Annuska Glas1, Leonie Delahaye1, Anke Witteveen1, Marie José Kersten1, Arno Velds1, Ron Kerkhoven1, Han van Krieken2, Robby Kibbelaar3, Laura van ‘t Veer1 and Daphne de Jong1
1The Netherlands Cancer Institute, Amsterdam, Nederland; 2University Hospital Nijmegen, Nijmegen, Nederland; 3Central Laboratories Friesland, Leeuwarden, Nederland
Follicular Lymphomas (FL) are the second most frequent lymphoma in adults. In the Netherlands, approximately 400 new cases of FL are seen yearly. Generally FL is indolent, but transformation to more aggressive stage is a common event. The prognosis after transformation to diffuse large B cell lymphoma (DLBCL) is poor and this forms a major cause of death in FL patients. Thus far, biological insights in the factors involved in transformation are limited to a few and individual genes. To improve the prognostic stratification of FL, microarray analysis was used for the development of a molecular diagnostic tool. This may also give insight in the biological pathways involved in transformation and may reveal possible targets for novel therapeutic approaches as well. In this study 24 patients with transformed FL were selected from the pathology files of three institutes in the Netherlands. Frozen tissue of both the indolent as well as the transformed stages (FL grade 3 or DLBCL) were available from these patients. Included were 18 cases of FL followed by DLBCL or FL grade 3 (median interval 47 months, range 12-228 months), 4 cases of DLBCL who relapsed as FL (median interval 24 months, range 12-50 months) and 2 cases of synchronous FL and DLBCL at different localizations. All cases were reviewed, classified and graded according to the WHO-classification. RNA was isolated, linearly amplified using T7 RNA polymerase, Cy5 or Cy3 labeled and co-hybridized with a RNA mixture of five tonsils to 18K human cDNA-arrays prepared at the KWF/NKI central microarray facility.
Cluster analysis using 120 genes, which were differentially expressed between the two phases of the disease, showed separation of the patient samples into four major clusters. Indolent FL cluster together in one branch and the aggressive lymphomas were separated into three groups (grade 3 FL, DLBCL and grade3/DLBCLmix).
In the transformed clusters, genes involved in cell turnover were up-regulated, while genes involved in B- and T-cell signaling were down-regulated. The groups could be further separated by differential expression of genes involved in intracellular signaling, proliferation and MHC class II genes. Two samples were misclassified as compared to the pathological diagnosis and the data suggests that an expression profile may provide a better stratification than the currently used histological data. Further studies are in progress to validate our findings that microarray analysis can separate indolent follicular lymphoma from transformed phase of the disease, using samples of patients who showed no transformation to aggressive disease (n=14), and 51 patients with transformed disease of whom only the indolent phase (n=17), or only the transformed phase (n=34) was available in the tissue banks.