Multiple Myeloma Molecular Cytogenetic Techniques And Cytogenetic Abnormalities

Part I Fluorescence In Situ Hybridization on Bone Marrow Slides for the Investigation of Cytogenetic Aberrations of Multiple MyelomaObjective To establish the technique of interphase fluorescence in situ hybridization (I-FISH) used directly on bone marrow smear, to develop a new method for detection of the molecular cytogenetic abnormalities in multiple myeloma (MM).Methods After a series of treatment,fixation and digestion, using the bone marrow smear as the carrier , the chromosome 8 centromere probe was used in interphase fluorescence in situ hybridization (I-FISH) for detection of molecular cytogenetic abnormalities. Specimens from patients with non-hemotologic malignancies were used as control. The results were compared with those of the conventional I-FISH. Two MM cell lines, Chromosome 8 abnormality in MM cell line including U266 and RPMI-8226, was also being investigated.Results There was no statistically significant difference of the proportion of various signals in non-hematologic malignancies detected with the two methods (P > 0.05). With bone marrow slides I-FISH, 8 out of 19 cases (42.1%) had chromosome 8 abnormalities, including 5 with ?8 (26.3%) and 3 with +8 (15.8%). The U266 cell line had no chromosome 8 number abnormality, RPMI-8226 had +8.Conclusion The I-FISH on the base of smear of bone marrow is characterized by convenience, economy and accuracy. Therefore, it can be used for molecular cytogenetic research in MM. Part II Fluorescence Immunophenotyping and Interphase Cytogenetics as a Tool for the Investigation of Cytogenetic Aberrations of Multiple MyelomaObjective To establish the technique of fluorescence immuno-phenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION) used on smear of bone marrow, to develop a new technique for detection of the molecular cytogenetic abnormalities in multiple myeloma (MM).Methods By using the bone marrow smear as the carries and the anti-CD138 antibody linked with FITC, direct fluorescence staining was applied to mark plasma cells(PCs) and differences were compared in the proportion of both PCs marked by fluorescence staining and PCs detected in morphology. At the same time, the chromosome 8 centromere probe were used in interphase fluorescence in situ hybridization (I-FISH) for detection of the chromosome 8 abnormalities in PCs marked by fluorescence staining. Normal control system was bone marrow smears from 5 non-hemotologic malignant diseases patients.Results There was no significant difference that the proportion of both PCs marked by fluorescence staining and PCs detected in morphology on smear were compared(P > 0.05). 4 out of 9 patients (44%) had the chromosome 8 abnormalities, including 3 cases with ?8(33%) and one case with +8(11% ).Conclusion The FICTION technique on the base of bone marrow smear is characterized by convenience, specificity and accuracy. Therefore , it can be used for molecular cytogenetic research in MM. Part III Immunofluorescence of the Cytoplasmic Light Chain in Situ Hybridization for the Investigation of Cytogenetic Aberrations of Multiple MyelomaObjective To establish the technique of immunofluorescence of the cytoplasmic light chain in situ hybridization (cIg-FISH) used on mononuclearcell smear of bone marrow, to develop a new technique for detection of the molecular cytogenetic abnormalities in multiple myeloma (MM).Methods By using the bone marrow smear as the carrier , the plasma cells(PCs) was applied in interphase fluorescence in situ hybridization (I-FISH) for detection of the chromosome 8 abnormalities and marked with fluorescence staining with either anti-human kappa or lambda light chain conjugated with 7-amino- 4-methylcoumarin-3-acetic acid(AMCA) and with second antibody conjugated with AMCA to enhance the intensity of fluorescence.Results There was visible blue fluorescence in PCs marked by cytoplasmic fluorescence staining after using fluorescence microscope. 7 out of 18 patients (38.9%) had the chromosome 8 abnormalities, including 5 cases with ?8(27.8%) and 2 caseS with +8(11.1% ).Conclusion The cIg-FISH technique on the base of bone marrow mononuclearcell smear is characterized by convenience, specificity and accuracy. Therefore , it can be used for molecular cytogenetic research in MM. Part IV 1q21/CKS1B Abnormality in Multiple MylomaObjective To investigate the 1q21/CKS1B abnormality in multiple myloma (MM).Methods The bone marrow plasma cells of MM patients were purified by anti-CD138 monoclonal antibody binded with magnetic cell sorting system, SpectrumOrange labeled probe for 1q21/ CKS1B and interphase fluorescence in situ hybridization (I-FISH) were applied to detect the abnormality of 1q21/CKS1B in sorted MM cells.Results Among 38 MM patients, the Amp1q21/ CKS1B was observed in 21(55.3%) cases, non-Amp1q21/CKS1B was observed in 17(44.7%)cases , The frequency of Amp1q21/CKS1B was 48% in newly diagnosed MM, and 69.2% in relapsed MM. Amp1q21/CKS1B was associated with bone marrow plasmacytosis (P=0.011), with Hb reduction(P=0.020). Newly diagnosed and relapsed MM had no less than 4 copies of Amp1q21 in 16% and 46.2% patients respectively, however, in those patients with Amp1q21, they had no less than 4 copies of Amp1q21 in 33.3% and 66.7% patients respectively. Amp1q21/ CKS1B was detected by I-FISH in RPMI-8226 and U266 human myeloma cell lines.Conclusion Amp1q21/CKS1B is associated with disease progression. The myeloma cells with at least 4 copies of 1q21 are potentially associated with a more aggressive condition. However, it need further investigation to confirm whether Amp 1q21/CKS1b is an independent prognostic factor.