P. Lichter

Genetics of chronic lymphocytic leukemia: Genomic aberrations and VH gene mutation status in pathogenesis and clinical course

The genetic characterization of chronic lymphocytic leukemia (CLL) has made significant progress over the past few years. While conventional cytogenetic analyses only detected chromosome aberrations in 40–50% of cases, new molecular cytogenetic methods, such as fluorescence in situ hybridization (FISH), have greatly enhanced our ability to detect chromosomal abnormalities in CLL. Today, genomic aberrations are detected in over 80% of CLL cases. Genes potentially involved in the pathogenesis were identified with ATMin a subset of cases with 11q deletion and p53 in cases with 17p13 deletion. For the most frequent aberration, the deletion 13q14, candidate genes have been isolated. Genetic subgroups with distinct clinical features have been identified. 11q deletion is associated with marked lymphadenopathy and rapid disease progression. 17p deletion predicts for treatment failure with alkylating agents, as well as fludarabine and short survival times. In multivariate analysis 11q and 17p deletions provided independent prognostic information. Recently, another important issue of genetic risk classification in CLL was identified with the mutation status of the immunoglobulin variable heavy chain genes (VH). CLL cases with unmutated VH show more rapid disease progression and shorter survival times. Whether CD38 expression can serve as a surrogate marker for VH mutation status is currently discussed controversially. VH mutation status and genomic abnormalities, such as 17p and 11q deletion, have recently been shown to be related to each other, but were of independent prognostic information in multivariate analysis. Moreover, genomic aberrations and VH mutation status appear to give prognostic information irrespective of the clinical stage and may therefore allow a risk assessment for individual patients early in the course of their disease.

Incidence and clinical significance of 6q deletions in B cell chronic lymphocytic leukemia.

Deletions affecting the long arm of chromosome 6 (6q) are among the most commonly observed chromosomal aberrations in lymphoid malignancies and have been identified as adverse prognostic factor in subsets of tumors. Whereas at least two regions of minimal deletion have been established, one in 6q21-q23 and another in 6q25-q27, no tumor suppressor gene that might be involved in the pathogenesis of lymphoid malignancies has been so far identified from these segments. For B cell chronic lymphocytic leukemia (B-CLL) conflicting data have been reported regarding the incidence and prognostic significance of 6q deletions. In the current study we have used two YAC clones mapping to deletion regions in bands 6q21 and 6q27 as probes for fluorescence in situ hybridization (FISH) in a large series of B-CLL cases to analyze the incidence, localization and clinical significance of 6q aberrations. Among 285 patients with B-CLL studied we identified 21 cases (7%) with 6q deletions. All deletions were found with the probe mapping to 6q21 while the 6q27 region was deleted only in a third of these cases. Analysis of the clinical characteristics and laboratory parameters showed that the patients with 6q deletions had higher white blood cell counts and more extensive lymphadenopathy. However, the overall survival and the treatment-free intervals were similar in the two groups. We conclude that deletions in 6q21 occur in 7% of B-CLL and identify a subgroup of patients characterized by a larger tumor mass but no inferior outcome.

Expression analysis of imbalanced genes in prostate carcinoma using tissue microarrays

To identify candidate genes relevant for prostate tumour prognosis and progression, we performed an exhaustive gene search in seven previously described genomic-profiling studies of 161 prostate tumours, and four expression profiling studies of 61 tumours. From the resulting list of candidate genes, six were selected for protein-expression analysis based on the availability of antibodies applicable to paraffinised tissue: fatty acid synthase (FASN), MYC, β-adrenergic receptor kinase 1 (BARK1, GRK2) the catalytic subunits of protein phosphatases PP1α (PPP1CA) and PP2A (PPP2CB) and metastasis suppressor NM23-H1. These candidates were analysed by immunohistochemistry (IHC) on a tissue microarray containing 651 cores of primary prostate cancer samples and benign prostatic hyperplasias (BPH) from 175 patients. In univariate analysis, expression of PP1α (P=0.001) was found to strongly correlate with Gleason score. MYC immunostaining negatively correlated with both pT-stage and Gleason score (P<0.001 each) in univariate as well as in multivariate analysis. Furthermore, a subgroup of patients with high Gleason scores was characterised by a complete loss of BARK1 protein (P=0.023). In conclusion, our study revealed novel molecular markers of potential diagnostic and therapeutic relevance for prostate carcinoma.

Heterogeneity of the API2-MALT1 gene rearrangement in MALT-type lymphoma.

The translocation t(11;18)(q21;q21), which is the most frequent chromosomal aberration in extranodal marginal zone B cell lymphomas of MALT-type, was characterised in a series of 34 biopsies, including 18 gastric non-Hodgkins lymphomas (NHL) of MALT-type, six MALT-type NHL of extragastral origin and 10 extranodal large B cell lymphomas (LBL). Based on fluorescence in situ hybridisation, STS-PCR analysis and screening of genomic PAC libraries, a physical map of contiguous DNA probes on chromosome 11 was constructed containing the anti-apoptotic genes API2 and API1 adjacent to the translocation breakpoint. RACE-PCR experiments revealed MALT1 the chromosome 18-derived fusion partner of API2, which has also been reported recently by other groups. RT-PCR analysis and DNA sequencing demonstrated the expression of an API2-MALT1 fusion transcript in 18/24 gastral and extragastral MALT-type lymphomas. In none of 10 LBLs was a translocation specific RT-PCR product detected. Five variants of the fusion transcript were identified and in all instances the open reading frame of the fused portion of the MALT1 gene was maintained. The molecular analysis of these variants allowed the design of optimised assays for the diagnosis of the API2-MALT1 gene rearrangement.

Further delineation of chromosomal consensus regions in primary mediastinal B-cell lymphomas: an analysis of 37 tumor samples using high-resolution genomic profiling (array-CGH).

Primary mediastinal B-cell lymphoma (PMBL) is an aggressive extranodal B-cell non-Hodgkins lymphoma with specific clinical, histopathological and genomic features. To characterize further the genotype of PMBL, we analyzed 37 tumor samples and PMBL cell lines Med-B1 and Karpas1106P using array-based comparative genomic hybridization (matrix- or array-CGH) to a 2.8k genomic microarray. Due to a higher genomic resolution, we identified altered chromosomal regions in much higher frequencies compared with standard CGH: for example, +9p24 (68%), +2p15 (51%), +7q22 (32%), +9q34 (32%), +11q23 (18%), +12q (30%) and +18q21 (24%). Moreover, previously unknown small interstitial chromosomal low copy number alterations (for example, −6p21, −11q13.3) and a total of 19 DNA amplifications were identified by array-CGH. For 17 chromosomal localizations (10 gains and 7 losses), which were altered in more than 10% of the analyzed cases, we delineated minimal consensus regions based on genomic base pair positions. These regions and selected immunohistochemistries point to candidate genes that are discussed in the context of NF-κB transcription activation, human leukocyte antigen class I/II defects, impaired apoptosis and Janus kinase/signal transducer and activator of transcription (JAK/STAT) activation. Our data confirm the genomic uniqueness of this tumor and provide physically mapped genomic regions of interest for focused candidate gene analysis.

Efficient nucleofection of primary human B cells and B-CLL cells induces apoptosis, which depends on the microenvironment and on the structure of transfected nucleic acids

Accumulation of neoplastic cells in B-cell chronic lymphocytic leukemia (B-CLL) is thought to be due to intrinsic defects in the apoptotic machinery of the leukemic cells or to an altered, survival-stimulating microenvironment in vivo. Despite their long survival in vivo, B-CLL cells undergo rapid spontaneous apoptosis ex vivo. To maintain survival in vitro, we established a coculture system using the human bone marrow-derived stromal cell line HS-5. The microenvironment in these cocultures lead to B-CLL cell survival for at least several months and therefore provided a tool for valid in vitro analysis, mimicking the in vivo situation. Although primary B lymphocytes are notoriously resistant to most gene transfer techniques, we achieved high transfection efficiency and cell viability in this coculture system by using a nucleofection-based strategy. Surprisingly, the introduction of circular plasmid DNA into B cells and B-CLL cells induced rapid apoptosis, which was independent of the type of transgene used, but dependent on the DNA concentration. However, transfection of these cells with mRNA was highly efficient and resulted in sustained cell viability and potent transgene expression. The described procedure represents a new approach to study gene function in primary B cells and B-CLL cells.

Molecular cytogenetic analysis of B-cell chronic lymphocytic leukemia

Abstractu2002The genetic alterations underlying the pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL) are difficult to assess. Cytogenetic studies are hindered by the low in vitro mitotic activity of the tumor cells and the limited resolution of chromosome banding. Molecular genetic analyses are hampered by nonclonal cells contained in the specimens and by the limited knowledge of candidate genes involved. As a complement to cytogenetic and molecular genetic techniques, fluorescence in situ hybridization (FISH) has proven powerful in the molecular cytogenetic analysis of B-CLL. FISH allows the detection of aberrations such as trisomies, deletions, and translocation breakpoints on the single cell level in dividing as well as nondividing cells without the prerequisite of detailed physical maps or knowledge of involved genes. As detected by the interphase cytogenetic FISH approach, the most common chromosome abnormalities of B-CLL are deletions in band 13q14, followed by deletions in 11q22-q23, trisomy 12, deletions in 17p13, and deletions in 6q21. Abnormalities in 17p13 seem to involve the TP53 tumor suppressor gene, but as yet no candidate genes have been identified for the other frequent aberrations. Toward the identification of such genes by positional cloning, FISH can be applied for detailed aberration mapping at the molecular level. Furthermore, the accurate detection of chromosome aberrations in B-CLL by FISH provides a valid basis for the evaluation of their prognostic significance. Inactivation of TP53 in 17p13 and deletions in 11q22-q23 have already been shown to be among the most important independent prognostic factors. Genetic abnormalities may eventually provide biological parameters, allowing a risk assessment for individual patients at the time of diagnosis of this clinically heterogeneous disease.

Protein expression analysis of chromosome 12 candidate genes in chronic lymphocytic leukemia (CLL)

The pathogenic role of trisomy 12 in chronic lymphocytic leukemia (CLL) remains unresolved, but recently an upregulated RNA expression level has been observed for chromosome 12 candidate genes. In the current study, the protein expression of chromosome 12 candidate genes was characterized by comparing CLL cases with (n=58) or without (n=16) trisomy 12, CD19+-B-cells and cell lines (JVM-2, EHEB, JURKAT). Immunoblotting was performed to quantify the levels of AID, APAF-1, ARF3, CCND2, CDK2, CKD4, GLI, MDM-2, p27, Smac/DIABLO and STAT6 (signal transducer and activator of transcription 6). The cell lines showed distinct expression patterns for CCND2, MDM-2, p27, Smac/DIABLO and STAT6, and displayed higher levels of CDK2 and CDK4 than the CLL cases. JURKAT and the CLL cases expressed uniformly high levels of p27, but low levels of CCND2. AID expression in the CLL cases was weak with slight variations regardless of the subgroup affiliation. The expression of the investigated proteins was independent of the trisomy 12 status as well as of the VH mutation status. The comparison of CD19+-B-cells with CLL revealed higher protein levels in CLL for CDK4, p27, Smac/DIABLO and STAT6. Further studies including protein expression experiments in genetic high-risk subgroups of CLL have to elucidate whether these proteins qualify as candidates for targeted CLL therapies.

DNA microarray analysis in malignant lymphomas

Recently, DNA microarray technology has opened new avenues for the understanding of lymphomas. By hybridization of cDNA to arrays containing >10,000 different DNA fragments, this approach allows the simultaneous evaluation of the mRNA expression of thousands of genes in a single experiment. Using sophisticated bioinformatic tools, the huge amount of raw data can be clustered resulting in (1) tumor subclassification, (2) identification of pathogenetically relevant genes, or (3) biological predictors for the clinical course. This approach already has provided novel insights into different entities of B-cell non-Hodgkins lymphomas. Genomic DNA chip hybridization (matrix-CGH) is a complementary approach focussing on genomic aberrations. In this review, we discuss the impact of this new technology both with regard to methodological aspects as well as to novel findings influencing our understanding of lymphomas.

BCL10 is not the gene inactivated by mutation in the 1p22 deletion region in mantle cell lymphoma

The BCL10 gene has recently been cloned from the 1p22 breakpoint of the translocation t(1;14)(p22;q32) observed in mucosa-associated lymphoid tissue (MALT) lymphoma. BCL10 was shown to be a proapoptotic-signaling gene encoding a protein that contains an amino-terminal caspase recruitment domain (CARD). Mutations within the BCL10 coding region resulting in truncated BCL10 proteins with loss of their proapoptotic function and preservation of their NF-κB activating function were detected in MALT lymphoma. Based on these findings it was proposed that BCL10 might have tumor suppressor function. Deletions involving 1p22 are commonly observed in mantle cell lymphoma (MCL). To investigate its role in MCL we have analyzed a series of 15 MCL for deletion and mutation of BCL10. Monoallelic 1p22 deletions were detected by fluorescence in situhybridization in five of the 15 cases and were shown to affect BCL10 in all cases. BCL10 was screened for mutations by DNA sequencing of RT-PCR amplified transcripts. In none of the 15 MCL cases studied were mutations found in the BCL10 coding region. a previously reported polymorphism exhibiting a silent 24cu2009>u2009g substitution was found in eight mcl cases and in four healthy probands. a missense mutation 13gu2009>T resulting in a substitution of a serine by an alanine was seen in one of the controls. Our results strongly suggest that BCL10 is not the candidate tumor suppressor gene inactivated by deletion or mutation in band 1p22 in MCL.