Genetic and micro-array based profiling of primary cutaneous B-cell lymphomas

Primary cutaneous large B-cell lymphomas (PCLBCL) account for 20% to 25% of all primary cutaneous lymphomas. Most PCLBCL are primary cutaneous follicle center cell lymphomas (PCFCCL) that present with nodules and tumors on a restricted area on the head and trunk. These lymphomas are highly responsive to radiotherapy and have an excellent prognosis (5-year survival 95%). In contrast, the PCLBCL of the leg have a high relapse rate and an unfavorable prognosis (5-year survival 58%).Comparative genome hybridization demonstrated that PCLBCL of the leg contain more chromosomal aberrations than PCFCCL (3,7 versus 1,3 per case). The most frequent type of aberration we detected was a gain of chromosome 18, found in 7/13 (54%) of PCLBCL of the leg, but not in any of the PCFCCL. Interestingly, the anti-apoptotic protein bcl2, encoded at chromosome 18, is abundantly expressed in PCLBCL of the leg but not by PCFCCL. However, using FISH and PCR we found that the expression of bcl-2 is not caused by the classical t(14;18) translocation. The second most frequent genetic alteration detected was a deletion of 6q, found in 4/13 (30%) of PCLBCL of the leg but not in PCFCCL. Earlier studies demonstrated frequent deletions of 6q in nodal diffuse large B-cell lymphoma and identified 6q deletions as a poor prognostic sign in follicular lymphoma (Tilly et al., Blood, 1994; 84:1043). Interestingly, in the PCLBCL of the leg we studied, 3 of 4 (75%) of the patients with 6q deletions died of their disease, whereas none of the patients without 6q deletions died.Aim: to identify diagnostic and prognostic markers as well as therapeutic targets in PCLBCL by investigating gene expression profiles and combining these data with detailed investigation of genetic alterations, in particular translocations.In a pilot study performed in collaboration with the Dept. of Human Genetics (Dr. den Dunnen) we determined gene expression profiles in biopsies from PCLBCL of the leg (n=7) and PCFCCL (n=4) containing more than 80% tumor cells using Affymetrix oligo-arrays (U95Av2, 12K). On average 55% of the genes were expressed in all biopsies. Using unsupervised hierarchical cluster analysis, PCLBCL of the leg (n=7) and PCFCCL (n=4) are classified in two distinct clusters. Within these separate clusters gene expression profiles are relatively homogeneous. More detailed analysis (supervised clustering) revealed increase of 67 genes and decrease of 84 genes in all PCLBCL of the leg compared to all PCFCCL. In patients with PCLBCL of the leg 38 genes are associated with a detrimental clinical outcome (13 increased and 25 decreased expression). Increased expression in PCLBCL of the leg versus PCFCCL included: immunoglobulin mu heavy chain, TRAF interacting protein, elongin A and TBC1 family member number 5 which all have been implicated in tumor development. In the proposed project results from our pilot study will be expanded by: 1. repeating our pilot-experiment on Affymetrix oligo-arrays (U133 A+B) thereby increasing the number of investigated gene transcripts to 39.000; 2. extending the experimental groups to 15 PCLBCL of the leg and 15 PCFCCL. Gene expression profiles will be analyzed by comparing results from: (i) PCLBCL of the leg with PCFCCL; and in collaboration with the Institute of Cancer Genetics Columbia University New York (Prof dr Dalla-Favera) also (ii) with normal B-cell subsets (Klein, J Exp Med, 2001) and (iii) with non-cutaneous diffuse large B-cell lymphomas (Shipp, Nat Med, 2002). In conjunction combined binary ratio labeling fluorescence in situ hybridization (COBRA-FISH) will be performed in collaboration with the Dept. Molecular Cell Biology (Prof dr AK Raap) to analyze chromosomal changes including translocations in PCLBCL. To investigate non-classical translocations of bcl2 we will perform interphase FISH and long-distance-(inverse) polymerase chain reaction (LD-IPCR). Earlier studies have used these techniques successfully to determine the diverse partners of BCL2, BCL6 and c-MYC translocation in nodal diffuse large B-cell lymphoma (Akasaka, Cancer Res, 2000; Vaandrager, Genes Chromosomes Cancer, 2000). The functional consequences of the detected chromosomal alterations, will be further investigated by evaluating gene expression profiles, quantitative-PCR and protein expression. The ultimate goal of these studies is to identify oncogenic proteins, which may serve as diagnostic or prognostic indicators as well as therapeutic targets in PCLBCL.