The course of chronic lymphocytic leukemia(CLL) is variable. In aggressive disease, the CLL usually express an unmutated immunoglobulin heavy-chain variable-region gene (IgVH) and the 70kD zeta-associated protein (ZAP-70), whereas in indolent diseases, the CLL cells usually express mutated IgVH but lack expression of ZAP-70, chronic lymphocytic leukemia upregulated gene1 (CLLU1), (first LLC-specific gene) and predictive instrument of LLC risk was used. Multiple myeloma oncogene-1 (MUM1) is instead a recent prognostic factor in the LLC, the expression correlates with a favourable clinical course and long survival. TNF- , IL4,and IL10 have also been suggested to be important for B-cells growth and survival. Aims of study. In the present study, we examined purified CLL-B cells to characterize the molecular alterations and gene rearrangements, to find biological markers in specific disease stages and to estimate the patient prognosis on the risk categories ( Binet stage B-C). Common polymorphisms in gene coding for cytokines implicated in the inflammatory response and TH1/Th2 balance might play a role in the development and prognosis of CLL. To test this hypothesis, we investigated 13 single nucleotide polymorphisms (SNPs) in nine of such genes. Materials and Methods We followed 23 patients with CLL between 2006-2008 from the Haematology “A. Businco” Cancer Hospital of Cagliari. Case age with a range from 40 to 75 years, and ratio M/W 2:1. Blood and bone marrow samples were collected from patients who satisfied diagnostic and immunophenotypic criteria for common B-cell CLL. All samples were assessed by flow cytometry analysis for CD5, CD79a and MUM1. RNA and DNA were isolated for molecular biology Real Time and sequence study. Besides 40 incident CLL cases and 113 population controls were available from case-control EPILYMPH study . A Taq man platform was exclusively used for genotyping. Sequence data and assay conditions for Taqman assay are available on the U.S. National Cancer Institute SNP500 project (http:/snp500cancer.nci. nih.gov). Results. 30% of the patients showed aggressive clinical course. The CD79b level was negative or weakly expressed in all cases (91%), only two cases were positive. 43% of the cases it were positive for CD38. Mutational status of immunoglobulin IgVH and CD38 correlation: 5% ZAP-70 +/ IgVH unmutated, 69% ZAP-70 -/ IgVH mutated, and absence of correlation in 26% of cases. Histopathology analysis has been performed with standard markers (CD5, CD79a, CD20, CD23) for eventually differentiation between classic CLL and its varying. The mutation status of IgVH was performed for VHL, VHFs, VHD specific families: IgVH extension molecular analysis showed 89% IgVH mutate. We examined a single IgVH subgroup, the most common of which was VH3 (52%), followed by VH4 (29%) and VH1(19%). ZAP-70 and CLLU1 were performed through Real-Time PCR and relative quantification expressed as (2^- CT). These data revealed that: 26% of patients was positive for ZAP- 70 and was not presented CLLU1 and IgVH correlation. Polymorphisms in IL1B and IL6 genes, but not the other tested interleukin SNPs, were associated with CLL risk. Individuals with aplotype including both variant alleles showed a 5.3-fold increase in CLL risk and individuals with SNP IL1B variant allele were significantly protect against CLL. Conclusions. In the IgVH analysis: discordant data respect European guideline and absence of VH3- 21 expression were obtained. The ZAP-70 values performed by molecular biology techniques remain the more important index regarding the IgVH mutation status. The MUM1 high expression has indicated patients with favourable clinical course and a longer survival. CLLU1, CD38, and ZAP-70 correlation has identified groups of patients to good and bad prognosis. Future research on prognostic indicators, such as clinical stage of disease, lymphocyte doubling time, CD38 and /or ZAP 70 expression levels, and IgVH mutational status, as related to the IL1B and IL6 gene polymorphisms and expression, would be important to provide molecular support to our findings. Poor statistical power is a major limitation in the present study, and caution is recommended in the interpretation of our findings.
Risk factors,new molecular and immunohistochemical markers in chronic lymphocytic C leukemia”
ENNAS, MARIA GRAZIA;
2009-01-01
Abstract
The course of chronic lymphocytic leukemia(CLL) is variable. In aggressive disease, the CLL usually express an unmutated immunoglobulin heavy-chain variable-region gene (IgVH) and the 70kD zeta-associated protein (ZAP-70), whereas in indolent diseases, the CLL cells usually express mutated IgVH but lack expression of ZAP-70, chronic lymphocytic leukemia upregulated gene1 (CLLU1), (first LLC-specific gene) and predictive instrument of LLC risk was used. Multiple myeloma oncogene-1 (MUM1) is instead a recent prognostic factor in the LLC, the expression correlates with a favourable clinical course and long survival. TNF- , IL4,and IL10 have also been suggested to be important for B-cells growth and survival. Aims of study. In the present study, we examined purified CLL-B cells to characterize the molecular alterations and gene rearrangements, to find biological markers in specific disease stages and to estimate the patient prognosis on the risk categories ( Binet stage B-C). Common polymorphisms in gene coding for cytokines implicated in the inflammatory response and TH1/Th2 balance might play a role in the development and prognosis of CLL. To test this hypothesis, we investigated 13 single nucleotide polymorphisms (SNPs) in nine of such genes. Materials and Methods We followed 23 patients with CLL between 2006-2008 from the Haematology “A. Businco” Cancer Hospital of Cagliari. Case age with a range from 40 to 75 years, and ratio M/W 2:1. Blood and bone marrow samples were collected from patients who satisfied diagnostic and immunophenotypic criteria for common B-cell CLL. All samples were assessed by flow cytometry analysis for CD5, CD79a and MUM1. RNA and DNA were isolated for molecular biology Real Time and sequence study. Besides 40 incident CLL cases and 113 population controls were available from case-control EPILYMPH study . A Taq man platform was exclusively used for genotyping. Sequence data and assay conditions for Taqman assay are available on the U.S. National Cancer Institute SNP500 project (http:/snp500cancer.nci. nih.gov). Results. 30% of the patients showed aggressive clinical course. The CD79b level was negative or weakly expressed in all cases (91%), only two cases were positive. 43% of the cases it were positive for CD38. Mutational status of immunoglobulin IgVH and CD38 correlation: 5% ZAP-70 +/ IgVH unmutated, 69% ZAP-70 -/ IgVH mutated, and absence of correlation in 26% of cases. Histopathology analysis has been performed with standard markers (CD5, CD79a, CD20, CD23) for eventually differentiation between classic CLL and its varying. The mutation status of IgVH was performed for VHL, VHFs, VHD specific families: IgVH extension molecular analysis showed 89% IgVH mutate. We examined a single IgVH subgroup, the most common of which was VH3 (52%), followed by VH4 (29%) and VH1(19%). ZAP-70 and CLLU1 were performed through Real-Time PCR and relative quantification expressed as (2^- CT). These data revealed that: 26% of patients was positive for ZAP- 70 and was not presented CLLU1 and IgVH correlation. Polymorphisms in IL1B and IL6 genes, but not the other tested interleukin SNPs, were associated with CLL risk. Individuals with aplotype including both variant alleles showed a 5.3-fold increase in CLL risk and individuals with SNP IL1B variant allele were significantly protect against CLL. Conclusions. In the IgVH analysis: discordant data respect European guideline and absence of VH3- 21 expression were obtained. The ZAP-70 values performed by molecular biology techniques remain the more important index regarding the IgVH mutation status. The MUM1 high expression has indicated patients with favourable clinical course and a longer survival. CLLU1, CD38, and ZAP-70 correlation has identified groups of patients to good and bad prognosis. Future research on prognostic indicators, such as clinical stage of disease, lymphocyte doubling time, CD38 and /or ZAP 70 expression levels, and IgVH mutational status, as related to the IL1B and IL6 gene polymorphisms and expression, would be important to provide molecular support to our findings. Poor statistical power is a major limitation in the present study, and caution is recommended in the interpretation of our findings.
I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
