Lilla Reiniger

Richter's and prolymphocytic transformation of chronic lymphocytic leukemia are associated with high mRNA expression of activation-induced cytidine deaminase and aberrant somatic hypermutation

Chronic lymphocytic leukemia (CLL) is an indolent B-cell non-Hodgkins lymphoma that may transform into higher-grade lymphoma. The transformation involves an increased number of prolymphocytic cells, termed prolymphocytic transformation (PLT) or the development of diffuse large B-cell lymphoma (DLBL), also referred to as Richters transformation (RT). To analyze whether activation-induced cytidine deaminase (AID), which is essential for somatic hypermutation (SHM) of normal B-cells, and malfunction of SHM termed aberrant somatic hypermutation (ASHM) are associated with higher-grade transformation of CLL, AID mRNA expression and the mutation pattern of c-MYC, PAX-5 and RhoH genes were analyzed in eight cases of CLL without transformation and in 21 cases that showed RT or PLT. Chronic lymphocytic leukemia cases, which showed no transformation or eventually transformed into higher-grade lymphoma, showed low levels of AID mRNA expression and low frequency of mutations of c-MYC, PAX-5 and RhoH genes. In both RT and PLT, high-levels of AID mRNA expression and high-frequency mutations of c-MYC, PAX-5 and RhoH genes were detected. These results indicate that AID expression and ASHM are associated with higher-grade transformation of CLL and provide further evidences that AID expression and ASHM may be activated during the clonal history of B-cell lymphomas.

ROR1 expression is not a unique marker of CLL.

Recent studies have identified receptor tyrosine kinase‐like orphan receptor 1 (ROR1) on the surface of chronic lymphoid leukaemia (CLL) cells. In order to determine whether ROR1 expression is a suitable surrogate marker for the diagnosis of CLL we analysed the mRNA level of ROR1 in different types of non‐Hodgkin lymphomas (NHL), and detected elevated levels of ROR1 compared to control peripheral mononuclear cells in several entities (CLL ≥ mantle cell lymphoma (MCL) > marginal zone lymphoma (MZL) >> diffuse large B‐cell lymphoma > follicular lymphoma). ROR1 protein was expressed intensely on the cell surface of lymphoma cells with leukaemic blood count detected by three colour immunofluorescence. Our results indicate that ROR1 expression is not limited to CLL cases, but it is more prevalent in NHLs, mainly in MCL where it is expressed intensely and MZL where it is expressed moderately, suggesting a general role of ROR1 in lymphoma genesis and/or maintenance. Copyright

Aberrant somatic hypermutation and expression of activation-induced cytidine deaminase mRNA in mediastinal large B-cell lymphoma.

To determine the possible role of aberrant somatic hypermutation (ASHM) and activation‐induced cytidine deaminase (AID) expression in the pathogenesis of mediastinal large B‐cell lymphoma (MBL), the mutational status of genes affected by ASHM, including c‐MYC, PAX‐5 and RhoH, was analysed, and the expression level of AID mRNA in tumour specimens from six patients with MBL was determined. Mutations in one or more genes and high expression of AID mRNA were detected in all the six cases of MBL. These results suggest that ASHM and AID expression may have a role in the pathogenesis of MBL.

Clonal selection in the bone marrow involvement of follicular lymphoma

To characterize the pathways of bone marrow (BM) involvement of follicular lymphoma (FL), we performed morphological and immunophenotypical analysis of tumor cells from lymph nodes (LNs) and corresponding BMs in 21 patients with FL. In three cases, genealogical trees were constructed based on the immunoglobulin variable region heavy chain (IgVH) gene sequences of tumor clones from LNs and BMs. Results showed that FLs within the BMs display identical or lower cytological grades than in the LNs. In the majority of cases, different proportions of tumor cells expressed bcl-2, CD10 and Ki67 in LNs and BMs. Tumor cells in the BM showed ongoing somatic hypermutation of the IgVH genes; the distribution of these mutations was highly consistent with antigen selection. The topology of the genealogical trees revealed that different subclones populate the LN and BM and BM infiltration may occur at different points of the clonal evolution of FL. Early descendants of the original tumor clone and derivatives of diversified tumor clones may invade the BM. These results suggest that the BM involvement of FL is associated with intensive clonal selection of tumor cells, and the BM provides a microenvironment similar to the germinal centers of LNs, where tumor cells retain their biological nature.

Lack of Angiogenesis in Experimental Brain Metastases

Angiogenesis is believed to be essential for the growth of metastatic tumors in the brain. We analyzed the vascularization of tumors formed by 4 epithelial cell lines (C38, ZR75, HT25, and H1650) and a fibrosarcoma (HT1080) cell line injected into the brains of mice. No peritumoral angiogenesis was observed. Tumors apparently acquired their vasculature by incorporation of native vessels. Vessel density was lower, but vessel diameter and vascular cell proliferation were higher within all tumors versus those in the peritumoral tissue. There was an inverse correlation between the number of incorporated vessels and vascular cell proliferation. Epithelial tumors with pushing growth patterns had lower vessel density and elevated vascular cell proliferation compared with invasive tumors. The incorporated vessels retained their normal structure, with the exception of astrocyte foot processes that were replaced by tumor cells. Attachment to the vascular basement membrane led to the differentiation of the ZR75 breast cancer cells. In the HT1080 metastases, there was intussusceptive angiogenesis, that is, the fibrosarcoma cells that were attached to the vessel caused lumen splitting and filled the developing pillars. Branching angiogenesis was not observed either in the tumors or in control cerebral wounds. These data suggest that sprouting angiogenesis is not needed for the incipient growth of cerebral metastases and that tumor growth in this model is a result of incorporation of host vessels.

High rate of neoplastic cells with genetic abnormalities in proliferation centers of chronic lymphocytic leukemia

In lymph nodes, chronic lymphocytic leukemia (CLL) cells (prolymphocytes and paraimmunoblasts) form proliferation centers (PCs), which are also known as pseudofollicles. To reveal whether PCs play a role in the accumulation of genetic alterations in CLL, we compared deletion at 11q22.3, 13q14.3, and 17p13.1 loci and trisomy 12 by the fluorescence in situ hybridization (FISH) technique in PCs versus surrounding small lymphocytes (SLs) in 12 formalin-fixed paraffin-embedded (FFPE) lymph nodes. The FFPE sections were stained with methylene blue and PCs were marked by laser beam. Subsequent FISH analysis was performed, relocalizing the previously defined regions. Loss of 11q was detected in five cases, loss of 13q in two cases, loss of 17p in two cases, and trisomy 12 in one case. In seven cases PCs contained a significantly higher ratio of cells with genetic alterations compared with the surrounding SLs. Our results show that CLL cells with genetic alterations tend to accumulate in PCs. The clonal expansion of the cell population carrying genetic alterations within PCs may contribute to CLL progression.

The cut-off levels of CD23 expression in the differential diagnosis of MCL and CLL

Flow cytometric analysis of CD23 expression in CD5‐positive B‐cells is a widely applied method in the differential diagnosis of chronic lymphocytic leukaemia (CLL) and mantle cell lymphoma (MCL). According to the most accepted criteria, the leukaemic cell population is CD19/CD5/CD23 triple positive in CLL but CD23‐negative in MCL. Recently, several groups have reported CD23‐positive MCL cases; however, these studies mostly analysed only CD23 positivity but not intensity. To determine the role and the cut‐off levels of CD23 positivity and intensity in the differential diagnosis of CLL and MCL, 26 cases of MCL and 84 cases of CLL were compared using flow cytometric analysis. Our results suggest that high values of CD23 positivity (>92.5%) and/or high fluorescence intensity (>44.5 mean fluorescence intensity (MFI)) of CD23 are related to CLL, whereas low CD23 positivity (<30%) is related to MCL. However, cases with intermediate CD23 positivity (between 30 and 92.5%) and lower intensity (<44.5 MFI) can either belong to CLL or MCL. In these cases, additional tests such as FISH analysis of the translocation t(11;14) or immunohistochemical detection of cyclin D1 overexpression are required to differentiate CLL from MCL. Copyright

Somatic hypermutation of IGVH genes and aberrant somatic hypermutation in follicular lymphoma without BCL-2 gene rearrangement and expression

The findings of this study suggest that follicular lymphoma with typical morphological and immunophenotypic features may develop even without the involvement of the BCL-2 gene. See related perspective article on page 1773. Background Follicular lymphoma is characterized by the t(14;18) translocation resulting in constitutive expression of BCL-2 protein; however approximately 10–15% of follicular lymphomas do not express BCL-2 protein, and a small fraction of these cases does not exhibit translocation of the BCL-2 gene either. It is highly debated whether cases of follicular lymphoma without BCL-2 gene rearrangement and expression represent a separate lymphoma entity with distinct biological characteristics, different from the BCL-2-positive cases. Design and Methods To further characterize follicular lymphomas without BCL-2 gene rearrangement and expression, we analyzed and compared the mutational status of IGVH genes as well as other genes (C-MYC, PAX-5 and RHOH) frequently involved in the specific type of genomic instability called aberrant somatic hypermutation in 11 cases of BCL-2-negative and 7 cases of BCL-2-positive follicular lymphomas. We also determined the levels of expression of activation-induced cytidine deaminase in these cases. Results The analyzed cases were grade 2 and grade 3A follicular lymphomas. Our findings demonstrate that follicular lymphomas without BCL-2 gene rearrangement and expression are associated with ongoing somatic hypermutation of the IGVH genes, low activity of aberrant somatic hypermutation and elevated activation-induced cytidine deaminase expression. These results were in concordance with the results found in the cases of BCL-2-positive follicular lymphoma. Conclusions Although, BCL-2 protein overexpression is considered to be a critical pathogenic event in the development of follicular lymphoma, our findings suggest that follicular lymphomas with the same morphology, immunophenotype, mutational pattern and activation-induced cytidine deaminase expression may develop without the involvement of BCL-2 gene. The present data support the hypothesis that BCL-2-positive and BCL-2-negative follicular lymphomas (grades 1-3A) represent a homogenous group with different initial but several common additional molecular pathways.

Quantitative miR analysis in chronic lymphocytic leukaemia/small lymphocytic lymphoma – proliferation centres are characterized by high miR-92a and miR-155 and low miR-150 expression

Proliferation centres (PCs) are histological hallmarks of lymph nodes in chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL). Chromosomal abnormalities have already been described to accumulate preferably in the PCs as opposed to the intervening small cell areas. To further characterize the pathogenic role of PCs, the expression levels of 17 selected miRs known to be involved in the development of CLL/SLL were compared in the PCs and the intervening small cell areas in lymph nodes of 15 patients with CLL/SLL. The miR expression levels were also compared to the cytogenetic alterations defined by FISH analysis. Our results show that two known oncomiRs, miR-155 and -92a were upregulated and the tumour suppressor miR-150 was downregulated in the PCs. Low expression of miR-150 was also associated with loss of 11q. In summary we found significantly higher expression of oncomiRs and lower expression of a tumour suppressor miR in PCs of CLL/SLL lymph nodes, which support the hypothesis that the PCs may drive the disease and play a role in progression.

Low CD23 expression correlates with high CD38 expression and the presence of trisomy 12 in CLL.

Chronic lymphocytic leukemia (CLL) is characterized by a neoplastic B‐cell population coexpressing CD5 and CD23; however, the expression of CD23 is variable. In human, two isotypes of CD23 have been identified and related to different functions. The aim of our study was to investigate the relative expression of the two CD23 isotypes in CLL and find possible correlation with other prognostic factors. The expression of CD23 isotypes was analyzed in 54 cases of CLL by polymerase chain reaction (PCR) and quantitative real‐time PCR. The immunophenotype of CLL cells was characterized by flow cytometry. We demonstrated a higher CD23a than CD23b expression of CLL cells. Our results also revealed two subsets of CLL cases with a distinct CD23 isotype expression pattern. Thirty‐two percent of the cases (group CLL1) showed both low mRNA level of CD23 isotypes and high protein levels of CD20 and CD38 in contrast to group CLL2 with high CD23 mRNA levels. By correlating these results to the presence of prognostic factors determined by fluorescence in situ hybridization, we found that the majority of the cases of group CLL1 (14/17) carried trisomy 12. In summary, our results confirm a high CD23a/CD23b ratio of the CLL cells and demonstrate that in a subset of CLL cases, low CD23 expression together with high CD20 and CD38 expressions may serve as a surrogate for trisomy 12. Copyright