor specimens. These performance characteristics were slightly better than for conventional multiplex PCR MSI testing (97% sensitivity and 95% specificity), which is based on comparison of microsatellite loci amplified from tumor and matched normal material, applied to the same specimen cohort. Because the assay uses an amplicon sequencing approach, it is rapid and appropriate for specimens with limited available material or fragmented DNA. This integrated testing strategy offers several advantages over existing methods, including a lack of need for matched normal material, sensitive and unbiased detection of variants in target genes, and an automated analysis pipeline enabling principled and reproducible identification of cancer-associated mutations and MSI status simultaneously. After initial diagnosis, the molecular characterization of colorectal cancers may require several separate clinical tests. Evaluation of microsatellite instability (MSI) status is recommended testing on all primary colon cancers from patients 50 years or younger (and in older patients if specific pathological features are present1Jenkins M.A. Hayashi S. O'Shea A.M. Burgart L.J. Smyrk T.C. Shimizu D. Waring P.M. Ruszkiewicz A.R. Pollett A.F. Redston M. Barker M.A. Baron J.A. Casey G.R. Dowty J.G. 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KRAS mutation testing of tumours in adults with metastatic colorectal cancer: a systematic review and cost-effectiveness analysis.Health Technol Assess. 2014; 18: 1-132Crossref Scopus (29) Google Scholar Next-generation sequencing (NGS) is becoming increasingly used by clinical laboratories as a cost-effective and scalable method to interrogate multiple genetic targets in parallel,29Pritchard C.C. Smith C. Salipante S.J. Lee M.K. Thornton A.M. Nord A.S. Gulden C. Kupfer S.S. Swisher E.M. Bennett R.L. Novetsky A.P. Jarvik G.P. Olopade O.I. Goodfellow P.J. King M.C. Tait J.F. Walsh T. ColoSeq provides comprehensive lynch and polyposis syndrome mutational analysis using massively parallel sequencing.J Mol Diagn. 2012; 14: 357-366Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 30Frampton G.M. Fichtenholtz A. Otto G.A. Wang K. Downing S.R. 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Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability.J Natl Cancer Inst. 2004; 96: 261-268Crossref PubMed Scopus (2461) Google Scholar used in current clinical MSI-PCR assays.16Bacher J.W. Flanagan L.A. Smalley R.L. Nassif N.A. Burgart L.J. Halberg R.B. Megid W.M. Thibodeau S.N. Development of a fluorescent multiplex assay for detection of MSI-High tumors.Dis Markers. 2004; 20: 237-250Crossref PubMed Scopus (195) Google Scholar Separate primers were designed to span exons containing relevant mutational hotspots in KRAS (exons 2, 3, and 4; codons 12, 13, 61, 117, and 147), NRAS (exons 2, 3, and 4; codons 12, 13, 61, 117, and 147), and BRAF (exon 15; codons 599, 600, and 601).Table 1Loci and Primer SequencesAssay stageTarget locusPrimer coordinatesRepeat typeForward primer sequenceReverse primer sequenceStage 1 PCRBat-25Chr4: 55598177-55598271(A)225′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTGGAGGATGACGAGTTGGCCCTAGAC-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCCCAAAGAGACAGCAGTTGGAACATGA-3′Stage 1 PCRBat-26Chr2: 47641524-47641622(T)195′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGTGGAGTGGAGGAGGGGAGAGAAA-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCTTGCAGTTTCATCACTGTCTGCGGT-3′Stage 1 PCRMONO-27Chr2: 39564859-39564957(T)285′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTACTGTCCTACTGTGCCTGGCTCC-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCCAGCCTGGGCAAGATAATGAGACCC-3′Stage 1 PCRNR-21Chr14: 23652311-23652403(A)225′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGGTGCACAGAGCAGAACCATCCT-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCGCAACCTCAAAAGCTGCCTCCCTTT-3′Stage 1 PCRNR-24Chr2: 95849327-95849419(T)245′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGTAGTCCCAGCTATTCGGGAGGC-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCAAATGACCCCTTCCTGCCCATCACT-3′Stage 1 PCRMSI-01Chr1: 201754376-201754446(T)175′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTTTGATGTCCTGCGTCTAGGGTCTGC-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCGACTGGAGCCTTGGACAGGTTGAGA-3′Stage 1 PCRMSI-03Chr2: 62063059-62063129(A)175′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCACTGCTATTTGAAAGAGTTGCTC-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCGCCACTGCTATTTGAAAGAGTTGCTC-3′Stage 1 PCRMSI-04Chr2: 108479588-108479658(T)185′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCCAAGATTCCTTCCCTGGCCACTC-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCACTGTCTGTAGTCCTGGCTTCGTGG-3′Stage 1 PCRMSI-06Chr5: 172421726-172421796(T)155′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGCAGCAAACTGAACAGGTCACCAAC-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCAGCAGCAAACTGAACAGGTCACCAAC-3′Stage 1 PCRMSI-07Chr6: 142691916-142691986(T)175′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCTGAAAGCAACCTAAGCTGTGGTGA-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCTGCTATAAGAGCTGAGCAGACGACA-3′Stage 1 PCRMSI-08Chr7: 1787485-1787555(A)175′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCAGCCCCCATGTACACTGTAGTCG-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCCCCACCCCAAGGCCAAAATCAGTAA-3′Stage 1 PCRMSI-09Chr7: 74608706-74608776(T)135′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTCTCGGCTACTTGGGAGGCTTAGG-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCCCGACTAAAGAGGTCATTCACTTGT-3′Stage 1 PCRMSI-11Chr11: 106695477-106695550(T)125′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGCATGTTTGCAGCCTTCTTCTGGA-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCAGCATGTTTGCAGCCTTCTTCTGGA-3′Stage 1 PCRMSI-12Chr15: 45897737-45897807(T)145′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCTGAGGCTAAACACTATCATGCCA-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCCAGAGGTTGCAGTGAGCCGAGATTG-3′Stage 1 PCRMSI-13Chr16: 18882625-18882695(A)155′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCTTCAGGTCAGGAAAACAGCTCG-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCTAATGACTTGGGCTTTGGAAGCAGC-3′Stage 1 PCRMSI-14Chr17: 19314883-19314953(T)185′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTCATTTCAACTGACCTGCCTGGCCTC-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCCTTGGCAAACGGGCAAGTCTTCAGT-3′Stage 1 PCRHSPH1-T17Chr13: 31722570-31722746(A)175′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGAAAAGGAACTGCATCTGTGACGG-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCTTTTCCTAATCCCCTGTGAAACCTGT-3′Stage 1 PCRBRAF exon 15Chr7: 140453095-140453431NA5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTACAACTGTTCAAACTGATGGGACC-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCCCTCATCCTAACACATTTCAAGCCCCA-3′Stage 1 PCRKRAS exon 4Chr12: 25378395-25378686NA5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTTTTCAGTGTTACTTACCTGTCTTGTC-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCTGACAAAAGTTGTGGACAGGT-3′Stage 1 PCRKRAS exon 3Chr12: 25380233-25380491NA5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCCAGTCCTCATGTACTGGTCCCT-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCCCGTCATCTTTGGAGCAGGAACA-3′Stage 1 PCRKRAS exon 2Chr12: 25398245-25398504NA5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGAATTAGCTGTATCGTCAAGGCACTC-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCACACGTCTGCAGTCAACTGGAATTT-3′Stage 1 PCRNRAS exon 4Chr1: 115252168-115252401NA5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTAATGCTGAAAGCTGTACCATACC-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCCCCAGCCTAATCTTGTTTTTCTT-3′Stage 1 PCRNRAS exon 3Chr1: 115258629-115258838NA5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGTGGCTCGCCAATTAACCCTG-3′5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTGTGTTTCTGAGAGACAGGATCAGGTCAGCGG-3′Stage 1 PCRNRAS exon 2Chr1: 115256475-115256731NA5′-ACAC

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