Gene expression profiles of tumor-positive sentinel lymph node biopsies from cutaneous melanoma patients. Homo sapiens

Sentinel lymph node, the first node draining the primary tumor, is a key component of tumor microenvironment promoting immune tolerance. In melanoma, sentinel node is one of the most important prognostic factors and the most frequent site of regional metastasis. To unravel the immunomodulatory pathways that are triggered by melanoma cells in the draining node that allow tumor spreading, transcriptional profiles associated to disease progression were analyzed in archival sentinel node biopsies (SNB). Gene expression profiles of melanoma-positive and negative SNB selected to maximize the differences in terms of disease stage and course, revealed subgroups correlating with regional node involvement and disease progression within positive biopsies. Transcriptional profiles revealed that genes showing differential expression between tumor-positive SNB with/without disease progression were mainly related to inflammatory response and that were mostly down regulated in patients with poor prognosis. TNFRSF8 encoding CD30 showing up regulation in SNB from patients with progressing disease displayed higher expression by immunohistochemical staining compared to SNB from non progressing patients. Subpopulations of CD30 positive CD4/CD8 double negative and CD4 Foxp3/PD-1 CD147 positive T cells were identified by flow cytometry analysis in metastatic nodes, suggesting a potential role of regulatory and tolerogenic T cells in melanoma progression. Overall design: Cutaneous melanoma patients undergoing SNB biopsy in 2001-2004 with available 5–year follow-up clinical data were selected. Data relative to 752 cases was extracted, 80% (n= 603) with a negative SNB and 20% (n=149) with SNB positivity for melanoma metastases. The latter underwent regional lymphadenectomy by CLND and 30% (n=44) resulted positive for melanoma metastases at non-sentinel regional lymph nodes while 70% (n=105) resulted negative. Analysis of follow-up data showed that disease recurrence at 5 yrs occurred in 9% SNB negative patients, 14% of the patients with positive SNB resulting negative at CLND, and in 57% of the patients with positive SNB resulting positive at CLND, consistent to the range of previously reported rates (Santinami M 2009, Balch CM 2009). In order to exploit gene expression profiles to unravel molecular modifications occurring in SNB from patients with progressing disease we selected two groups of cases representing the extremes of the survey, i.e. patients positive at CLND (stage IIIB-C) recurring within 5 years follow-up (SNB-PP), and patients negative at CLND (stage IIIA-B) and non-relapsing at 5 years (SNB-PN). In addition, a group of negative SNB patients without disease recurrence at 5 years was selected and analysed for comparison as tumor negative SNB (SNB-N).

前哨淋巴结（Sentinel lymph node）是引流原发肿瘤的首个淋巴结，是促进免疫耐受的肿瘤微环境关键组成部分。在黑色素瘤中，前哨淋巴结是最重要的预后因素之一，也是区域转移最常见的部位。为阐明黑色素瘤细胞在引流淋巴结中触发的、促进肿瘤扩散的免疫调节通路，研究人员对存档的前哨淋巴结活检（sentinel node biopsies, SNB）样本中与疾病进展相关的转录组谱进行了分析。
选取旨在最大化疾病分期与病程差异的黑色素瘤阳性与阴性SNB的基因表达谱，鉴定出了阳性活检样本中与区域淋巴结受累及疾病进展相关的亚群。转录组谱分析显示，伴与不伴疾病进展的肿瘤阳性SNB之间的差异表达基因主要与炎症应答相关，且在预后不良的患者中大多呈下调表达。编码CD30的TNFRSF8在疾病进展患者的SNB中呈上调表达，免疫组化染色结果显示其表达水平高于无疾病进展患者的SNB。通过流式细胞术，研究人员在转移性淋巴结中鉴定出CD30阳性CD4/CD8双阴性T细胞与CD4 Foxp3/PD-1 CD147阳性T细胞亚群，提示调节性与耐受原性T细胞可能在黑色素瘤进展中发挥潜在作用。
总体实验设计：选取2001-2004年间接受前哨淋巴结活检、且具备5年随访临床数据的皮肤黑色素瘤患者。最终纳入752例病例的相关数据，其中80%（n=603）为SNB阴性患者，20%（n=149）为SNB黑色素瘤转移阳性患者。后者接受了区域淋巴结清扫术（complete lymph node dissection, CLND），其中30%（n=44）在非前哨区域淋巴结检出黑色素瘤转移，70%（n=105）未检出。随访数据分析显示，5年内疾病复发率在SNB阴性患者中为9%，在SNB阳性但区域淋巴结清扫术后阴性的患者中为14%，在SNB阳性且区域淋巴结清扫术后仍阳性的患者中为57%，这与此前报道的发生率范围相符（Santinami M 2009, Balch CM 2009）。为借助基因表达谱阐明疾病进展患者SNB中发生的分子改变，研究人员选取了研究队列中的两个极端表型组：即区域淋巴结清扫术后阳性且处于ⅢB-C期、在5年随访期内复发的患者（SNB-PP），以及区域淋巴结清扫术后阴性且处于ⅢA-B期、5年内未复发的患者（SNB-PN）。此外，还选取了一组SNB阴性且5年内无疾病复发的患者作为肿瘤阴性SNB对照（SNB-N）进行分析。