RAS testing for colorectal cancer patients is reliable in European laboratories that pass external quality assessment.

Wild-type status of KRAS and the NRAS gene (exon 2, 3, and 4) in the tumor should be determined before treatment of metastatic colorectal cancer (mCRC) patients with EGFR-targeting agents. There is a large variation in test methods to determine RAS status, and more sensitive detection methods were recently introduced. Data from quality assessment programs indicate substantial error rates. This study assessed the completeness and correctness of RAS testing in European laboratories that successfully passed external quality assessment (EQA). Participants were requested to send material of their most recent ten patients with mCRC who had been tested for RAS status. Isolated DNA, a hematoxylin and eosin stained tissue slide with a marked area for macrodissection and accompanying patient reports were requested. Samples were reevaluated in a reference laboratory by using a next-generation sequencing approach. In total, 31 laboratories sent in the requested material (n = 309). Despite regulations for anti-EGFR therapy, one institute did not perform full RAS testing. Reanalysis was possible for 274 samples with sufficient DNA available. In the hotspot codons of KRAS and NRAS, seven discordant results were obtained in total, five of them leading to a different prediction of anti-EGFR therapy efficacy (2%; n = 274). Results show that oncologists can rely on the quality of laboratories with good performance in EQA. Oncologists need to be aware that the testing laboratory participates successfully in EQA programs. Some EQA providers list the good performing laboratories on their website.

In situ detection of antigen-specific T cells in cryo-sections using MHC class I tetramers after dendritic cell vaccination of melanoma patients.

Application of tetrameric MHC class I-peptide complexes has significantly improved the monitoring of antigen-specific T cell immune responses in mouse models as well as in clinical studies. Especially MHC class I tetramer analysis of tumor-specific T cells in suspension or on thick vibratome sections from viable tissue has been proven extremely useful. Using the well-characterized mouse tyrosinase-related-protein-2 specific cytotoxic T cell (CTL) clone LP9, we now developed a method that allows for specific identification of T cells with MHC class I tetramers in 8 mum thick, chemically fixed cryosections. The protocol was validated in a murine influenza virus-infection model. Moreover, analysis of delayed type hypersensitivity (DTH) skin biopsies from melanoma patients vaccinated with peptide-loaded mature dendritic cells, revealed the presence and location of anti-tumor CTLs. The specificity of the CTLs detected in situ correlated with both the DTH challenge specificity and reactivity of cell suspensions derived from the same biopsies. Collectively, our data demonstrate that in situ MHC class I tetramer staining provides a valuable tool to reveal the presence and anatomical location of specific CTLs in frozen tissue following immune-based treatment strategies in cancer patients.

Improved resolution by mounting of tissue sections for laser microdissection.

BACKGROUND: Laser microbeam microdissection has greatly facilitated the procurement of specific cell populations from tissue sections. However, the fact that a coverslip is not used means that the morphology of the tissue sections is often poor. AIMS: To develop a mounting method that greatly improves the morphological quality of tissue sections for laser microbeam microdissection purposes so that the identification of target cells can be facilitated. METHODS: Fresh frozen tissue and formalin fixed, paraffin wax embedded tissue specimens were used to test the morphological quality of mounted and unmounted tissue. The mounting solution consisted of an adhesive gum and blue ink diluted in water. Interference of the mounting solution with DNA quality was analysed by the polymerase chain reaction using 10-2000 cells isolated by microdissection from mounted and unmounted tissue. RESULTS: The mounting solution greatly improved the morphology of tissue sections for laser microdissection purposes and had no detrimental effects on the isolation and efficiency of amplification of DNA. One disadvantage was that the mounting solution reduced the cutting efficiency of the ultraviolet laser. To minimise this effect, the mounting solution should be diluted as much as possible. Furthermore, the addition of blue ink to the mounting medium restores the cutting efficiency of the laser. CONCLUSIONS: The mounting solution is easy to prepare and apply and can be combined with various staining methods without compromising the quality of the DNA extracted.