Molecular approaches to the diagnosis and evaluation of follicular lymphoma
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Background: Molecular studies of immunoglobulin (IG) genes have provided insights into the pathogenesis of different lymphoid malignancies and allowed the development of useful diagnostic tools as well as powerful prognostic markers. This study concerns the role of molecular analysis of IG genes in the diagnosis and evaluation of follicular lymphoma (FL).
Aims: The aims of the present study were a) to investigate the application of PCRbased clonality analysis of IG genes on formalin-fixed, paraffin-embedded (FFPE) tumour samples, b) to determine the value of such analysis in bone marrow staging and c) to analyze the use of IG heavy chain variable (IGHV) genes and mutational status in relation to prognosis in FL patients.
Materials and methods: The thesis is based on three papers (Paper I-III). In Paper I, DNA from FFPE samples of 118 patients diagnosed with FL in the period 1998- 2008 was used. PCR-based clonality was assessed by IG heavy (IGH), kappa (IGK) and lambda (IGL) primers in multiplexed reactions, and by a PCR procedure that was optimized for FFPE tissue. In Paper II, DNA was obtained from fresh bone marrow aspirates of 96 FL patients and subjected to PCR-based clonality analysis. The PCR results were controlled by analysis of the primary tumour and related to morphological detection of bone marrow involvement. In 71 patients, the results were also compared with data from concurrent flow cytometric immunophenotyping. In Paper III, 106 patients with FL were included. IGHV gene sequences were determined using DNA from FFPE tumour samples and direct sequencing with forward and reverse primers.
Results: In Paper I, the highest clonality detection rates were reached when IGH and IGK assays were combined (94.9%). FFPE samples stored for 6-11 years did not perform significantly worse than those stored for 1-5 years with respect to clonality detection. In Paper II, bone marrow involvement by PCR-based clonality was found in 34.4% (33/96) of patients. PCR-positive patients had a significantly poorer survival than PCR-negative patients (P=0.001, log-rank test). Thirteen patients positive by PCR but without morphological BM involvement, had significantly poorer survival than patients with negative morphology and negative PCR result (P=0.002). The poor survival associated with PCR-based bone marrow involvement was independent of high FLIPI score by multivariate analysis (P=0.007). Bone marrow involvement by morphology or flow cytometry did not show prognostic impact on survival. In Paper III, 104 productive rearranged IGH sequences were obtained from FFPE tumour samples of 99 patients. The IGHV3, IGHV1 and IGHV4 were the most frequently encountered subgroups while the IGHV3-23 was the most frequently encountered gene. Patients with the IGHV5 subgroup (P=0.013, log-rank) or more than one IGHV subgroups (P<0.001, log-rank) in their tumours showed significantly poorer survival than patients with other IGHV subgroups. IGHV5/>1 IGHV subgroup usage was of independent prognostic importance in multivariate analysis (P=0.005). Unmutated sequences, showing >98% homology to the closest IGHV gene, were detected in 15.2% of cases. Unmutated IGHV genes were associated with age >60 years at diagnosis, but not with survival.
Conclusions: An improved PCR protocol for detection of clonality in FFPE samples was presented, and a combination of IGH and IGK analyses was recommended for diagnostic purposes (Paper I). PCR-based clonality analysis significantly improved the prognostic value of bone marrow staging, and the inclusion of PCR-based analysis in bone marrow examination of FL patients was suggested (Paper II). The presence of the IGHV5 subgroup or more than one IGHV subgroup identified patients with shorter survival, indicating that IGHV sequence analysis could aid in predicting prognosis for FL (Paper III).