Contributions
Abstract: PB2140
Type: Publication Only
Background
Chromosomal abnormalities play an important role in prognostic stratification of Multiple Myeloma (MM). Current technology used to evaluate such genetic abnormalities is Fluorescence In Situ Hybridization (FISH). Nevertheless, it is difficult for FISH to screen all lesions simultaneously, due to high costs and technique limitations. Multiplex Ligation-dependent Probe Amplification (MLPA) is a polymerase chain reaction that permits the amplification of multiple targets by a single reaction. This technique is useful to detect Copy Number Variations (CNVs), but it is unable to find out balanced translocations. Furthermore, MLPA analysis detects abnormalities only if they are present in 30-40% of pathological cells.
Aims
In this study we evaluated the MLPA technique in the identification of chromosomal abnormalities occurring in MM patients, comparing the results with those obtained by FISH.
Methods
Cohort study included 24 MM patients (male to female ratio 15/9, median age 69 years, range 53-85 years). Bone marrow samples were taken in line with good medical practice. After evaluating the presence and percentage of plasma cells with optical microscopy, a sample part of bone marrow was used for the FISH assay. MLPA analysis (SALSA MLPA P425-B1 MM probemix, MRC-Holland, Amsterdam, Netherlands) was conducted on DNA extracted from marrow aspirates with commercially kits. PCs were purified from MM samples using CD138 microbeads and magnet-assisted cell sorting (MiltenyiBiotec).We enriched 7/24 (29%) samples. The PCR products were initially analyzed using ABI 3130 Genetic analyzer (Applied Biosystems), later using SeqStudio Genetic Analyzer (Applied Biosystems).
Results
FISH analysis detected genetic alterations in 20 of 24 patients tested for assay: 13q deletion was the most frequently genetic aberration (9/20) followed by 17p deletion (3/20) and 1q amplification (3/20). Other genetic alterations included 1p deletion in 1patient and a deletion of Y chromosome in another one. Out of these 20 patients, 16 were positive for IGH rearrangements. Overall, out of 24 patients, 12 cases were positive for CNVs. We could identify genetic alteration in 10/24 multiple samples by using MLPA. The most frequently genetic aberrations included 13q deletion (6/10) followed by 1q amplification (4/10). There was 1 positive case for chromosome 1p deletion, while the chromosome 17p deletion was positive in 1 patient. Interestingly, we also found 2 patients with chromosome 5q amplification, 2 with 15q amplification, 1 with 12p deletion and 1 with Y chromosome deletion. Overall, concordant results were detected in 18/24 (75%) cases. The discordant results were 6/24 (25%). Out of these 6 cases, MLPA analysis were conducted on DNA isolated from CD138-enriched plasma cells in 2 patients, while MLPA tests were made on DNA isolated from whole bone marrow mononuclear cells in 4 patients.
Conclusion
In this study there was a consistent concordance between FISH and MLPA for detection of CNVs. However, some discrepancies emerged, which were probably attributable to the non-isolation of CD138+ cells in some samples. MLPA can’t detect balanced aberrations, but identified some additional cytogenetic abnormalities that are not usually investigated by a standard FISH approach; therefore, taken together, MLPA and FISH represent complementary techniques, both useful to find cytogenetic aberrations in MM patients. The study is still recruiting new patients.
Session topic: 13. Myeloma and other monoclonal gammopathies – Biology & Translational Research
Keyword(s): Cytogenetics, FISH, Multiple Myeloma
Abstract: PB2140
Type: Publication Only
Background
Chromosomal abnormalities play an important role in prognostic stratification of Multiple Myeloma (MM). Current technology used to evaluate such genetic abnormalities is Fluorescence In Situ Hybridization (FISH). Nevertheless, it is difficult for FISH to screen all lesions simultaneously, due to high costs and technique limitations. Multiplex Ligation-dependent Probe Amplification (MLPA) is a polymerase chain reaction that permits the amplification of multiple targets by a single reaction. This technique is useful to detect Copy Number Variations (CNVs), but it is unable to find out balanced translocations. Furthermore, MLPA analysis detects abnormalities only if they are present in 30-40% of pathological cells.
Aims
In this study we evaluated the MLPA technique in the identification of chromosomal abnormalities occurring in MM patients, comparing the results with those obtained by FISH.
Methods
Cohort study included 24 MM patients (male to female ratio 15/9, median age 69 years, range 53-85 years). Bone marrow samples were taken in line with good medical practice. After evaluating the presence and percentage of plasma cells with optical microscopy, a sample part of bone marrow was used for the FISH assay. MLPA analysis (SALSA MLPA P425-B1 MM probemix, MRC-Holland, Amsterdam, Netherlands) was conducted on DNA extracted from marrow aspirates with commercially kits. PCs were purified from MM samples using CD138 microbeads and magnet-assisted cell sorting (MiltenyiBiotec).We enriched 7/24 (29%) samples. The PCR products were initially analyzed using ABI 3130 Genetic analyzer (Applied Biosystems), later using SeqStudio Genetic Analyzer (Applied Biosystems).
Results
FISH analysis detected genetic alterations in 20 of 24 patients tested for assay: 13q deletion was the most frequently genetic aberration (9/20) followed by 17p deletion (3/20) and 1q amplification (3/20). Other genetic alterations included 1p deletion in 1patient and a deletion of Y chromosome in another one. Out of these 20 patients, 16 were positive for IGH rearrangements. Overall, out of 24 patients, 12 cases were positive for CNVs. We could identify genetic alteration in 10/24 multiple samples by using MLPA. The most frequently genetic aberrations included 13q deletion (6/10) followed by 1q amplification (4/10). There was 1 positive case for chromosome 1p deletion, while the chromosome 17p deletion was positive in 1 patient. Interestingly, we also found 2 patients with chromosome 5q amplification, 2 with 15q amplification, 1 with 12p deletion and 1 with Y chromosome deletion. Overall, concordant results were detected in 18/24 (75%) cases. The discordant results were 6/24 (25%). Out of these 6 cases, MLPA analysis were conducted on DNA isolated from CD138-enriched plasma cells in 2 patients, while MLPA tests were made on DNA isolated from whole bone marrow mononuclear cells in 4 patients.
Conclusion
In this study there was a consistent concordance between FISH and MLPA for detection of CNVs. However, some discrepancies emerged, which were probably attributable to the non-isolation of CD138+ cells in some samples. MLPA can’t detect balanced aberrations, but identified some additional cytogenetic abnormalities that are not usually investigated by a standard FISH approach; therefore, taken together, MLPA and FISH represent complementary techniques, both useful to find cytogenetic aberrations in MM patients. The study is still recruiting new patients.
Session topic: 13. Myeloma and other monoclonal gammopathies – Biology & Translational Research
Keyword(s): Cytogenetics, FISH, Multiple Myeloma