Evaluation of 13q14 Status in Multiple Myeloma by Digital Single Nucleotide Polymorphism Technology

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 malignancies¹ and has an extremely variable prognosis, with survival ranging from a few months to more than 10 years.² 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.³ 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,⁴ and translocations involving 14q32.⁵ Deletion of chromosome 13q is one of the most frequent cytogenetic abnormalities associated with MM, occurring in 40% to 50% of cases.^4,6,7 The majority of deletions involve entire chromosomes or chromosome arms,^8,9 although partial and interstitial deletions have also been described.^10,11 Where interstitial deletions are present, they most commonly involve chromosome band 13q14 to 13q 21.^10,12The presence of chromosome 13q deletions has been suggested to be an adverse prognostic factor in MM.^6,10 This has lead several groups to hypothesize that the 13q14 region may harbor one or more as yet uncharacterized tumor suppressor genes.^6,10 This region of chromosome 13 has not only been associated with tumorigenesis in MM⁶ but also in chronic lymphocytic leukemia,¹³ myelofibrosis,¹⁴ melanoma,¹⁵ prostate cancer,¹⁶ laryngeal cancer,¹⁷ colon cancer¹⁸ and non-squamous cell lung cancer.¹⁹Several methods for the detection and characterization of chromosome 13q deletions have been described. These include metaphase cytogenetics,¹⁰ interphase fluorescence in situ hybridization (FISH),²⁰ comparative genome hybridization (CGH)⁴ and array-based CGH.²¹ 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.²² FISH and CGH have a higher detection rate,^4,20 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.^23,24 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) analysis²⁵ 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 testing²⁶ 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.