A multicentre validation of Metasin: a molecular assay for the intraoperative assessment of sentinel lymph nodes from breast cancer patients.

Aims: Treatment strategies for breast cancer continue to evolve. No uniformity exists in the UK for the management of node-positive breast cancer patients. Most centres continue to use conventional histopathology of sampled sentinel lymph nodes (SLNs), which requires delayed axillary clearance in up to 25% of patients.

Measuring gene expression from cell cultures by quantitative reverse-transcriptase polymerase chain reaction.

Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) offers a robust method for the measurement of RNA levels for any gene within cells harvested at any point before or during cell culture. The key elements of RNA extraction followed by a two-step qRT-PCR method (reverse transcription and PCR) are described, followed by a brief section on analysis of the results. There are a number of excellent kits available commercially for much of this work, but it is essential to ensure that the quality and quantity of cDNA produced is adequate for the standard PCR or array to be used.

The molecular basis of the chemosensitivity of metastatic cutaneous melanoma to chemotherapy.

Background: Chemotherapy benefits relatively few patients with cutaneous melanoma. The assessment of tumour chemosensitivity by the ATP-based tumour chemosensitivity assay (ATP-TCA) has shown strong correlation with outcome in cutaneous melanoma, but requires fresh tissue and dedicated laboratory facilities.

Aim: To examine whether the results of the ATP-TCA correlate with the expression of genes known to be involved in resistance to chemotherapy, based on the hypothesis that the molecular basis of chemosensitivity lies within known drug resistance mechanisms.

Resistance gene expression determines the in vitro chemosensitivity of non-small cell lung cancer (NSCLC).

Background: NSCLC exhibits considerable heterogeneity in its sensitivity to chemotherapy and similar heterogeneity is noted in vitro in a variety of model systems. This study has tested the hypothesis that the molecular basis of the observed in vitro chemosensitivity of NSCLC lies within the known resistance mechanisms inherent to these patients' tumors.

Methods: The chemosensitivity of a series of 49 NSCLC tumors was assessed using the ATP-based tumor chemosensitivity assay (ATP-TCA) and compared with quantitative expression of resistance genes measured by RT-PCR in a Taqman Array following extraction of RNA from formalin-fixed paraffin-embedded (FFPE) tissue.

Activation of tonsil dendritic cells with immuno-adjuvants.

Background: Dendritic cells (DC) play the key role in directing antigen-specific immune responses and manipulating their function may be a useful tool for immunotherapy. The balance between immune stimulation and tolerance is particularly important at mucosal interfaces, where discrimination between dangerous pathogens and innocuous antigens takes place. In humans, although much is known about the responses of monocyte derived DC, relatively little is known about effect of immuno-stimulatory adjuvants on DC found in tonsil.

Screening autoantibody profiles in systemic rheumatic disease with a diagnostic protein microarray that uses a filtration-assisted nanodot array luminometric immunoassay (NALIA).

Background: We developed a cost-efficient modular system for multiplex analysis of the multiple autoantibodies that characterize systemic rheumatoid diseases.

Methods: The nanodot array luminometric immunoassay (NALIA) system consists of conventional 96-well membrane-bottomed plates in which antigens or antibodies are adsorbed onto the underside of the membrane. Current arrays use a 5 x 5 format (25 dots/well), which allows 10 analytes to be measured in duplicate: double-stranded DNA (dsDNA), centromere protein B (CENP-B), PCNA, Sm, Sm ribonucleoprotein (Sm-RNP), U1-snRNP, Scl70, SSA/Ro, SSB/La, Jo-1, and controls.

Cancer cell adaptation to chemotherapy.

Background: Tumor resistance to chemotherapy may be present at the beginning of treatment, develop during treatment, or become apparent on re-treatment of the patient. The mechanisms involved are usually inferred from experiments with cell lines, as studies in tumor-derived cells are difficult. Studies of human tumors show that cells adapt to chemotherapy, but it has been largely assumed that clonal selection leads to the resistance of recurrent tumors.

Rapid up-regulation of cyclooxygenase-2 by 5-fluorouracil in human solid tumors.

Inhibition of cyclooxygenase (COX)-2 has been associated with reduced growth of malignant cells. Current therapy of gastrointestinal carcinomas involves the use of 5-fluorouracil (5-FU)-based chemotherapy and we have therefore studied the effect of this agent on the expression of COX-2. COX-2 expression was measured by quantitative RT-PCR in biopsies from a series of 14 esophageal carcinomas, six of which had paired samples taken before and after chemotherapy, and in tumor-derived cells exposed to 5-FU in vitro from a series of 44 tumors, including breast, ovarian, esophageal and colonic carcinomas.