Fluorescence In Situ Hybridization on Enriched CD138-Positive Cells in Plasma Cell Myeloma2015-07-01 00:01:25
ACTA MEDICA INTERNATIONAL; July 2015: DOI:10.5530/ami.2015.5.0
Yu Shi, Charalambos Solomides, Gerald Gong, Zi-Xuan (Zoe) Wang, GuldeepUppa, Vandi Ly, Stephen Peiper, Peter Herbut, Renu Bajaj
To validate plasma cell enrichment technique for improving the detection of cytogenetic abnormalities in the Plasma cell myeloma (PCM)/multiple myeloma (MM). We compared the abnormality detection rate for overnight unstimulated bone marrow cultures to that for the plasma cell enriched fractions obtained with the use of CD138-coated immunomagnetic beads. Average enrichment factor (EF) was 11. One or more abnormalities were detected in 90% of enriched samples vs. 65% of non-enriched samples, thus resulting in a significantly higher detection rate of total cytogenetic abnormalities in enriched plasma cells (p=0.0038). Additional findings of RB1 deletion, TP53-, 1p-, 1q+ and IGH@ rearrangement seen in the 25% of enriched samples could contribute to the altered risk in the patient. One of the three cases with plasma cells as low as 1% by morphology was positive for a residual disease marker in the enriched sample and negative in the non-enriched sample. The plasma cell enrichment technique increased the detection rate of diagnostic and prognostic markers and is a very sensitive method for detecting minimal residual disease.
Plasma Cell Myeloma/multiple myeloma (PCM/MM), is a bone marrow based multifocal plasma cell neoplasm associated with an M protein in serum or urine and
disseminated marrow involvement. Myeloma is the second most common hematopoietic malignancy with 22,350 new cases and 10,710 deaths in US in 2013 according to data of National Cancer Institute. Myeloma spans a clinical spectrum from asymptomatic to highly aggressive disease. Staging for myeloma does not use the tumor size, lymph node, metastasis (TNM) system. The International Myeloma Working Group diagnostic criteria is commonly used. The criteria for Symptomatic myeloma include: M–protein ≥ 30 g/L and/or bone marrow clonal cells ≥ 10% and must have evidence of end-organ damage that can be attributed to the plasma cell proliferative process; manifested by CRAB (calcium, renal failure, anemia, and bone lesions). The immunophenotype for myeloma (malignant) cells are CD38+, CD138+, CD56+ and CD19–, CD45– or CD45lo, which are different from healthy (benign) bone marrow plasma cells with CD38+, CD138+, CD19+, CD45+, CD56–. Despite recent therapeutic advances, myeloma remains as an incurable tumor with median survival of 6 years. Cytogenetic studies have revealed various chromosome abnormalities which contribute to stratify myeloma into standard and high-risk for drug resistance. There are several diagnostic challenges in identifying cytogenetic abnormalities by interphase FISH, including small sample size and low proportion of disease PCs in the hemodilute BM that can lead to false negative results. In accordance with the International Myeloma Working group (IMWG) recommendations, FISH should be carried out on nuclei from purified plasma cells. There are two available methods to isolate cells: fluorescence activated cell sorting (FACS) and magnetic activated cell sorting (MACS) using surface cell markers. Four studies have been published to use isolation technique enriching plasma cells with improved detection of genetic abnormalities in myeloma bone marrow samples.
Among them, one study is based on FACS, the latest 3 studies have switched to MACS instead. Compared to FACS, MACS is a convenient, high-throughput, time-saving, and less costly method for sample enrichment.
In this study we investigated to validate an immunomagnetic positive cell selection protocol for the isolation of CD138 positive myeloma plasma cells to enhance diagnostic sensitivity of interphase FISH analysis by concentrating plasma cells in the sample.
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Myeloma | plasma cell | cytogenetic | enrichment | Fluorescence In Situ Hybridization (FISH)