Differential expression of cyclin-dependent kinase 6 in cortical thymocytes and T-cell lymphoblastic lymphoma/leukemia.

Cyclin-dependent kinase-6 (CDK6) is the earliest inducible member of the CDK family in human T lymphocytes, involved in growth factor stimulation and cell cycle progression. CDK6 is one of the targets of p16 and p15, CDK inhibitors encoded by MTS1 and MTS2, two tumor suppressor genes that are frequently deleted in T-cell leukemia. In this study we have investigated CDK6 expression in normal and neoplastic lymphoid tissues using immunohistochemistry and flow cytometry. In normal (six samples) and hyperplastic (four samples) thymuses, strong CDK6 expression was observed in a discrete proportion of cortical thymocytes (10 to 15%), mainly located in the peripheral (subcapsular) zone of the cortex. All tested cases of T-cell lymphoblastic lymphoma/leukemia (T-LBL/ALL) showed strong CDK6 expression in the majority (up to 100%) of neoplastic lymphoid cells. Western blot analysis confirmed the expected CDK6 protein size (40 kd). According to Southern blot analysis, CDK6 overexpression in neoplastic T lymphoblasts was not due to gene amplification. In all other lymphomas investigated (28 peripheral T-cell non-Hodgkin's lympohomas (T-NHLs), 7 CD30+ anaplastic NHLs, 22 high-grade B-NHLs, 15 low-grade B-NHLs, 25 B-cell precursor ALLs), CDK6 was not expressed or expressed at low levels, with the only exception of three nasal angiocentric T-NHLs, all exhibiting CDK6 immunoreactivity comparable to that observed in T-LBL/ALL. These data provide evidence that CDK6 is abnormally expressed in T-LBL/ALL and may be involved in the pathogenesis of this malignancy. In addition, the quantitative difference of CDK6 expression between neoplastic and non-neoplastic cortical thymocytes can be potentially useful in the differential diagnosis of thymic neoplasms on histological and cytological specimens.     

Differential reactivity of a novel monoclonal antibody (DF3) with human malignant versus benign breast tumors

We have defined a human breast tumor associated antigen using a murine monoclonal antibody (MAb DF3) prepared against a membrane-enriched fraction of a human breast carcinoma. This antigen has a MW of 290 kD and is detectable on the cell surface of human breast carcinoma cells using a live cell radioimmunoassay and fluorescence flow cytometry. More important, immunoperoxidase staining with MAb DF3 clearly distinguishes malignant and benign breast lesions. A cytoplasmic staining pattern has been observed with 40 of 51 (78%) breast carcinomas, but only one of 13 fibroadenoma or fibrocystic disease specimens. In contrast, reactivity of benign breast lesions with MAb DF3 primarily occurs along apical borders on ductules. These results demonstrate that the DF3 antigen is present on apical borders of more differentiated secretory mammary epithelial cells and in the cytosol of less differentiated cells.     

Molecular events underlying interleukin‐6 independence in a subclone of the CMA‐03 multiple myeloma cell line

We explored the molecular mechanisms involved in the establishement of CMA‐03/06, an IL‐6‐independent variant of the multiple myeloma cell line CMA‐03 previously generated in our Institution. CMA‐03/06 cells grow in the absence of IL‐6 with a doubling time comparable with that of CMA‐03 cells; neither the addition of IL6 (IL‐6) to the culture medium nor co‐culture with multipotent mesenchymal stromal cells increases the proliferation rate, although they maintain the responsiveness to IL‐6 stimulation as demonstrated by STAT1, STAT3, and STAT5 induction. IL‐6 independence of CMA‐03/06 cells is not apparently due to the development of an autocrine IL‐6 loop, nor to the observed moderate constitutive activation of STAT5 and STAT3, since STAT3 silencing does not affect cell viability or proliferation. When compared to the parental cell line, CMA‐03/06 cells showed an activated pattern of the NF‐κB pathway. This finding is supported by gene expression profiling (GEP) analysis identifying an appreciable fraction of modulated genes (28/308) in the CMA‐03/06 subclone reported to be involved in this pathway. Furthermore, although more resistant to apoptotic stimuli compared to the parental cell line, CMA‐03/06 cells display a higher sensibility to NF‐κB inhibition induced by bortezomib. Finally, GEP analysis suggests an involvement of a number of cytokines, which might contribute to IL‐6 independence of CMA‐03/06 by stimulating growth and antiapoptotic processes. In conclusion, the parental cell‐line CMA‐03 and its variant CMA‐03/06 represent a suitable model to further investigate molecular mechanisms involved in the IL‐6‐independent growth of myeloma cells. © 2013 Wiley Periodicals, Inc.     

Simple genetic diagnosis of hairy cell leukemia by sensitive detection of the BRAF-V600E mutation

Hairy cell leukemia (HCL) is a distinct clinicopathologic entity that responds well to purine analogs but is sometimes difficult to differentiate from HCL-like disorders (e.g., splenic marginal zone lymphoma and HCL variant). We recently identified the BRAF-V600E mutation as the disease-defining genetic event in HCL. In this study, we describe a new, simple, and inexpensive test for genetics-based diagnosis of HCL in whole-blood samples that detects BRAF-V600E through a sensitive allele-specific PCR qualitative assay followed by agarose-gel electrophoresis. This approach detected BRAF-V600E in all 123 leukemic HCL samples investigated containing as few as 0.1% leukemic cells. BRAF-V600E was detected at different time points during the disease course, even after therapy, pointing to its pivotal role in HCL pathogenesis and maintenance of the leukemic clone. Conversely, 115 non-HCL chronic B-cell neoplasms, including 79 HCL-like disorders, were invariably negative for BRAF-V600E. This molecular assay is a powerful tool for improving the diagnostic accuracy in HCL.     

Phenotypic and functional characterization of T-BAM (CD40 ligand)+ T- cell non-Hodgkin's lymphoma

The precise mechanisms regulating T-helper function have been intensively investigated. We and others have recently identified a new T-cell-B-cell-activating molecule called T-BAM that directs B-cell differentiation by interacting with the CD40 molecule on B cells. Using a specific monoclonal antibody against T-BAM (5C8), we have previously shown that T-BAM expressing T cells are predominantly CD4+CD8- and in normal lymphoid tissue have a unique distribution. However, no information has been obtained regarding the phenotype and functional properties of human neoplastic T cells. Therefore, we investigated T- BAM expression immunohistochemically in 87 well-characterized T-cell non-Hodgkin's lymphomas and lymphoid leukemias (LL). We found that 21/81 neoplasms expressed detectable T-BAM and these positive tumors belong almost exclusively to the CD4+CD8- subtype. In addition, to determine whether T-BAM expression could be induced on T-BAM-LL cells, we activated T-BAM-LLs in vitro and showed that T-BAM could be upregulated only in CD4+CD8- tumors. Our studies clearly show that T- BAM is constitutively expressed in a large number of T-cell neoplasms with a relative mature phenotype (CD4+CD8-) and that only CD4+ neoplastic T cells can be induced in vitro to express this molecule. Additional studies are necessary to identify the biologic significance of T-BAM expression and its potential and clinical implications.     

Structural and functional analysis of oncogenes and tumor suppressor genes in adult T-cell leukemia/lymphoma shows frequent p53 mutations.

The human T-cell lymphotropic virus type I (HTLV-I) is capable of inducing adult T-cell leukemia/lymphoma (ATLL). However, the long latency period between infection and development of ATLL, as well as the small fraction of the infected population that actually develops this disease, suggest that additional factors are involved in its pathogenesis. Therefore, we performed a molecular analysis of 10 cases of ATLL presenting in a nonendemic area that were shown to have HTLV-I sequences by polymerase chain reaction as well as clonal T-cell receptor beta gene rearrangements. We analyzed these cases for alterations in some of the oncogenes and tumor suppressor genes frequently involved in hematopoietic neoplasia. Specifically, we used a single-strand conformation polymorphism assay to determine the presence of mutations in the p53 tumor suppressor gene, as well as the K-RAS, N-RAS, H-RAS, and c-myc oncogenes. In addition, we studied the c-myc gene for rearrangements by Southern blotting and assessed expression of the retinoblastoma (Rb) and p53 genes by immunostaining. Analysis of the c-myc gene and the RAS family of oncogenes did not show any alterations. Also, the Rb gene was expressed in all cases analyzed. However, we found mutations of the p53 gene in 3 of the 10 cases and these results were confirmed by sequence analysis. In two of these cases, we showed by restriction fragment length polymorphism analysis of chromosome 17p sequences that the p53 mutations were accompanied by a loss of heterozygocity. Also, these mutations correlated with an altered pattern of p53 expression. Thus, mutations in the p53 locus may be a cofactor for the development of ATLL in some cases, whereas the c-myc, Rb, and RAS genes do not appear to be involved in these neoplasms.