We have developed a new assay, ISET (isolation by size of epithelial tumor cells), which allows the counting and the immunomorphological and molecular characterization of circulating tumor cells in patients with carcinoma, using peripheral blood sample volumes as small as 1 ml. Using this assay, epithelial tumor cells can be isolated individually by filtration because of their larger size when compared to peripheral blood leukocytes. ISET parameters were defined using peripheral blood spiked with tumor cell lines (HepG2, Hep3B, MCF-7, HeLa, and LNCaP). ISET can detect a single, micropipetted tumor cell, added to 1 ml of blood. We also demonstrate that fluorescence in situ hybridization can be used to perform chromosomal analyses on tumor cells collected using ISET. Polymerase chain reaction-based genetic analyses can be applied to ISET-isolated cells, and, as an example, we demonstrate homozygous p53 deletion in single Hep3B cells after filtration and laser microdissection. Finally, we provide evidence for the in vivo feasibility of ISET in patients with hepatocellular carcinoma undergoing tumor resection. ISET, but not reverse transcriptase-polymerase chain reaction, allowed analysis of cell morphology, counting of tumor cells, and demonstration of tumor microemboli spread into peripheral blood during surgery. Overall, ISET constitutes a novel approach that should open new perpectives in molecular medicine.

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In recent years the growing interest in quantitative applications of the polymerase chain reaction (PCR) has favoured the development of a large number of assay procedures suitable for this purpose. In this paper we review some basic principles of quantitative PCR and in particular the role of reference materials and calibrators and the different strategies adopted for nucleic acid quantification. We focus on two methodological approaches for quantitative PCR in this review: competitive PCR and real-time quantitative PCR based on the use of fluorogenic probes. The first is one of the most common methods of quantitative PCR and we discuss the structure of the competitors and the various assay procedures. The second section is dedicated to a recent promising technology for quantitative PCR in which the use of fluorogenic probes and dedicated instrumentation allows the development of homogeneous methods. Assay performance of these methods in terms of practicability and reliability indicates that these kinds of technologies will have a widespread use in the clinical laboratory in the near future.

High-resolution melting analysis (HRMA) provides a valid approach to efficiently detect DNA genetic and somatic mutations. In this study, HRMA was used for the screening of 116 colorectal cancers (CRCs) to detect hot-spot mutations in the KRAS and BRAF oncogenes. Mutational hot spots on the PIK3CA gene, exons 9 and 20, were also screened. Direct sequencing was used to confirm and characterize HRMA results. HRMA revealed abnormal melting profiles in 65 CRCs (56.0%), 16 of them harboring mutations in 2 different genes simultaneously. The frequency of mutations was 17.2% for PIK3CA (11.2% in exon 9 and 6.0% in exon 20), 43.1% for KRAS exon 2, and 9.5% in exon 15 of the BRAF gene. We found a significant association between PIK3CA and KRAS mutations (P = .008), whereas KRAS and BRAF mutations were mutually exclusive (P = .001). This report describes a novel approach for the detection of PIK3CA somatic mutations by HRMA.

Analysis of circulating tumor cells (CTC) in the peripheral blood of cutaneous melanoma patients provides information on the metastatic process and potentially improves patient management. The isolation by size of epithelial tumor cells (ISET) is a direct method for CTC identification in which tumor cells are collected by filtration as a result of their large size. So far, ISET has been applied only to CTC detection from epithelial cancer patients, and the technique has never been applied to cutaneous melanoma patients. We herein investigated the presence of CTC by ISET in the peripheral blood of 140 subjects (87 with cutaneous melanomas, 10 subjects undergoing surgery for melanocytic nevi, 5 patients with non-melanoma skin tumors, and 38 healthy volunteers). The identification of the cells trapped in filters as CTC was supported by positivity for immunohistochemical markers and for tyrosinase mRNA by real-time RT-PCR. CTC were neither detected in the controls nor in the in situ melanoma group. In contrast, CTC were shown in 29% of patients with primary invasive melanoma and in 62.5% of metastatic melanoma patients (P<0.01). CTC detection correlated with the presence of mRNA tyrosinase in blood samples, assayed by real-time RT-PCR (P=0.001). CTC detection corroborated by suitable molecular characterization may assist in the identification and monitoring of more appropriate therapies in melanoma patients.