Loss Of Heterozygosity Analysis At 6q16-q23 In Pathogenesis Of Non-Hodgkin Lymphomas

Deletion or inactivation of tumor suppressor genes (TSGs) plays a significant role in the tumorgenesis. Cytogenetic researches indicate that non-random deletions of chromosomal segments, where tumor-related genes may harbor, happen in almost all tumor cells. Loss of heterozygosity (LOH) involves the mutation of one allele together with the loss of the second allele in a biallelic locus. LOH of tumor suppressor genes may results in the development of cancers. LOH analysis with high polymorphic microsatellite markers on chromosome is a powerful technique for identifying chromosomal deletion and tumor suppressor genes. It is well reported that deletions of the long arm of chromosome 6 (6q) are frequent chromosomal aberrations in several solid tumors including ovarian carcinoma (6ql3, 6q21, 6q24-27), hepatic carcinoma (6ql4-22, 6q26-27), glioma (6ql6.3-21, 6qtel). Deletions of 6q are also frequent in hematologic neoplasms such as lymphoma and are associated with an adverse clinical outcome. It implicates the presence of tumor suppressor genes within this chromosomal segment. Three regions of minimal deletion (RMD) have been identified at 6q21, 6q23 and 6q25-27 in lymphoma. This study focused on LOH analysis at 6ql6-q23 with eight microsatellite markers in an attempt to identify candidate tumor suppressor genes within this region involved in the development of non-Hodgkin lymphoma (NHL). Meanwhile, protein expressions of FYN, CX43 and CD24 genes located at 6ql6-q23 and lymphoma-related genes Bcl-2 and Bcl-6 were detected by immunohistochemistrical method. Cell proliferate index of Ki-67 was also calculated. Then we analysed relationship between LOH and known genes at 6ql6-q23, explored unknown genes and evaluated their possible roles in the pathogenesis of NHLs.Materials and Methods1. Materials: Cases of NHL were obtained from our affiliated hospitals and other local hospitals from the year of 1997 to 2003. There were 39 cases used for detection of LOH and 61 cases (including 35 case in formal 39 case sample) used for analysis of immunohistochemistry. All specimens were fixed in 10% formalin and then embedded in paraffin. Sections were stained by hemotoxylin and eosin and by immunohistochemistry. All cases were divided into T- and B- NHL on the basis of their immunophenotypes and morphologies according to the WHO classification. Of 39 and 61 case sample of NHLs respectively, there were 15 and 27 extranodal marginal zone B-cell lymphomas of MALT type (MALT-MZL), 13 and 19 diffuse large B-cell lymphomas (DLBCL), 6 and 7 follicular lymphomas (FL). 5 and 7 cases were T-cell NHLs (T-NHL).The selected immune markers were as follows: ① markers of lymphocyte immunophenotypes: LCA(2B11, DAKO), CD20(L26, DAKO), CD45RO(OPD4, DAKO); ② markers of 6q16-q23-related genes: FYN(F9500-04, Santa Cruz), CD24(C-20, Santa Cruz), CX43(D-7, Chemincom) ; ③ markers of lymphoma-related genes: Bcl-2(124, DAKO), Bcl-6(PG-B6P, Santa Cruz); ④cell proliferate marker: Ki-67(Ki-S5, DAKO). 2. Methods: (1) Genomic DNAs from tumor tissues and corresponding normal tissues were prepared by classical proteinase K digestion and phenol-chloroform extraction method. (2) Eight microsatellite markers spanning 6ql 6-q23 were selected. Optimized PCRs were performed using proper primers. Then the PCR products were pre-denaturated at 95℃ for 5 minutes and applied onto 8%urea-formamide-polyacrylamide gels for electrophoresis. The gels were stained by silver. Intensities of biallelic stain in tumor tissues were observed by comparing that in corresponding normal tissues to recognize LOH. (3) Those genes located near or between LOH-positive markers were selected for evaluation of protein expression in tumor tissues and corresponding normal tissues. The selected genes could be detected in paraffin-embedded tissues by immunohistochemistry and they were FYN, CD24 and CX43. (4) Furthermore, expression of Bcl-2, Bcl-6, and Ki-67 proteins was detected by immunohistochemistrical staining with S-P method in 61 cases. The protocols were performed under the direction of kit instru...