Project 2: Molecular Approaches for Assessing Metastasis and Disease Relapse

项目 2：评估转移和疾病复发的分子方法

• 批准号: 7728757
• 负责人: Dave S B Hoon
• 金额: $ 16.08万
• 依托单位: SAINT JOHN'S CANCER INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7728757
• 关键词: Alleles; Antimony; Area; Australia; Biological Assay; Biological Markers; Biopsy; Blinded; Blood; Clinical; Concentration measurement; Coupled; CpG Island Methylator Phenotype; CpG Islands; Cutaneous Melanoma; Cytokine Inducible SH2-Containing Protein; DNA; DNA Markers; Data; Deposition; Development; Diagnostic Neoplasm Staging; Dimensions; Discipline of Nuclear Medicine; Disease Outcome; Dopachrome isomerase; Epigenetic Process; Evaluable Disease; Failure; Family; Functional RNA; Functional disorder; GATA4 transcription factor; Genes; Genomics; Genotype; Histology; Histopathology; Immunohistochemistry; Link; Lymph Node Dissections; Lymph node excision; MALDI-TOF Mass Spectrometry; Malignant Neoplasms; Malignant neoplasm of gastrointestinal tract; Mass Spectrum Analysis; Messenger RNA; Metastatic Melanoma; Methods; Methylation; Modality; Molecular; Monitor; Neoplasm Metastasis; Nodal; Operative Surgical Procedures; Outcome; Paraffin Embedding; Pathologist; Patients; Pattern; Physicians; Plasma; Polymerase Chain Reaction; Primary Neoplasm; Prognostic Factor; Promoter Regions; Publications; Recurrence; Recurrent disease; Reverse Transcriptase Polymerase Chain Reaction; Risk; Sentinel; Sentinel Lymph Node; Sentinel Lymph Node Biopsy; Serum; Site; Specimen; Staging; Surgeon; Time; Tissues; Tumor Markers; Tumor Suppressor Proteins; Tumor stage; bisulfite; clinically significant; cohort; improved; lymph nodes; melanoma; outcome forecast; prognostic; programs; retinoic acid receptor beta 2; tissue-factor-pathway inhibitor 2; tumor; tumor progression

The overall studies have been to develop and monitor the molecular prognosis and upstaging of tumordraining lymph nodes and primary melanoma tumors. In addition, the other major program is development of DNA prognostic biomarkers for primary melanomas and serum. Aims I and II: It is clear that a successful sentinel node biopsy (SNB) requires a collaborative effort between the nuclear medicine physician, the surgeon and the pathologist, and a failure in any of these areas may result in an unsatisfactory outcome. The aim of the study was to investigate a cohort of patients with false negative (FN) sentinel node (SNs) to identify deficiencies that may have caused the FN result. A FN SN was defined as a patient who had a negative SNB result but subsequently developed their first recurrence within the biopsied nodal field. In a major collaborative study with the Sydney Melanoma Unit (SMU, Australia); we investigated a cohort of melanoma patients with FN SN biopsies to identify possible reasons for the FN result. Seventy-four SNs from 33 patients found to have had a FN SNB were analyzed by reviewing the lymphoseintigraphy, surgical data and histopathology, and assessing nodal tissue using multimarker real-time quantitative reverse transcriptase polymerase chain reaction (qRT), and antimony concentration measurements (as a marker of "true" SN status) using inductively coupled plasma mass spectroscopy. The qRT studies were performed on archival paraffin-embedded (PE) tissues from SMU. A multimarker real-time qRT assay with 5 mRNA markers (MAGE 3, MART-1, GalNAc-T, PAX-3 and TRP-2) was used as is for the current MLST-II study. Specimens were blinded to the qRT assay performer and clinician. Nine SNs (12%) from 9 patients (27%) were found to have evidence of melanoma on histopathologic review. 12 SNs (16%) from 10 patients (30%) were found to be qRT(+). Four of these 12 SNs were positive on histopathology; 8 were negative. Four patients (12%) were upstaged by qRT. Sixteen patients had their SNB histology, lymphoseintigraphy and surgical data reviewed. Identifiable causes of the FN SNBs were not found after review of all modalities in 4 patients . SNs from all 4 patients had antimony levels indicative of a SN. Of the SNs evaluable by qRT, 1 was qRT(+) and 7 SNs from 2 patients were qRT(-).10 SNs (14%) from 9 of the 33 patients (27%) were found to have evidence of metastatic melanoma on histopathologic review and/or IHC analysis. Of the 8 evaluable by qRT, 5 of these SNs were qRT(+) and 3 were qRT(-). 2 of the qRT(-) SNs with histopathologic/IHC evidence of melanoma had tiny deposits (0.1mm and 0.15mm in maximum dimension). A FN SN can occur because of deficiencies in nuclear medicine, surgery or histopathology. qRT can detect "occult" metastatic melanoma in SNs that were identified as negative by histopathology. These studies confer that qRT upstaging on PE tissue sections can be of clinical utility (Ann Surg, in press, see ref. 10). Currently, we are tracking MLST-I patients from the multicenter site in which patients had SLN(-) that recurred as well as those that had SLN(+) with complete lymph node dissection (CLND) that had negative NSLN. The objective will be to determine if our qRT multimarkers can upstage these different patient cohorts. Tracking of these patients specimens have been started with JWCI and SMU. This year, we will be retrieving these lymph node blocks to perform qRT. Aims III: This Aim is focused on developing genomic prognostic biomarkers in primary tumors and blood. We have focused on developing epigenetic biomarkers examining methylation of gene promoter regions of CpG islands. Several types of assays have been developed such as real time PCR used on a real-time thermocycler, absolute quantitative allele methylation assay (AQAMA) also used on a real-time thermocycler, and Sequnom (MALDI-TOF) mass spectrometry. The approach with the latter two assays is absolute quantitative analysis of genomic DMA from PE tissues and blood. We have been able to develop these assays. In the PE analysis we have assessed 7 MINT markers which are methylated tumor-related loci (non-coding) in PE primary and metastatic tissues. We have demonstrated the prognostic utility of MINT 31 and 17. Along with these markers we have assessed multiple tumor-related genes such as GATA4, GATA binding protein 4; RARbeta2, retinoic acid receptor-beta 2, RASSF1A, Ras association domain family 1A; SOCS-1, suppressor of cytokine signaling-1; TFPI-2, tissue factor pathway inhibitor-2; and WIF-1, Wnt inhibitory factor-1. The CpG island methylator phenotype (CIMP) may be associated with development of malignancy through coordinated inactivation of tumor-suppressor and tumor-related genes (TRGs) and methylation of multiple non-coding, methylated-in-tumor (MINT) loci. These epigenetic changes create a distinct CIMP pattern that has been linked to recurrence and survival in gastrointestinal cancers. Because epigenetic inactivation of TRGs also has been implicated in development and progression of malignant melanoma, we hypothesized the existence of a clinically significant CIMP in cutaneous melanoma. We examined the methylation status of the CpG island promoter region of TRGs related to melanoma pathophysiology and a panel of MINT loci (MINT-1, 2, 3, 12, 17, 25, and 31) in primary and metastatic tumors of different clinical stages. We showed an increase in the extent of methylation of the TRGs WIF-1, TFPI-2, RASSF1A, and SOCS-1 with advancing clinical tumor stage. Furthermore, we find a significant positive association between the methylation status of MINT-17, MINT-31, and TRGs. These findings demonstrate the significance of a CIMP pattern that is associated with advancing clinical stage of malignant melanoma, and may be used to identify primary melanomas that have a high risk of metastasis or recurrence. The study has been submitted for publication. This is the first major finding on epigenetic changes in primary melanomas related to tumor progression. The studies will be further expanded to examine the clinical utility of the CIMP as a prognostic genotype of primary tumors. Studies on circulating DNA are being performed using methylated TRGs and non-coding genomic loci. Methods to improved DNA isolation serum and bisulfite are being revised. We are focusing developing assays to assess non-coding region DNA in the serum.

总体研究是开发和监测肿瘤的分子预后和升级 淋巴结和原发性黑色素瘤肿瘤。此外，另一个主要计划是开发 原发性黑色素瘤和血清的DNA预后生物标志物。 目标I和II：很明显，成功的前哨节点活检（SNB）需要协作努力 在核医学医师，外科医生和病理学家之间以及这些领域的任何一个失败 可能会导致结果不令人满意。该研究的目的是调查一群假患者 负（FN）哨兵节点（SNS）以识别可能导致FN结果的缺陷。一个fn s是 定义为患者的患者，其结果为负结果，但随后在 活检的淋巴结场。在与悉尼黑色素瘤部门（澳大利亚SMU）的一项重大合作研究中；我们 研究了一组FN SN活检的黑色素瘤患者，以确定FN结果的可能原因。 来自33名患者的74例SNS通过审查了FN SNB。 淋巴言语，手术数据和组织病理学以及使用多标记实时评估淋巴结组织 定量逆转录酶聚合酶链反应（QRT）和锑浓度 使用电感耦合等离子体质谱法测量（作为“真” SN状态的标记）。这 QRT研究对SMU的档案石蜡（PE）组织进行。多标记实时 使用5个mRNA标记（法师3，Mart-1，GalNAC-T，PAX-3和TRP-2）的QRT测定法 当前的MLST-II研究。标本对QRT分析的表演者和临床医生视而不见。九个SNS（12％） 发现从9例患者（27％）有组织病理学评论的黑色素瘤证据。 12 SNS（16％） 发现10名患者（30％）为QRT（+）。这12个SN中有四个对组织病理学呈阳性。 8 负面。 QRT升级了四名患者（12％）。 16名患者患有SNB组织学， 审查了淋巴含量和手术数据。未在 审查4例患者的所有方式。来自所有4例患者的SNs均具有矩阵水平，指示SN。的 QRT可评估的SNS是QRT（+）的1个，来自2例患者的7个SN是QRT（ - ）。来自33个中9个的SNS（14％）。 发现患者（27％）有组织病理学检查和/或IHC的转移性黑色素瘤的证据 分析。在QRT可评估的8个中，其中5个SN为QRT（+），3为QRT（ - ）。 qrt（ - ）sn的2个 黑色素瘤的组织病理学/IHC证据的沉积物很小（最大尺寸为0.1mm，0.15mm）。 由于核医学，手术或组织病理学缺乏，可能会发生FN SN。 QRT可以 在SNS中检测“神秘”转移性黑色素瘤，这些转移性黑色素瘤被组织病理学鉴定为阴性。这些研究 允许在PE组织切片上升级的QRT可以是临床效用（Ann Surg，在印刷中，请参见参考文献10）。 目前，我们正在跟踪来自多中心部位的MLST-I患者，其中患者患有SLN（ - ） 复发以及具有完全淋巴结解剖（CLND）的SLN（+）的那些 NSLN。目的是确定我们的QRT多标志物是否可以上升这些不同的患者队列。 JWCI和SMU的这些患者标本的跟踪已经开始。今年，我们将检索 这些淋巴结块要执行QRT。 AIMS III：该目标集中在原发性肿瘤和血液中开发基因组预后生物标志物。 我们的重点是开发研究表观遗传生物标志物，研究了研究基因启动子区域的甲基化 CPG群岛。已经开发了几种类型的测定，例如实时使用的实时PCR 热环生，绝对定量等位基因甲基化测定法（AQAMA）也用于实时热环体， 和Sequnom（Maldi-TOF）质谱法。后两个测定的方法是绝对的 对PE组织和血液中基因组DMA的定量分析。我们已经能够开发这些 测定。在PE分析中，我们评估了7种甲基化肿瘤相关基因座的薄荷标记 （非编码）在PE原发和转移组织中。我们已经证明了薄荷的预后效用31 和17。与这些标记一起，我们评估了多个与肿瘤相关的基因，例如GATA4，GATA 结合蛋白4； rarbeta2，视黄酸受体-beta 2，Rassf1a，Ras关联域家族1A； SOCS-1，抑制细胞因子信号1； TFPI-2，组织因子途径抑制剂2；和Wif-1，Wnt 抑制因子1。 CpG岛甲基表型（CIMP）可能与恶性肿瘤的发展有关 通过协调肿瘤抑制剂和肿瘤相关基因（TRG）和甲基化的灭活 多个非编码，甲基化肿瘤（薄荷）基因座。这些表观遗传变化产生了独特的cimp 与胃肠道癌的复发和生存有关的模式。因为表观遗传学 TRG的失活也与恶性黑色素瘤的发育和进展有关，我们 假设在皮肤黑色素瘤中存在临床意义的CIMP。我们检查了 与黑色素瘤病理生理学和A相关的CpG岛启动子区域的甲基化状态 薄荷位点（Mint-1、2、3、12、17、25和31）的薄荷位置和转移性肿瘤中的面板 阶段。我们显示了TRGS WIF-1，TFPI-2，RASSF1A和 SOCS-1具有前进的临床肿瘤阶段。此外，我们发现 MINT-17，MINT-31和TRG的甲基化状态。这些发现证明了 与恶性黑色素瘤的临床阶段有关的CIMP模式，可用于 识别具有转移或复发风险高风险的原发性黑色素瘤。该研究已提交 出版。这是与肿瘤相关的原发性黑色素瘤表观遗传变化的第一个主要发现 进展。这些研究将进一步扩展，以检查CIMP作为预后的临床实用性 原发性肿瘤的基因型。 正在使用甲基化的TRG和非编码基因组基因座进行循环DNA的研究。 正在修改改善DNA分离血清和硫酸含量的方法。我们集中于开发测定法 评估血清中的非编码区域DNA。