Wang X, Heller R, VanVoorhis N, Cruse C W, Glass F, Fenske N, Berman C, Leo-Messina J, Rappaport D, Wells K
Cutaneous Oncology Program, Moffitt Cancer Center, University of South Florida, Tampa.
Ann Surg. 1994 Dec;220(6):768-74. doi: 10.1097/00000658-199412000-00010.
The presence or absence of lymph node metastases in patients with malignant melanoma is the most powerful prognostic factor for predicting survival. If regional nodal metastases are found, the 5-year survival for the patient decreases approximately 50%. If the presence or absence of regional nodal metastases will determine which patients receive formal dissections or which patients enter adjuvant trials, then a technique is needed to accurately screen lymph node samples for occult disease. Routine histopathologic examination routinely underestimates the number of patients with metastases. This study was initiated to develop a highly sensitive clinically applicable method to detect micrometastases by examining lymph nodes for the presence of tyrosinase messenger RNA (mRNA). The hypothesis was that if mRNA for tyrosinase is found in the lymph node preparation, that finding is good evidence that metastatic melanoma cells are present.
The assay is accomplished using the combination of reverse transcription and double-round polymerase chain reaction (RT-PCR). The amplified samples are examined on a 2% agarose gel and tyrosinase cDNA is seen as a 207 base pair fragment. Lymph node preparations from 29 patients who were clinically stage I and II and undergoing elective node dissections were analyzed both by standard pathologic staining and RT-PCR.
Eleven of 29 lymph node (38%) samples from 29 patients with intermediate thickness melanoma were pathologically positive. Nineteen of the 29 lymph node preparations (66%) were RT-PCR-positive, and these included all of the pathologically positive samples, so that the false-negative rate was 0. In a spiking experiment, one SK-Mel-28 melanoma cell in a background of one million normal lymphocytes could be detected, thus indicating the sensitivity of this method. In addition, analysis by restriction enzyme mapping showed that the amplified 207-bp PCR product produced is part of the tyrosinase gene sequence.
The RT-PCR method is an extremely sensitive, reproducible, and efficient technique for the identification of micrometastases in patients with melanoma and could be widely applicable. If clinical correlation is obtained, staging of the melanoma patient becomes more accurate, and treatment becomes more standardized and rational, because all those patients who have evidence of nodal disease can be identified so that they may benefit from more extensive surgery (formal node dissections) or adjuvant therapies. Based on these results, RT-PCR could be a powerful tool to detect micrometastatic melanoma.
恶性黑色素瘤患者有无淋巴结转移是预测生存的最有力预后因素。如果发现区域淋巴结转移，患者的5年生存率会降低约50%。如果区域淋巴结转移的有无将决定哪些患者接受根治性清扫或哪些患者进入辅助治疗试验，那么就需要一种技术来准确筛查淋巴结样本中的隐匿性疾病。常规组织病理学检查常常低估有转移的患者数量。本研究旨在开发一种高度敏感的临床适用方法，通过检测淋巴结中酪氨酸酶信使核糖核酸（mRNA）的存在来检测微转移。假设是如果在淋巴结标本中发现酪氨酸酶的mRNA，那么这一发现就是存在转移性黑色素瘤细胞的有力证据。
该检测通过逆转录和双轮聚合酶链反应（RT-PCR）相结合来完成。扩增后的样本在2%琼脂糖凝胶上进行检测，酪氨酸酶互补脱氧核糖核酸（cDNA）表现为一个207碱基对的片段。对29例临床分期为I期和II期且正在接受选择性淋巴结清扫的患者的淋巴结标本进行标准病理染色和RT-PCR分析。
29例中等厚度黑色素瘤患者的29个淋巴结样本中，有11个（38%）病理检查呈阳性。29个淋巴结标本中有19个（66%）RT-PCR呈阳性，且这些标本包括了所有病理检查呈阳性的样本，因此假阴性率为0。在一个加样实验中，在一百万个正常淋巴细胞背景下的一个SK-Mel-28黑色素瘤细胞能够被检测到，从而表明了该方法的敏感性。此外，通过限制性酶切图谱分析表明，所产生的扩增207碱基对PCR产物是酪氨酸酶基因序列的一部分。
RT-PCR方法是一种极其敏感、可重复且高效的技术，用于识别黑色素瘤患者中的微转移，并且可能具有广泛的适用性。如果获得临床相关性，黑色素瘤患者的分期将变得更加准确，治疗将变得更加标准化和合理，因为所有有淋巴结疾病证据的患者都能被识别出来，以便他们能从更广泛的手术（根治性淋巴结清扫）或辅助治疗中获益。基于这些结果，RT-PCR可能是检测微转移性黑色素瘤的有力工具。
