A simple method for PCR based analyses of immunohistochemically stained, microdissected, formalin fixed, paraffin wax embedded material.

Microdissection was performed on sections cut from formalin fixed, paraffin wax embedded archival material, which had been subjected to conventional immunohistochemistry. Crude DNA extracts, which were obtained from these microdissected samples by a simple microwave step, were then added directly to amplification reactions. Analyses using a range of polymerase chain reaction (PCR) based techniques, including microsatellite repeat polymorphism analysis at the NM23-H1 locus and sequencing of exons 5, 7, and 8 of the p53 gene, were performed successfully. Universal PCR amplification was also carried out on the microdissected material and probes suitable for use in comparative genomic hybridisation (CGH) were obtained in all cases. This technique will enable a range of effective genetic analyses to be carried out on specific subsets of cells that have been characterised previously by immunohistochemistry.

Copy number gain at 12q12-14 may be important in the transformation from follicular lymphoma to diffuse large B cell lymphoma.

The purpose of this study was to identify novel areas of genomic copy number change associated with transformation from follicular lymphoma (FL) to diffuse large B cell lymphoma (DLBL). DNA was extracted from tumour cells micro-dissected from paraffin- embedded tissue sections in 24 patients with FL and subsequent transformation to DLBL and 18 patients with de novo DLBL. Tumour DNA was compared to reference DNA using comparative genomic hybridization. Abnormalities common to all 3 groups were gains on chromosomes 4q, 5q, 7q, 11q and X and losses on 3p, 8p and 10q. Copy number changes seen in both transformed and de novo DLBL and not seen in FL were gains on 2p and losses on 1q, 15q and Xq. Gains on 2q, 6p, 7p and 17q and losses on 5p and 8q were specific to transformed DLBL cases. Gain on 12q12-14 was found in 52% of the transformed DLBL cases and was never seen in its follicular counterpart. Patterns of genomic copy number change associated with specific clinical events in NHL have been demonstrated and suggest that gains on 2q, 6p, 7p, 12q and 17q and losses on 5p and 8q may be important in the transformation from low to high-grade disease.