Technical reproducibility of single-nucleotide and size-based DNA biomarker assessment using DNA extracted from formalin-fixed, paraffin-embedded tissues.

DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues has been used in the past to analyze genetic polymorphisms. We evaluated the technical reproducibility of different types of assays for gene polymorphisms using DNA extracted from FFPE material. By using the MassARRAY iPLEX system, we investigated polymorphisms in DPYD (rs1801159 and rs3918290), UMPS (rs1801019), ERCC1 (rs11615), ERCC1 (rs3212986), and ERCC2 (rs13181) in 56 FFPE DNA samples. By using PCR, followed by size-based gel electrophoresis, we also examined TYMS 5' untranslated region 2R/3R repeats and GSTT1 deletions in 50 FFPE DNA samples and 34 DNAs extracted from fresh-frozen tissues and cell lines. Each polymorphism was analyzed by two independent runs. We found that iPLEX biomarker assays measuring single-nucleotide polymorphisms provided consistent concordant results. However, by using FFPE DNA, size-based PCR biomarkers (GSTT1 and TYMS 5' untranslated region) were discrepant in 32.7% (16/49, with exact 95% CI, 19.9%-47.5%; exact binomial confidence limit test) and 4.2% (2/48, with exact 95% CI, 0.5%-14.3%) of cases, respectively, whereas no discrepancies were observed using intact genomic DNA. Our findings suggest that DNA from FFPE material can be used to reliably test single-nucleotide polymorphisms. However, results based on size-based PCR biomarkers, and particularly GSTT1 deletions, using FFPE DNA need to be interpreted with caution. Independent repeated assays should be performed on all cases to assess potential discrepancies.

Current perspective: bevacizumab in colorectal cancer--a time for reappraisal?

Bevacizumab was the first anti-angiogenic drug to be licensed in malignant disease, based on the results of a randomised trial in advanced colorectal cancer, in which the addition of bevacizumab to chemotherapy with irinotecan plus fluorouracil/leucovorin (IFL) significantly improved tumour response, progression-free survival (PFS) and overall survival (median 15.6-20.3 months, p<0.001). A subsequent randomised trial of bevacizumab combined with fluoropyrimidine and oxaliplatin (FOLFOX or CAPOX) confirmed an improvement in PFS, but without a survival benefit, probably due to the limited duration of bevacizumab treatment. However, in the second-line setting a randomised trial of bevacizumab combined with FOLFOX showed a significant improvement in survival, similar to that observed with IFL in the first-line. A benefit from the use of bevacizumab plus chemotherapy beyond progression remains unproven but data from non-randomised trials are encouraging. In contrast, bevacizumab monotherapy has limited efficacy in advanced disease and currently there are no data to support maintenance monotherapy. Bevacizumab is recognised to cause hypertension, arterial and venous thrombosis, intestinal perforation and impairment of wound healing but can be safely used in patients undergoing surgery, particularly when the timing of surgery is controlled. At the 2009 ASCO annual meeting, the first adjuvant study to report its primary end-point, NSABP protocol C-08, failed to demonstrate an improvement in 3-year disease-free survival from the addition of bevacizumab to modified FOLFOX6 for resected stage II/III disease. Health economics have unfortunately limited the universal use of bevacizumab, but it is hoped that the future identification of predictive biomarkers may enhance the benefits and thereby improve cost-effectiveness.