Signature patterns of human papillomavirus type 16 in invasive anal carcinoma.

Many studies have reported that most invasive anal carcinomas contain high-risk human papillomaviruses (HPVs), HPV16 being the most prevalent type. This study aimed to investigate HPV status and cellular biomarkers in invasive anal cancers. HPV genotype distribution was determined in 76 anal cancers by the INNO-LiPA assay (Innogenetics, Gent, Belgium). HPV16-positive samples were then tested for viral load and physical state with type-specific real-time polymerase chain reaction targeting E6, E2, and albumin genes. Samples were also subjected to immunohistochemical analysis of p16, Ki-67, p53, and p21. Of the analyzable tumors, 98.6% were positive for α-HPV DNA. HPV16 was the most prevalent genotype (89.0%), followed by HPV39 (4.1%) and HPV33 (2.7%). HPV16 viral load was high, ranging from 2.1 × 10(3) to 1.5 × 10(7) copies/10(3) cells. Integration of HPV16 estimated by the E2/E6 ratio was detected in 77.8% of cases, among which 70.4% were mixed integrated and episomal DNA cases and 7.4% were fully integrated DNA cases. The latter cases were associated with a low HPV16 load compared with cases containing either episomes or mixed integrated and episomal DNA. As expected, most HPV16-positive tumors expressed p16 (92.6%) with a high proliferative index, whereas a minority of them overexpressed p53 (10.3%). p21 expression did not appear to correlate with p53 expression. Although HPV16 was almost exclusively detected, high viral load and differences in DNA integration have been identified in the present series of anal cancers. HPV features assessed in conjunction with expression of cell-cycle regulators could be helpful, as joint biomarkers, in predicting clinical outcome.

A multiplex SNaPshot assay as a rapid method for detecting KRAS and BRAF mutations in advanced colorectal cancers.

The analysis of KRAS mutations has become a prerequisite for anti-epidermal growth factor receptor therapy in patients with metastatic colorectal cancers. KRAS mutations are associated with resistance to treatment by monoclonal antibodies such as cetuximab and panitumumab and thus are correlated with a shorter progression-free survival. BRAF mutations also may play a role in treatment decisions. The widespread use of these targeted therapies has generated the need to develop cost-effective methods for routine KRAS and BRAF analysis. The aim of this study was to compare a multiplex SNaPshot assay with DNA sequencing and high-resolution melting analysis for identifying KRAS codons 12 and 13 and BRAF codon 600 mutations. Thus 110 routinely formalin-fixed and paraffin-embedded tissue blocks were tested by each method. The SNaPshot analysis detected KRAS and BRAF codon 600 mutations in, respectively, 34.5% (n = 38) and 10% (n = 11) of these tissue blocks. These results were confirmed by direct DNA sequencing and by high-resolution melting analysis. The costs and time constraints of each detection method were compared at the same time. In conclusion, our newly designed multiplex SNaPshot assay is a fast, inexpensive, sensitive, and robust technique for molecular diagnostic practices and patient selection.