Detection of neuroblastoma cells in bone marrow and peripheral blood at diagnosis by the reverse transcriptase-polymerase chain reaction for tyrosine hydroxylase mRNA

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Abstract
Background. Bone marrow metastasis often occurs in patients with neuroblastoma; therefore, a sensitive assay to detect occult neuroblastoma cells in bone marrow (BM) and peripheral blood (PB) is needed. The feasibility and clinical value of using the reverse transcriptase- (RT) polymerase chain reaction (PCR) to amplify mRNA for tyrosine hydroxylase (TH), the first enzyme of catecholamine synthesis, was evaluated to detect neuroblastoma cells in patient samples. Methods. Thirty-eight patients with Stages I to IV neuroblastoma and eight healthy donors were included in this study. Bone marrow and PB samples obtained at diagnosis were examined for TH mRNA. After preparation of complementary DNA, the PCR was performed to amplify the TH gene. Results. Tyrosine hydroxylase mRNA was detected in neuroblastoma samples including a cell line and tumor tissues, but was not detected in normal BM or PB mononuclear cells. Neuroblastoma cells were detected at a level of 1 per 105-6 normal PB mononuclear cells by this method. Tyrosine hydroxylase mRNA was detected in 18 of 38 BM samples, and all 12 BM samples with cytologic evidence of tumor cells were positive for TH mRNA by the RT-PCR. Six of 26 patients without cytologic evidence of tumor cells in the BM were also positive for TH mRNA. TH mRNA was detected in BM samples from 1 of 14 patients with Stage I disease, 2 of 7 patients with Stage II disease, 1 of 3 patients with Stage III disease and all patients with Stage IV (11 patients) and IVS (3 patients) diseases. Tyrosine hydroxylase mRNA also was detected in 8 of 14 PB samples (one of five patients in Stages I, II or III and 7 of 9 in Stage IV or IVS). Conclusions. Reverse transcriptase-polymerase chain reaction amplification of TH mRNA was a sensitive and specific method of detecting occult neuroblastoma cells in BM and PB samples. Neuroblastoma cells could be detected by this method in some BM samples that had no cytologic evidence of tumor cells and in some PB samples at the time of diagnosis. The clinical significance of these very low levels of neuroblastoma cells detected by RT-PCR requires further investigation. Cancer 1995;75 2757–61.