Evaluation of 13q14 Status in Multiple Myeloma by Digital Single Nucleotide Polymorphism Technology
2009; Elsevier BV; Volume: 11; Issue: 5 Linguagem: Inglês
10.2353/jmoldx.2009.090027
ISSN1943-7811
AutoresKaty Hanlon, Lorna W. Harries, Sian Ellard, Claudius Rudin,
Tópico(s)Chronic Lymphocytic Leukemia Research
ResumoChromosome 13q deletions are common in multiple myeloma and other cancers, demonstrating the importance of this region in tumorigenesis. We used a novel single nucleotide polymorphism (SNP)-based technique, digital SNP (dSNP), to identify loss of heterozygosity (LOH) at chromosome 13q in paraffin-embedded bone marrow biopsies from 22 patients with multiple myeloma. We analyzed heterozygous SNPs at 13q for the presence of allelic imbalances and examined the results by sequential probability ratio analysis. Where possible, dSNP results were confirmed by fluorescence in situ hybridization. Using dSNP, we identified 13q LOH in 16/18 (89%) (95% Confidence Interval; 65%, 99%) patients without the need for neoplastic cell enrichment. In 8/16 (50%) cases, either partial or interstitial patterns of LOH were observed. Both fluorescence in situ hybridization and dSNP data proved concordant in just 3/9 cases. Five of the six discrepancies showed LOH by dSNP occurring beyond the boundaries of the fluorescence in situ hybridization probes. Our findings show that dSNP represents a useful technique for the analysis of LOH in archival tissue with minimal infiltration of neoplastic cells. The high-resolution screening afforded by the dSNP technology allowed for the identification of complex chromosomal rearrangements, resulting in either partial or interstitial LOH. Digital SNP represents an attractive approach for the investigation of tumors not suitable for genomic-array analysis. Chromosome 13q deletions are common in multiple myeloma and other cancers, demonstrating the importance of this region in tumorigenesis. We used a novel single nucleotide polymorphism (SNP)-based technique, digital SNP (dSNP), to identify loss of heterozygosity (LOH) at chromosome 13q in paraffin-embedded bone marrow biopsies from 22 patients with multiple myeloma. We analyzed heterozygous SNPs at 13q for the presence of allelic imbalances and examined the results by sequential probability ratio analysis. Where possible, dSNP results were confirmed by fluorescence in situ hybridization. Using dSNP, we identified 13q LOH in 16/18 (89%) (95% Confidence Interval; 65%, 99%) patients without the need for neoplastic cell enrichment. In 8/16 (50%) cases, either partial or interstitial patterns of LOH were observed. Both fluorescence in situ hybridization and dSNP data proved concordant in just 3/9 cases. Five of the six discrepancies showed LOH by dSNP occurring beyond the boundaries of the fluorescence in situ hybridization probes. Our findings show that dSNP represents a useful technique for the analysis of LOH in archival tissue with minimal infiltration of neoplastic cells. The high-resolution screening afforded by the dSNP technology allowed for the identification of complex chromosomal rearrangements, resulting in either partial or interstitial LOH. Digital SNP represents an attractive approach for the investigation of tumors not suitable for genomic-array analysis. Multiple myeloma (MM) is a post germinal center B-cell malignancy characterized by the accumulation of plasma cells in the bone marrow, chromosomal instability, and chromosomal translocations involving the immunoglobulin heavy chain locus. This disorder is estimated to account for 10% of all hematological malignancies1Phekoo KJ Schey SA Richards MA Bevan DH Bell S Gillett D Moller H A population study to define the incidence and survival of multiple myeloma in a National Health Service Region in UK.Br J Haematol. 2004; 127: 299-304Crossref PubMed Scopus (121) Google Scholar and has an extremely variable prognosis, with survival ranging from a few months to more than 10 years.2Kyle RA Rajkumar SV Multiple myeloma.N Engl J Med. 2004; 351: 1860-1873Crossref PubMed Scopus (1247) Google Scholar Despite recent advances in gene expression profiling, the molecular mechanisms underlying the development of MM remain unclear.Cytogenetic instability is a key feature of MM, and chromosomal abnormalities are detectable in the majority of cases.3Perez-Simon JA Garcia-Sanz R Tabernero MD Almeida J Gonzalez M Fernandez-Calvo J Moro MJ Hernandez JM San Miguel JF Orfao A Prognostic value of numerical chromosome aberrations in multiple myeloma: a FISH analysis of 15 different chromosomes.Blood. 1998; 91: 3366-3371PubMed Google Scholar Several recurrent abnormalities have emerged that appear to influence disease development and progression. These include gains of chromosome regions 1q, 9q, and 11q, losses of chromosome regions 6q, Xp, and Xq,4Liebisch P Viardot A Bassermann N Wendl C Roth K Goldschmidt H Einsele H Straka C Stilgenbauer S Dohner H Bentz M Value of comparative genomic hybridization and fluorescence in situ hybridization for molecular diagnostics in multiple myeloma.Br J Haematol. 2003; 122: 193-201Crossref PubMed Scopus (44) Google Scholar and translocations involving 14q32.5Avet-Loiseau H Facon T Grosbois B Magrangeas F Rapp MJ Harousseau JL Minvielle S Bataille R Oncogenesis of multiple myeloma: 14q32 and 13q chromosomal abnormalities are not randomly distributed, but correlate with natural history, immunological features, and clinical presentation.Blood. 2002; 99: 2185-2191Crossref PubMed Scopus (308) Google Scholar Deletion of chromosome 13q is one of the most frequent cytogenetic abnormalities associated with MM, occurring in 40% to 50% of cases.4Liebisch P Viardot A Bassermann N Wendl C Roth K Goldschmidt H Einsele H Straka C Stilgenbauer S Dohner H Bentz M Value of comparative genomic hybridization and fluorescence in situ hybridization for molecular diagnostics in multiple myeloma.Br J Haematol. 2003; 122: 193-201Crossref PubMed Scopus (44) Google Scholar,6Zojer N Konigsberg R Ackermann J Fritz E Dallinger S Kromer E Kaufmann H Riedl L Gisslinger H Schreiber S Heinz R Ludwig H Huber H Drach J Deletion of 13q14 remains an independent adverse prognostic variable in multiple myeloma despite its frequent detection by interphase fluorescence in situ hybridization.Blood. 2000; 95: 1925-1930PubMed Google Scholar,7Fonseca R Harrington D Oken MM Dewald GW Bailey RJ Van Wier SA Henderson KJ Blood EA Rajkumar SV Kay NE Van Ness B Greipp PR Biological and prognostic significance of interphase fluorescence in situ hybridization detection of chromosome 13 abnormalities (delta13) in multiple myeloma: an eastern cooperative oncology group study.Cancer Res. 2002; 62: 715-720PubMed Google Scholar The majority of deletions involve entire chromosomes or chromosome arms,8Avet-Loiseau H Daviet A Saunier S Bataille R Chromosome 13 abnormalities in multiple myeloma are mostly monosomy 13.Br J Haematol. 2000; 111: 1116-1117Crossref PubMed Scopus (95) Google Scholar,9Fonseca R Oken MM Harrington D Bailey RJ Van Wier SA Henderson KJ Kay NE Van Ness B Greipp PR Dewald GW Deletions of chromosome 13 in multiple myeloma identified by interphase FISH usually denote large deletions of the q arm or monosomy.Leukemia. 2001; 15: 981-986Crossref PubMed Scopus (116) Google Scholar although partial and interstitial deletions have also been described.10Tricot G Barlogie B Jagannath S Bracy D Mattox S Vesole DH Naucke S Sawyer JR Poor prognosis in multiple myeloma is associated only with partial or complete deletions of chromosome 13 or abnormalities involving 11q and not with other karyotype abnormalities.Blood. 1995; 86: 4250-4256PubMed Google Scholar,11Nomdedeu JF Lasa A Ubeda J Saglio G Bellido M Casas S Carnicer MJ Aventin A Sureda A Sierra J Baiget M Interstitial deletions at the long arm of chromosome 13 may be as common as monosomies in multiple myeloma. A genotypic study.Haematologica. 2002; 87: 828-835PubMed Google Scholar Where interstitial deletions are present, they most commonly involve chromosome band 13q14 to 13q 21.10Tricot G Barlogie B Jagannath S Bracy D Mattox S Vesole DH Naucke S Sawyer JR Poor prognosis in multiple myeloma is associated only with partial or complete deletions of chromosome 13 or abnormalities involving 11q and not with other karyotype abnormalities.Blood. 1995; 86: 4250-4256PubMed Google Scholar,12Elnenaei MO Hamoudi RA Swansbury J Gruszka-Westwood AM Brito-Babapulle V Matutes E Catovsky D Delineation of the minimal region of loss at 13q14 in multiple myeloma.Genes Chromosomes Cancer. 2003; 36: 99-106Crossref PubMed Scopus (65) Google ScholarThe presence of chromosome 13q deletions has been suggested to be an adverse prognostic factor in MM.6Zojer N Konigsberg R Ackermann J Fritz E Dallinger S Kromer E Kaufmann H Riedl L Gisslinger H Schreiber S Heinz R Ludwig H Huber H Drach J Deletion of 13q14 remains an independent adverse prognostic variable in multiple myeloma despite its frequent detection by interphase fluorescence in situ hybridization.Blood. 2000; 95: 1925-1930PubMed Google Scholar,10Tricot G Barlogie B Jagannath S Bracy D Mattox S Vesole DH Naucke S Sawyer JR Poor prognosis in multiple myeloma is associated only with partial or complete deletions of chromosome 13 or abnormalities involving 11q and not with other karyotype abnormalities.Blood. 1995; 86: 4250-4256PubMed Google Scholar This has lead several groups to hypothesize that the 13q14 region may harbor one or more as yet uncharacterized tumor suppressor genes.6Zojer N Konigsberg R Ackermann J Fritz E Dallinger S Kromer E Kaufmann H Riedl L Gisslinger H Schreiber S Heinz R Ludwig H Huber H Drach J Deletion of 13q14 remains an independent adverse prognostic variable in multiple myeloma despite its frequent detection by interphase fluorescence in situ hybridization.Blood. 2000; 95: 1925-1930PubMed Google Scholar,10Tricot G Barlogie B Jagannath S Bracy D Mattox S Vesole DH Naucke S Sawyer JR Poor prognosis in multiple myeloma is associated only with partial or complete deletions of chromosome 13 or abnormalities involving 11q and not with other karyotype abnormalities.Blood. 1995; 86: 4250-4256PubMed Google Scholar This region of chromosome 13 has not only been associated with tumorigenesis in MM6Zojer N Konigsberg R Ackermann J Fritz E Dallinger S Kromer E Kaufmann H Riedl L Gisslinger H Schreiber S Heinz R Ludwig H Huber H Drach J Deletion of 13q14 remains an independent adverse prognostic variable in multiple myeloma despite its frequent detection by interphase fluorescence in situ hybridization.Blood. 2000; 95: 1925-1930PubMed Google Scholar but also in chronic lymphocytic leukemia,13van Everdink WJ Baranova A Lummen C Tyazhelova T Looman MW Ivanov D Verlind E Pestova A Faber H van der Veen AY Yankovsky N Vellenga E Buys CH RFP2, c13ORF1, and FAM10A4 are the most likely tumor suppressor gene candidates for B-cell chronic lymphocytic leukemia.Cancer Genet Cytogenet. 2003; 146: 48-57Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar myelofibrosis,14Reilly JT Cytogenetic and molecular genetic aspects of idiopathic myelofibrosis.Acta Haematol. 2002; 108: 113-119Crossref PubMed Scopus (21) Google Scholar melanoma,15Calin GA Trapasso F Shimizu M Dumitru CD Yendamuri S Godwin AK Ferracin M Bernardi G Chatterjee D Baldassarre G Rattan S Alder H Mabuchi H Shiraishi T Hansen LL Overgaard J Herlea V Mauro FR Dighiero G Movsas B Rassenti L Kipps T Baffa R Fusco A Mori M Russo G Liu CG Neuberg D Bullrich F Negrini M Croce CM Familial cancer associated with a polymorphism in ARLTS1.N Engl J Med. 2005; 352: 1667-1676Crossref PubMed Scopus (105) Google Scholar prostate cancer,16Lu W Takahashi H Furusato M Maekawa S Nakano M Meng C Kikuchi Y Sudo A Hano H Allelotyping analysis at chromosome 13q of high-grade prostatic intraepithelial neoplasia and clinically insignificant and significant prostate cancers.Prostate. 2006; 66: 405-412Crossref PubMed Scopus (14) Google Scholar laryngeal cancer,17Schlade-Bartusiak K Stembalska A Ramsey D Significant involvement of chromosome 13q deletions in progression of larynx cancer, detected by comparative genomic hybridization.J Appl Genet. 2005; 46: 407-413PubMed Google Scholar colon cancer18Diep CB Kleivi K Ribeiro FR Teixeira MR Lindgjaerde OC Lothe RA The order of genetic events associated with colorectal cancer progression inferred from meta-analysis of copy number changes.Genes Chromosomes Cancer. 2006; 45: 31-41Crossref PubMed Scopus (147) Google Scholar and non-squamous cell lung cancer.19Huang Y Yang HJ Jin Y Li HM Fu SB [13q14 aberration is related to the metastatic potential of human NSCLC].Yi Chuan. 2005; 27: 531-534PubMed Google ScholarSeveral methods for the detection and characterization of chromosome 13q deletions have been described. These include metaphase cytogenetics,10Tricot G Barlogie B Jagannath S Bracy D Mattox S Vesole DH Naucke S Sawyer JR Poor prognosis in multiple myeloma is associated only with partial or complete deletions of chromosome 13 or abnormalities involving 11q and not with other karyotype abnormalities.Blood. 1995; 86: 4250-4256PubMed Google Scholar interphase fluorescence in situ hybridization (FISH),20Kaufmann H Kromer E Nosslinger T Weltermann A Ackermann J Reisner R Bernhart M Drach J Both chromosome 13 abnormalities by metaphase cytogenetics and deletion of 13q by interphase FISH only are prognostically relevant in multiple myeloma.Eur J Haematol. 2003; 71: 179-183Crossref PubMed Scopus (35) Google Scholar comparative genome hybridization (CGH)4Liebisch P Viardot A Bassermann N Wendl C Roth K Goldschmidt H Einsele H Straka C Stilgenbauer S Dohner H Bentz M Value of comparative genomic hybridization and fluorescence in situ hybridization for molecular diagnostics in multiple myeloma.Br J Haematol. 2003; 122: 193-201Crossref PubMed Scopus (44) Google Scholar and array-based CGH.21Largo C Saez B Alvarez S Suela J Ferreira B Blesa D Prosper F Calasanz MJ Cigudosa JC Multiple myeloma primary cells show a highly rearranged unbalanced genome with amplifications and homozygous deletions irrespective of the presence of immunoglobulin-related chromosome translocations.Haematologica. 2007; 92: 795-802Crossref PubMed Scopus (34) Google Scholar These techniques, although useful, have several limitations. Metaphase cytogenetics requires the presence of a dividing cell population. Since terminally differentiated plasma cells have a low proliferative capacity, conventional cytogenetics is only informative in approximately one third of cases.22Sawyer JR Waldron JA Jagannath S Barlogie B Cytogenetic findings in 200 patients with multiple myeloma.Cancer Genet Cytogenet. 1995; 82: 41-49Abstract Full Text PDF PubMed Scopus (331) Google Scholar FISH and CGH have a higher detection rate,4Liebisch P Viardot A Bassermann N Wendl C Roth K Goldschmidt H Einsele H Straka C Stilgenbauer S Dohner H Bentz M Value of comparative genomic hybridization and fluorescence in situ hybridization for molecular diagnostics in multiple myeloma.Br J Haematol. 2003; 122: 193-201Crossref PubMed Scopus (44) Google Scholar,20Kaufmann H Kromer E Nosslinger T Weltermann A Ackermann J Reisner R Bernhart M Drach J Both chromosome 13 abnormalities by metaphase cytogenetics and deletion of 13q by interphase FISH only are prognostically relevant in multiple myeloma.Eur J Haematol. 2003; 71: 179-183Crossref PubMed Scopus (35) Google Scholar but may not identify cases where the affected area of chromosome is very small. Array-CGH is a highly sensitive technique that can detect very small regions of deletion, however, it is acknowledged to have limited sensitivity for the detection of aberrations present in a low percentage of cells.23Oostlander AE Meijer GA Ylstra B Microarray-based comparative genomic hybridization and its applications in human genetics.Clin Genet. 2004; 66: 488-495Crossref PubMed Scopus (149) Google Scholar,24Schwaenen C Nessling M Wessendorf S Salvi T Wrobel G Radlwimmer B Kestler HA Haslinger C Stilgenbauer S Dohner H Bentz M Lichter P Automated array-based genomic profiling in chronic lymphocytic leukemia: development of a clinical tool and discovery of recurrent genomic alterations.Proc Natl Acad Sci USA. 2004; 101: 1039-1044Crossref PubMed Scopus (202) Google Scholar Since MM samples typically consist of a heterogeneous population of normal and malignant cells, the identified techniques may not be suitable in cases where the myeloma load is low. In MM, therefore, the minimally deleted region on 13q has hitherto remained poorly defined.We report here the novel application of digital single nucleotide polymorphism (dSNP) analysis25Zhou W Goodman SN Galizia G Lieto E Ferraraccio F Pignatelli C Purdie CA Piris J Morris R Harrison DJ Paty PB Culliford A Romans KE Montgomery EA Choti MA Kinzler KW Vogelstein B Counting alleles to predict recurrence of early-stage colorectal cancers.Lancet. 2002; 359: 219-225Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar for the characterization of 13q LOH status in a panel of 22 MM cases. We have previously shown that dSNP compares well with established molecular techniques, including FISH and multiplex ligation-dependent probe amplification, for the detection of the chromosome aberrations resulting in LOH (In press: JMD08-0167). Digital SNP allows detection of LOH through the direct counting of alleles. The technique relies on the identification of heterozygous single nucleotide polymorphisms (SNPs) in patient samples and the subsequent separation of the heterozygous alleles through the serial dilution of patient DNA. PCR amplification of the heterozygous alleles is performed and the allelic frequencies counted. Deviation from the expected 50:50 ratio for heterozygous alleles represents LOH at that specific locus and is highly suggestive of a deletion. Sequential probability ratio testing26Piantadosi SS Clinical Trials. Wiley-Interscience, New York1997: 236-237Google Scholar confirms the significance of such deviations. Digital SNP technology is applicable to both fresh and archival material, and can be used on samples regardless of proliferative state.The aim of this study was to employ a panel of SNP probes to characterize 13q14 LOH status in archival tissue from a cohort of 22 patients with MM.Materials and MethodsSubjectsTwenty-two patients with MM, diagnosed and treated at the Royal Devon and Exeter Hospital were included in this study. The study protocol had received prior approval by the local research ethics committee and all patients provided written informed consent in accordance with the Declaration of Helsinki. The panel of 22 MM anonymized cases (MM01 to MM22) was collected from 2002 to 2007. All patients were over 16 years old and had an established diagnosis of MM with documented bone marrow involvement.Assessment of Myeloma LoadThe myeloma load in each sample was determined by CD138 staining of two 2 μmol/L sections framing the ten thick sections (10 μmol/L) used for DNA extraction. Assessment of the myeloma load was performed by a hematopathologist.DNA Extraction and QuantificationDNA was extracted from 10 paraffin sections of 10 μmol/L thickness using a modified protocol of the Qiagen QIAamp tissue kit (Qiagen, Crawley, UK).27Wickham CL Boyce M Joyner MV Sarsfield P Wilkins BS Jones DB Ellard S Amplification of PCR products in excess of 600 base pairs using DNA extracted from decalcified, paraffin wax embedded bone marrow trephine biopsies.Mol Pathol. 2000; 53: 19-23Crossref PubMed Scopus (52) Google Scholar DNA samples were quantified using a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington) and assessed for integrity using the BIOMED 2 specimen control size ladder, which yields products of 84, 96, 200, 300, 400, and 600 bp (Invivoscribe, San Diego, CA).Assay Design and ValidationA panel of 11 SNP markers that covered the region of 13q12 to 13q34 was chosen on the basis of high population heterozygosity (0.3 to 0.5). Probes and primers for these SNPs were designed by the Applied Biosystems Assays-on-Demand service (Applied Biosystems, Foster City, CA) (Table 1). In each case, allele specific probes were labeled 5′ with either 6-FAM or VIC and 3′ with a minor groove binding protein serving as a non-fluorescent quencher. Assays were validated using standard curves generated from seven (1:2) serial dilutions of heterozygous DNA to ensure equivalent amplification and probing efficiency, and by amplification of homozygous individuals of both genotypes to ensure that background fluorescence was within acceptable limits.Table 1Sequences of Probes and Primers Used for Digital SNP AnalysisNameSequenceLabelsRB-1 F5′-GTTGCTGGACAGCCTATGGAT-3′NONERB-1 R5′-AAGGAATTATACCAAAGCAGCTAACTGAA-3′NONERB-1 P(1)5′-TCAGGTGACTATCTTTTGT-3′5′-VIC,3′-MGBRB-1 P(2)5′-CAGGTGACTATGTTTTGT-3′5′6-FAM,-3′- MGBARLTS1 F5′-AGCCCCTCCCCAGTGA-3′NONEARLTS1 R5′-CAGGCTGAGTCTAGAGAGATTCCA-3′NONEARLTS1 P(1)5′-TCCCAGCAGTGGTC-3′5′-VIC,3′-MGBARLTS1 P(2)5′-CCCAGCGGTGGTC-3′5′-6-FAM,3′-MGBEBPL F5′-CCCACCCTTCTACTTTTCTGGATTT-3′NONEEBPL R5′-CACCAGCCGGCTTAAAGC-3′NONEEBPL P(1)5′-AAATGGAAAGATTGTCAGAGTA-3′5′-VIC,3′-MGBEBPL P(2)5′-ATGGAAAGATTGTCGGAGTA-3′5′-6-FAM,3′-MGBC13Orf1 F5′-CTCATTAGATCCAATCCCACTGCTT-3′NONEC13Orf1 R5′-CCACTGTAAATGACTAAATTTGAACTGCTT-3′NONEC13Orf1 P(1)5′-AAACTCCCAAACAAAG-3′5′-VIC,3′-MGBC13Orf1 P(2)5′-AACTCCCAGACAAAG-3′5′-6-FAM,3′-MGBKCNRG F5′-GATCTCTCAAAAATTGATGAGTGTTGGTT-3′NONEKCNRG R5′-TCGAGACCTTGCTGTGATTCTG-3′NONEKCNRG P(1)5′-AAAGTCATTTGAATTCC-3′5′-VIC,3′-MGBKCNRG P(2)5′-AAGTCATTTCAATTCC-3′5′-6-FAM,3′-MGBDLEU2 F5′-ACTTCCCTACCTATTCTCCTGGAAAA-3′NONEDLEU2 R5′-GGAGCTAGAGGCATTTTGTAAGCAA-3′NONEDLEU2 P(1)5′-AATCAAGAAATGACACTTT-3′5′-VIC,3′-MGBDLEU2 P(2)5′-CAATCAAGAAATTACACTTT-3′5′-6-FAM,3′-MGBDLEU1 F5′-GCCAGTGTCTAAACTCCAAACAAC-3′NONEDLEU1 R5′-GGCGGTTTTAAATGCACGTGTATC-3′NONEDLEU1 P(1)5′-CAATGAGACCTAGTATATG-3′5′-VIC,3′-MGBDLEU1 P(2)5′-CAATGAGACCTTGTATATG-3′5′-6-FAM,3′-MGBFAM10A4 F5′-CTACCAGCAGGTGTCATTATGGA-3′NONEFAM10A4 R5′-TTTCTTTCTCACAGCTAGTTCCTACAAC-3′NONEFAM10A4 P(1)5′-TCTGGAATTTACAGTATTTT-3′5′-VIC,3′-MGBFAM10A4 P(2)5′-CTGGAATTTACACTATTTT-3′5′-6-FAM,3′-MGBDLEU7 F5′-GGGTCTTGAAAGAAAAGTACAGAGCTA-3′NONEDLEU7 R5′-CCACTCAGTTTTCCCACACCTAA-3′NONEDLEU7 P(1)5′-AAAGAAAGGCATCCCCCCAG-3′5′-VIC,3′- MGBDLEU7 P(2)5′-AAAGGCATCGCCCCAG-3′5′-6-FAM,3′- MGBSLC15A1 F5′-GCTGCTGATTTCAGTGGAGACA-3′NONESLC15A1 R5′-TTTTGGATTAATCACCTCCCAGCTT-3′NONESLC15A1 P(1)5′-TCTAGATGCAAGTATCTG-3′5′-VIC,3′-MGBSLC15A1 P(2)5′-TAGATGCAAATATCTG-3′5′-6-FAM,3′-MGBUBAC2 F5′-GAATCCTAAGATGCTTAATTTTGTAAGTTTGCA-3′NONEUBAC2 R5′-GGTGCATGATGAGCAGTGAAAA-3′NONEUBAC2 P(1)5′-CTCGGATCATATTTAG-3′5′-VIC,3′-MGBUBAC2 P(2)5′-CTCGGATCGTATTTAG-3′5′-6-FAM,3′-MGBProbes are labeled 5′ with 6-FAM or VIC and 3′with a minor groove binding protein (MGB) to enhance probe specificity. Nucleotides representing the SNPs under investigation are highlighted in bold. Open table in a new tab dSNP ValidationTo determine the validity of the approach, an artificial cell mixture was made using known amounts of homozygous DNA diluted in DNA from an unrelated heterozygous individual. Briefly, DNA samples were diluted to 2.5 mmol/L, and plated out on a 96 well real-time PCR plate. PCR reactions were performed in a total volume of 8 μl including 4 μl universal master mix (no AMPerase) (Applied Biosystems), assay mix (0.9 μmol/L probe, 1.8 μmol/L each primer) and 2.5pg/μl DNA. PCR cycles were 50°C for 2 minutes and 95°C for 10 minutes, followed by 60 cycles of 95°C for 15 seconds, and 60°C for 1 minute. Reactions were performed on the ABI Prism 7000 platform. Following real-time amplification, appropriate baseline and threshold levels were set and samples were designated as allele 1, allele 2, allele 1 + 2 or no product detected.Initial Assessment of Sample GenotypeAll 22 MM samples were amplified by real-time PCR to identify individuals heterozygous for each SNP. PCR was performed in 10 μl reactions including 5 μl universal master mix, no AMPerase) (Applied Biosystems), assay mix (0.9 μmol/L probe, 1.8 μmol/L each primer) and 40 ng of DNA. PCR cycles were 50°C for 2 minutes and 95°C for 10 minutes, followed by 60 cycles of 95°C for 15 seconds and 60°C for 1 minute. Following amplification, the genotype of each sample was determined for each SNP as outlined above.Calculation of Optimal DNA DilutionThe optimal DNA concentration to yield good separation of alleles was calculated from the average molecular weight of a mole of base (342 g), the number of molecules in a mole of substance (the Avogadro constant; 6.023 × 1023) and the number of bases in the human genome (3 × 109). However, since this figure takes no account of the presence of PCR inhibitors or DNA degradation in the sample, optimal dilution factors were further determined empirically.dSNP AnalysisOptimally diluted DNA samples were plated out in ABI optical plates and amplified on the ABI Prism 7000 platform. PCR reactions were performed in a total volume of 8 μl including 4 μl universal master mix (no AMPerase) (Applied Biosystems), assay mix (0.9 μmol/L probe, 1.8 μmol/L each primer) and 2 μl appropriately diluted DNA. PCR cycles were 50°C for 2 minutes and 95°C for 10 minutes, followed by 60 cycles of 95°C for 15 seconds and 60°C for 1 minute. Following amplification, each well was genotyped as outlined above.Sequential Probability AnalysisLikelihood analysis was used to assess the strength of evidence for deviation from a 50:50 distribution of alleles. The specific method used was the sequential probability ratio testing, as previously described.25Zhou W Goodman SN Galizia G Lieto E Ferraraccio F Pignatelli C Purdie CA Piris J Morris R Harrison DJ Paty PB Culliford A Romans KE Montgomery EA Choti MA Kinzler KW Vogelstein B Counting alleles to predict recurrence of early-stage colorectal cancers.Lancet. 2002; 359: 219-225Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar This technique allows two probabilistic hypotheses to be compared as data accumulates. A sample with no LOH is expected to show allelic balance, with the two heterozygous alleles appearing at approximately the same frequency (null hypothesis H0) (Figure 1A). A sample with LOH will have allelic imbalance (alternative hypothesis H1) with over-representation of one allele (Figure 1B). The degree of imbalance will be directly proportional to the number of clonal plasma cells that are present in the sample and that harbor the aberration. Threshold curves representing the two hypotheses are generated for a specific tumor load based on a predetermined likelihood ratio. Analysis is concluded by plotting the number of informative alleles tested against the major allele ratio (Figure 2). If the point is plotted above the upper threshold curve, it is considered as evidence of allelic imbalance and hence LOH at that locus. Points plotted below the lower threshold curve are categorized as having allelic balance and therefore no LOH. Points falling between the threshold curves were analyzed as previously described.28Zhou W Galizia G Lieto E Goodman SN Romans KE Kinzler KW Vogelstein B Choti MA Montgomery EA Counting alleles reveals a connection between chromosome 18q loss and vascular invasion.Nature Biotechnol. 2001; 19: 78-81Crossref Scopus (670) Google Scholar Briefly, dSNP experiments were performed until the number of informative alleles exceeded 150. The mid-point value between the two hypotheses was then calculated for the given tumor load. When the major allele ratio exceeded the mid-point value it was considered as evidence of LOH, while ratios falling below the mid-point value were assigned to the no LOH category.Figure 2Sequential Probability analysis for detection of loss of heterozygosity (LOH) at 13q. The statistical evidence that an observed skew in allele frequency represents LOH is assessed by plotting the major allele frequency on the y axis against the number of alleles tested on the x axis. Threshold curves are calculated for a given tumor load based on the likelihood ratio of 8. Points plotted above the upper curve represent LOH, while those below the lower curve represent no LOH (see text for further explanation). The different curves represent different ratios of non-neoplastic to neoplastic cells as follows. (A) = 50:50 mixture; (B) = 60:40 mixture; (C) = 70:30 mixture; (D) = 80:20 mixture.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A likelihood ratio of >8 was accepted as evidence of LOH since it provides a posttest probability of LOH of 89%, if the pre-test probability of the presence of LOH at 13q is assumed to be 50%. Since sequential probability ratio testing allows for repeat testing, the posttest probability of LOH can be increased further by testing each sample with several allelic probes.The proportions P of the major allele for a likelihood ratio of 8 for the presence of LOH in test samples with varying myeloma cell infiltrations were calculated according to the formula P = log (16)/(n*log[(100-m)/100]) + log([100-m]/[100-m/2])/log([100-m]/100), where n = total number of alleles counted and m = the percentage of myeloma infiltration in the biopsy. The results of the equation were tabulated with Microsoft Excel 2000 software.Confirmation of Results with FISHIn nine cases, suitable samples were available for FISH analysis of chromosome 13q. FISH analysis was performed by Wessex Regional Cytogenetics Laboratory, according to the methods outlined by Ross et al.29Ross FM Ibrahim AH Vilain-Holmes A Winfield MO Chiecchio L Protheroe RK Strike P Gunasekera JL Jones A Harrison CJ Morgan GJ Cross NC Age has a profound effect on the incidence and significance of chromosome abnormalities in myeloma.Leukemia. 2005; 19: 1634-1642Crossref PubMed Scopus (65) Google Scholar The method employs two probes (RB1 and D13S319) mapping at chromosome band 13q14.ResultsDNA Extraction and QuantificationDNA was extracted from paraffin sections as described above. The DNA samples were shown to be suitable for PCR by the amplification of the 400 ± 600 bp product
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