Quantitative real-time RT-PCR in sentinel lymph nodes from melanoma patients. Detection of melanocytic mRNA predicts disease-free survival.

Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) for specific melanoma markers is more sensitive than histology for detecting cells of melanocytic origin in sentinel lymph nodes (SLNs) in cutaneous melanoma. The clinical significance of a positive qRT-PCR analysis is unclear. We performed qRT-PCR for the presence of MART-1 and tyrosinase in SLNs from 93 melanoma patients, and then followed these patients clinically (median follow-up time 43.

Pathologic assessment of melanoma sentinel nodes: a role for molecular analysis using quantitative real-time reverse transcription-PCR for MART-1 and tyrosinase messenger RNA.

Purpose: Molecular analysis of melanoma sentinel nodes (SN) is sensitive, but poorly specific because metastases cannot be distinguished from benign nevus inclusions (BNI). We investigated whether quantitative reverse transcription-PCR (RT-PCR) detection of MART-1 and tyrosinase mRNAs could improve this specificity and contribute to SN assessment.

Experimental Design: Two hundred twenty SNs from 95 melanoma patients analyzed by extensive immunohistopathology and real-time quantitative RT-PCR.

The influence of immunohistochemistry on mRNA recovery from microdissected frozen and formalin-fixed, paraffin-embedded sections.

Laser-assisted microdissection (LAM) is now widely used to obtain specific cell populations from heterogeneous tissues. A major disadvantage of LAM is poor tissue morphology during microscopy, in part because coverslips are not used. Immunohistochemical labeling can improve identification of target cells but may affect the subsequent analysis of the microdissected tissue.

Quantification of melanoma mRNA markers in sentinel nodes: pre-clinical evaluation of a single-step real-time reverse transcriptase-polymerase chain reaction assay.

Although reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of melanocyte-associated mRNA can detect sentinel node melanoma metastases, most published assays are semi-quantitative methods of unknown sensitivity and precision, unsuitable for clinical use. We describe a single-step real-time quantitative RT-PCR assay for MART-1 and tyrosinase mRNAs, suitable for sentinel node analysis in a clinical setting. Using serial dilutions of melanoma cell line SK-MEL-28 RNA in water as a calibrator, we obtained linear calibration curves covering the range 0.

Sentinel lymph nodes in malignant melanoma: extended histopathologic evaluation improves diagnostic precision.

Background: The optimal technique for sentinel lymph node (SN) assessment in patients with melanoma is controversial. Molecular analysis (reverse transcriptase-polymerase chain reaction) detects significantly greater numbers of SNs with suspected micrometastases (up to 71%) than does routine histopathology (approximately 20%). The authors sought to identify possible reasons for this discrepancy and to determine whether using an extended histopathologic protocol could improve diagnostic precision.

Towards quantitative mRNA analysis in paraffin-embedded tissues using real-time reverse transcriptase-polymerase chain reaction: a methodological study on lymph nodes from melanoma patients.

Improved extraction techniques combined with sensitive real-time reverse transcriptase-polymerase chain reaction may allow detection of mRNA in formalin-fixed, paraffin-embedded (FFPE) materials, but the factors affecting mRNA quantification in clinical material using these methods have not been systematically analyzed. We designed analyses using real-time reverse transcriptase-polymerase chain reaction for quantification of MART-1, beta-actin, and beta(2)-microglobulin mRNAs. The analytical intra- and interassay imprecision (coefficient of variation) was in the range 10 to 20% for all three genes studied.