Combination Of Conventional Cytogenetics And Multiplex Fluorescence In Situ Hybridization To Detect Complex Chromosomal Aberrations In Acute Leukemia

Objective:(1) To set up the technical system of multiplex fluorescence in situ hybridization (M-FISH) technique. (2) To explore the value of combination of conventional cytogenetics, locus specific FISH and M-FISH in the detection of the complex chromosomal aberrations (CCAs) and marker chromosomes in acute leukemia (AL). (3) To find out unreported or very rare abnormalities by reviewing literature and checking the Mitelman database of Chromosome Aberrations in Cancer and to discuss the possible prognostic relevance of these abnormalities.Methods:(1) Bone marrow specimens of patients with acute leukemia were collected, cultured, prepared and R-banded for conventional cytogenetic analysis. (2) M-FISH was performed on specimens of the 11 AL cases whose conventional cytogenetic analysis showed CCAs or marker chromosomes in order to define the unrecognized chromosomal aberrations and the constitution of marker chromosomes, and to identify small and cryptic translocations. (3) Locus specific FISH with specific probes was performed on specimens whose M-FISH results showed or suspected involvement of specific genes, such as MLL amplification or P53 deletion, for verification or exclusion.Results:(1) The M-FISH technical system was successfully established, with all of the 11 M-FISH experiments being successful and with good images. (2) Conventional cytogenetics (CC) showed CCAs and/or marker chromosomes in the 11 AL cases studied. Altogether CC detected 27 numerical and 41 structural chromosomal abnormalities, including 17 marker chromosomes, whose constitution and resources were undetermined. (3) M-FISH analysis determined the resources and chatacteristics of the structural abnormalities undetermined by CC. Among the 27 numerical and 41 structural chromosomal abnormalities detected by CC,3 chromosome gains and 9 chromosome losses as well as 12 structural abnormalities were confirmed by M-FISH. The other 15 chromosome losses detected by CC were revised by M-FISH as derivative chromosomes. M-FISH also detected 3 chromosome gains that were undetected by CC. The other 29 structural abnormalities including 17 marker chromosomes were characterized by M-FISH. The locus specific FISH performed on certain patients further verified the M-FISH results. (4) Results of literature reviewing and database searching:A total of 33 structural abnormalities were detected by M-FISH, among which 6 have never been reported before, i.e. t(5q-;16)(?ql4;q24), der(9)(Y::9::Y::9), der(7) (7::8::9), ins(20;21), der(11) (11::21::20) and der(3)t(3p-;13)(3p-;q21) and most of them resulted from unbalanced translocations. Some of the abnormalities are very rare, with no more than 100 cases reported world wide up to now, including der(15)t(Y;15), dic(16;17), der(12)t(8;12), t(2;4), der(10)t(10;17), der(14)t(14;15), der(12)t(6;12), der(1)t(1;5), der(15)t(1;15), der(17)t(5;17), der(3)t(3p-;13), t(9;15) and der(13)t(10;13). (5) Analyzing the distribution of chromosomes involved in CCA of AL:Almost all chromosomes were involved in CCAs, the more common ones were chromosome 17,5,7,15,11 in AML and 8,9,14,22 in ALL.Conclusion:Not only can M-FISH verify the abnormalities determined by CC, but the technique can also determine the complex chromosomal abnormalities and recognize the resources and constitution of marker chromosomes undetermined by CC. Combination of these two kinds of technique can also help further determine the breakpoint in some patients, raise resolution of karyotyping, and help discover unreported abnormalities and very rare abnormalities, which justifies its clinical application for the detection of CCAs and marker chromosomes in AL.