KRAS mutation detection in paired frozen and Formalin-Fixed Paraffin-Embedded (FFPE) colorectal cancer tissues.

KRAS mutation has been unambiguously identified as a marker of resistance to cetuximab-based treatment in metastatic colorectal cancer (mCRC) patients. However, most studies of KRAS mutation analysis have been performed using homogenously archived CRC specimens, and studies that compare freshly frozen specimens and formalin-fixed paraffin-embedded (FFPE) specimens of CRC are lacking. The aim of the present study was to evaluate the impact of tissue preservation on the determination of KRAS mutational status. A series of 131 mCRC fresh-frozen tissues were first analyzed using both high-resolution melting (HRM) and direct sequencing. KRAS mutations were found in 47/131 (35.8%) using both approaches. Out of the 47 samples that were positive for KRAS mutations, 33 had available matched FFPE specimens. Using HRM, 2/33 (6%) demonstrated suboptimal template amplification, and 2/33 (6%) expressed an erroneous wild-type KRAS profile. Using direct sequencing, 6/33 (18.1%) displayed a wild-type KRAS status, and 3/33 (9.1%) showed discordant mutations. Finally, the detection of KRAS mutations was lower among the FFPE samples compared with the freshly frozen samples, demonstrating that tissue processing clearly impacts the accuracy of KRAS genotyping.

High ratio of interfollicular CD8/FOXP3-positive regulatory T cells is associated with a high FLIPI index and poor overall survival in follicular lymphoma.

Several studies have highlighted the importance of the microenvironment in the behaviour of follicular lymphoma (FL). We conducted an immunohistochemical analysis to assess the role of different cell subpopulations, i.e. CD8, CD68 and forkhead box protein P3 (FOXP3)-positive regulatory T cells (Treg cells) in 84 lymph nodes of 58 patients with FL (58 at diagnosis, 26 at relapse). Since the overall effect of Tregs is considered to depend on their number in relation to CD8(+) effector T cells, we determined the corresponding ratio for each patient and correlated the results with clinical parameters. The interfollicular CD8/FOXP3(+) cell ratio was significantly higher in patients with histological grade 3 tumours (2.04 vs. 1.63) and with a high risk FLIPI index (2.99 vs. 1.53) compared to those with grade 1-2 tumours or a low-intermediate FLIPI index. Similar results were obtained for the follicular CD8(+)/FOXP3(+) cell ratio. The interfollicular CD8/FOXP3 ratio was found to have prognostic value [a 5-year overall survival (OS) of 82 vs. 59% for a ratio of ±1.68]. In addition, an interfollicular FOXP3(+) cell number of more than 86 cells/mm(2) was correlated with a more favourable outcome (p=0.03). When patients at diagnosis and relapse were compared, a significant difference (p=0.05) in the localization (interfollicular vs. intrafollicular) of FOXP3(+) cells was observed. The CD8/FOXP3 ratio in the interfollicular areas was significantly different (1.66 at diagnosis vs. 2.2 at relapse, p=0.05). The presence of a small number of FOXP3(+) cells with a high CD8/FOXP3 ratio is probably the indicator of an active immune response during tumour development, with lymphoma cells acting as targets or bystanders.