Assays For Occult Metastases Rtpcr

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A study was initiated to develop a highly sensitive method to detect micrometas-tases by examining lymph nodes for the presence of tyrosinase messenger RNA (mRNA) [9]. The assay is based on the biosynthetic pathway of melanin. It is known that tyrosine is converted to melanin in the melanocyte or melanoma cell. The key is that the first two steps of the synthesis are catalyzed by the enzyme tyrosinase. Tyrosinase is a mono-oxygenase that catalyzes the conversion of tyro-sine to 3,4-dihydroxyphenylalanine (DOPA) and of DOPA to dopaquinone. Tyro-sinase is one of the most specific markers of melanocytic differentiation. All cells of the body will have the gene for tyrosinase, but only cells that are actively producing pigment, such as melanoma cells or melanocytes, will express the mRNA for the tyrosinase gene. If this gene product is found in the lymph node preparation, in the peripheral blood, or in the bone marrow, then that finding is good evidence that metastatic melanoma cells are present in that compartment.

The test was modified and refined for lymph node work from an assay originally described by Smith and colleagues for peripheral blood using the combination of reverse transcription and two rounds of RT-PCR [10]. mRNA from the node is converted into a cDNA copy so that a stable product is obtained. The amplified samples were separated on a 2% agarose gel to examine for the presence of a 207-base pair (bp) fragment representing tyrosinase cDNA (Fig. 3). In a spiking experiment, one SK-Mel-28 melanoma cell in 1 million normal lymphocytes could be detected, indicating that the sensitivity of this method is two orders of magnitude greater than routine H&E examination.

In an initial study, sentinel nodes from 29 patients were analyzed by standard pathological staining and RT-PCR. Eleven of 29 lymph nodes samples (38%) from 29 patients with intermediate thickness melanoma were histologi-cally positive. Nineteen of the 29 lymph node preparations (66%) were RT-PCR

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Figure 3 Gel of the tyrosinase RT-PCR assay with a positive signal being the 207-bp signal. Lane 9 is a melanoma control cell line that acts as the positive control. Lanes 2-4 are a breast cancer cell line, a colon cancer cell line, and normal lymph node, respectively, all of which are negative. Lanes 5-8 are from mRNA preparations of the sentinel nodes of three patients who were called histologically negative by the pathologist. In 75%, there is evidence of missed micrometastatic disease.

Figure 3 Gel of the tyrosinase RT-PCR assay with a positive signal being the 207-bp signal. Lane 9 is a melanoma control cell line that acts as the positive control. Lanes 2-4 are a breast cancer cell line, a colon cancer cell line, and normal lymph node, respectively, all of which are negative. Lanes 5-8 are from mRNA preparations of the sentinel nodes of three patients who were called histologically negative by the pathologist. In 75%, there is evidence of missed micrometastatic disease.

positive and these included all of the pathologically positive samples. Restriction enzyme analysis showed that the amplified 207-bp RT-PCR product was a part of the tyrosinase gene sequence. These data suggested that the RT-PCR method was an extremely sensitive, reproducible, and efficient technique for the identification of micrometastases in patients with melanoma [9].

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