Fish Coinbilled PCR-Gonescan And Mutational Analysis In The Study Of Molecular Oathological Characteristics Of B-NHL And PRCC

Lymphoma is one of the most common malignant tumors with complex subtypes and variable response to the therapy and prognosis worldwide.The classification diagnosis of lymphoma is always difficult. With the developing of molecular technology,《Pathology&Genetics-Tumours of Haematopoietic and Lymphoid Tissues)) was published in2001and revised as《Tumours of Haematopoietic and Lymphoid Tissues》(4th)by the world healthy organization (WHO) in2008.It is used as the guideline for the clinical and pathological diagnosis of lymphoma, and also offers scientific evidences for the targeted therapy.Immunoglobulin heavy chain (IgH) gene is normally located in14q32.3,expresses in mature B cells with1250kb. In the development of normal B lymphocyte, the four separate coding sequence of IgH gene, including variable area (V), diversity (D), joint (J) and constant region (C) rearrange randomly to form a structural gene.In theory, each B cell carries its unique IgH gene due to the randomness of rearrangement and insert bases. So, the IgH genes from reactive lymphonodes rearrange in polyclonal form, while the rearrangement of IgH gene from malignant B cell lymphoma is monoclonal.Therefore, the monoclone rearrangement of IgH gene can be used as a tumor marker for the differential diagnosis of B cell lymphoma, or the detection of minimal residual lymphoma cells.PCR based GeneScan technique can analyze the length of rearrangement fragments of IgH gene by using fluorescent labeled primers and special analysis software in computer. It is sensitive to detect0.5-1%of lymphocytes carrying clonal rearrangement IgH. Meanwhile, it can distinguish the different length of PCR products by1bp.Extensive data have been collected indicating the association of genetic abnormalities, including chromosomal transpositions, deletions, amplifications and mutations.Most of these abnormities led to the activation of proto-oncogene, disruption of a tumor suppressing-gene, or generation a fusion gene with gain of functional proto-oncogene activity. In most mature B cell tumor, the chromosomal transposition could led to Juxtaposes of oncogenes and the regulatory sequences of normal expressed gene (mostly Ig gene). And this Juxtaposes led to deregulation of translation. For example, t (14,18) IgH/BCL2translocation is characteristic of the majority of follicular lymphoma (FL), which causes BCL-2apoptosis inhibitory protein expression in germinal center-like structure of FL;in DLBCL,variant of chromosomal abnormalities can be seen,40%of which is related to BCL-6gene and20-30%is related to BCL-2gene; and about30%of MALT lymphoma is related to the MALT1gene caused by t(11,18). Fluorescence in situ hybridization (FISH) is a visual and sensitive technique to detect disease-related gene abnormalities, including mutations, deletions and translocations.We detected IgH rearrangement of44B-NHL samples by PCR-agarose gel electrophoresis, of which39cases(88.6%) were detected to have a single band of IgH rearrangement by FR3A primers；28samples (67.6%) were detected as a single band by FR2A primers;40samples (90.9%) were detected as a single band with FR3A and/or FR2A primers. By using PCR-based GcneScan clonal rearrangement comprised56.4%in our study(14monoclonal rearrangement and8biclonal rearrangement) with FR3A primers, non-clonal rearrangement comprises43.6%(7oligoclonal rearrangement and10polyclonal rearrangement).For FR2A primers, clonal rearrangement rate was55.6%(12monoclonal rearrangement and3biclonal rearrangement), and non-clonal rearrangement rate was44.4%(4oligoclonal rearrangement and7polyclonal rearrangement).Combined FR3A and FR2A primers, the clonal rearrangement rate raised to61.6%(17monoclonal rearrangement and7biclonal rearrangement), and non-clonal rearrangement was about38.5%(7oligoclonal rearrangement and8polyclonal rearrangement).We found22cases in35DLBCL were detected carrying IgH broken gene by FISH, the positive rate was62.9%, of which5(5/10) cases was in GCB subtype and17(17/25) cases in non-GCB subtype. BCL-6broken gene were detected in9cases, the positive rate was25.7%, including3cases of GCB subtype (3/10) and6cases of non-GCB subtype (6/25).BCL-2gene break and translocation were detected only in one same case, the positive rate was2.9%. At the same time, we found43%DLBCL cases carrying the gain of BCL-2gene.In addition, MALT lymphoma were detected the MALT1broken gene in25%.And one of two FL samples was detected BCL-2broken gene.Combined the FISH and PCR-GeneScan techniques, we found in15cases with IgH oligoclonal rearrangement or polyclonal rearrangement,8cases were detected the gene abnormality,7of11DLBCL patients carried IgH broken gene, one FL with polyclonal rearrangement was detected the BCL-2broken gene. In addition,4cases of DLBCL which were negative for the detection of IgH rearrangement by PCR, carried IgH broken gene,3of them carried BCL-6broken gene and2patients carried the gain of BCL-2gene. Thus, the positive rate of molecular diagnosis of lymphoma raises from61.5%to81.8%by using combined these two technologies.In conclusion, PCR-agarose gel electrophoresis may use as the first step to detection of IgH rearrangement, and PCR-GeneScan techniques may further define clonal rearrangement types of B-NHL.FISH could use the dual color breakapart probes for the chromosomal abnormalities screening, because of the independence of information of translocation partners, and dual color fusion probes are helpful in the further diagnosis of subtypes of lymphoma. The low rate of IGH/BCL2translocation and the high rate of BCL-2gene amplification of DLBCL may be associated with the low rate of GCB subtype in Chinese DLBCL patients.By FISH and PCR-GeneScan techniques, the positive rate of molecular diagnosis improves from61.5%to81.8%.The combined method is more sensitive than single detection of chromosome abnormality by FISH or single detection of IgH rearrangements.It provides more accurate and scientific evidences for lymphoma diagnosis especially the difficult cases. Anyway, molecular diagnostic techniques must be combined with the traditional morphological, immunohistochemical method and other indicators. Papillary renal cell carcinoma (PRCC) is an independent subtype of kidney cancer, characterized by special morphological and cytological features. PRCC comprise about7%-15%of kidney malignancies.In China, the reported proportion is only5%. Histologically PRCC is characterized by the presence of papillary configuration (>50%).Previous molecular cytogenetic analysis of PRCC demonstrates trisomy of chromosomes7,16, and17and deletion of the Y chromosome are characteristic of PRCC.Occasionally, trisomy of chromosome12,16or20occurs.Several mutations in the tyrosine kinase domain of the met proto-oncogene (MET), which is the receptor of hepatocyte growth factor (HGF), are associated with hereditary and sporadic PRCC. The missense mutations (V11101and H1112R) in MET exon16have been reported in North American and Italy families. These mutations gathered in clusters, which region is critical to tyrosine-kinase regulation.So far, little has been reported about the molecular cytogenetic analysis in a Chinese family with PRCC.In our research, we applied FISH technique to detect the abnormal of chromosome in three patients.Both trisomy7and trisomy17were found in the tumor tissues from all three patients. The gain of chromosomes7and17occurred in about50%-70%of tumor cells, while the percentages of chromosome alteration were <10%in normal peripheral tissues. We analyzed the exonl4,16-19of MET in the tumor tissue of three patients in a Chinese family.In exon16of MET a G to A mutation at nt3522was identified by direct sequencing of the PCR products, with an amino acid change from valine to isoleucine (V11101), while exon14,17-19were normal. Further, we detected the blood samples of12family members including the three patients, and found that the three patients and four of their children carry the same mutation.In conclusion, we present three PRCC patients in a Chinese family carrying trisomy7and17and a missense mutation (V11101) in Exon16of MET. Four of seven children in the next generation carry the same mutation without symptoms as yet, indicating that V11101may be an early predictor of familial PRCC and a preferential therapeutic target in the Chinese population.