Identifying Diagnostic Markers for Cervical, Breast, and Prostate Cancer

识别宫颈癌、乳腺癌和前列腺癌的诊断标志物

• 批准号: 8349194
• 负责人: Thomas Ried
• 金额: $ 42.25万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349194
• 关键词: Aneuploidy; Benign; Biological; Biological Assay; Biopsy; Body Weight decreased; Breast Carcinoma; Carcinoma; Cell Nucleus; Cell physiology; Cells; Centers for Disease Control and Prevention (U.S.); Centromere; Cervical; Cervical Adenocarcinoma; Cervical Cancer Screening; Cervical Intraepithelial Neoplasia; Cervical dysplasia; Cervix carcinoma; Chromosomal Instability; Chromosome abnormality; Chromosomes; Chromosomes, Human, Pair 3; Classification; Clinical; Collaborations; Color; Colposcopy; Complement; Cytogenetics; Cytology; DNA Probes; Data; Data Set; Detection; Diagnosis; Diagnostic; Diagnostic tests; Disease; Dysplasia; ERBB2 gene; Early Detection Research Network; Early Diagnosis; Epithelial; Event; Failure; Fine needle aspiration biopsy; Gene Expression Profile; Gene Expression Profiling; Genes; Genetic screening method; Genome; Genomic Instability; Genomics; Glandular Cell; Goals; Hemorrhage; Histocompatibility Testing; Hospitals; Human; Human Papillomavirus; Imagery; Individual; Infection; Interphase; Interphase Cell; Lesion; Link; Liquid substance; Loop electrosurgical excision procedure; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Malignant neoplasm of prostate; Mammography; Measurement; Messenger RNA; Methods; Molecular Profiling; Operative Surgical Procedures; Outcome; PSA screening; Pain; Pap smear; Paris, France; Patients; Play; Premalignant; Preparation; Procedures; Prostate; Prostate carcinoma; Publishing; Receiver Operating Characteristics; Recurrent Malignant Neoplasm; Risk; Role; Sampling; Screening for cancer; Screening procedure; Sensitivity and Specificity; Series; Severities; Signal Transduction; Solid Neoplasm; Specimen; Staining method; Stains; Stratification; Sweden; Symptoms; TERC gene; Telomerase; Testing; Therapeutic Intervention; Time; Tissues; Translating; Validation; Woman; base; cancer cell; cancer diagnosis; cancer genome; cancer therapy; clinical practice; design; disorder prevention; follow-up; high risk; immunocytochemistry; improved; intraepithelial; malignant breast neoplasm; molecular marker; outcome forecast; prognostic; programs; prospective; tool; trait; tumor; tumor progression; tumorigenesis; validation studies

Cervical cytology (Pap-smears) CGH analyses of human tumors have revealed that chromosomal aberrations result in genomic imbalances specific for diverse tissue types. Furthermore, these changes define discrete steps in the progression of epithelial tumors. More than 95% of cervical carcinomas carry extra copies of chromosome 3, which results in the genomic amplification of the human telomerase gene TERC. It is therefore logical to apply the visualization of these recurring and specific chromosomal aberrations to complement and enhance the cytomorphological diagnosis of human cancers and their precursor lesions. This can be achieved using interphase cytogenetics with fluorescently tagged DNA probes that recognize specific chromosomal target regions directly in interphase cells. The application of this genetic test to routinely collected cytological specimens proved that high-grade intraepithelial lesions (HSIL, which are comprised of CIN2 and CIN3) could be discerned from normal samples, ASCUS and low-grade intraepithelial lesions (LSIL, or CIN1) with a sensitivity and specificity exceeding 90%, independent of the cytomorphological assessment. In order to explore whether gain of 3q and genomic amplification of hTERC increases the propensity of progression from LSIL to HSIL and invasive carcinoma, we have applied the probe set to a series of previously stained PAP-smears. The samples included (i) CIN1 and CIN2 lesions that progressed to CIN3, (ii) CIN1 and CIN2 lesions that regressed spontaneously, and (iii) normal PAP-smears from women who subsequently developed CIN3 or cervical cancer. Our data suggest that genomic amplification of hTERC is required for the transition from CIN1 and CIN2 to CIN3 and that it predicts progression. Of note, hTERC amplification was found in 30% of cytologically normal PAP-smears from women who were diagnosed with CIN3 or invasive cervical carcinomas after a short latency. We conclude that the detection of genomic amplification of hTERC in routinely collected PAP-smears can assist in identifying low-grade lesions with a high progression risk and in reduction of false negative individual cytological screenings. In an independent study we were curious as to whether the amplification of the human telomerase gene could also be used to diagnose another type of cervical cancer, i.e., cervical adenocarcinomas. Interphase nuclei prepared from archival material of 12 primary cervical adenocarcinomas were hybridized with a triple colour probe-set specific for centromeres of chromosomes 3 and 7 and the TERC gene. The findings were correlated with HPV infection. We observed high proportions of nuclei with increased copy numbers for TERC in both HPV positive and HPV negative tumors. The absolute number of TERC signals was increased in HPV positive (mean 3.7) and HPV negative (mean 5.0) tumors. Amplification of the human telomerase gene TERC is a consistent aberration in cervical adenocarcinomas and is independent of the presence of HPV genomes. Therefore, application of our probe set may provide an objective genetic test for the assessment of glandular cells in Pap smears and hence for the diagnosis of cervical adenocarcinomas. Based on these results, we designed a large-scale validation study in which the presence of TERC amplification in Pap smears was correlated to the histological diagnosis after colposcopy and biopsy. In this study, we used a four-color FISH assay by Abbott/Vysis based on our previous results that included a biotinylated DNA probe for the detection of HPV (provided to us through a MTA with that company). In collaboration with the Karolinska Hospital in Stockholm, Sweden, we collected 78 liquid based cytology (LBC) samples from women referred to the Karolinska Hospital because of a suspicious Pap smear. At the time of LBC sampling, all patients were examined by colposcopy, at which time a biopsy was taken. For most patients a subsequent histological diagnosis after loop electrosurgical excision procedure (LEEP) and a follow-up Pap smear were available. The LBC samples were also analyzed by several HPV tests (line blot, Quantovir HPV test, HPV mRNA detection using the NorChip assay), and for the expression levels of p16 using immunocytochemistry. The histological diagnosis revealed 14 benign cases, 17 samples rated as cervical intraepithelial neoplasia (CIN) grade 1, 22 CIN2, 23 CIN3, and two carcinomas. As expected, the number of TERC positive cases increased with the severity of the dysplasia. We then compared, using Receiver Operating Characteristic (ROC) curves, which of the tests rendered the highest sensitivity and specificity for the discernment of benign lesions and CIN1 from CIN2, CIN3, and cancer. We showed that detection of genomic amplification of TERC clearly outperforms any other test, including detection of HPV, as a diagnostic test for the presence of high-grade cervical dysplasia (CIN2 and CIN3) and cancer in routinely collected LBC samples. The correlation with the histological classification, the standard for a diagnosis, was excellent. For further validation, we have engaged in a prospective trial organized through NCIs Early Detection Research Network (EDRN) and the Centers for Disease Control and Prevention (CDC). The goal is to ultimately establish whether detection of genomic amplification of chromosome 3q in Pap smears could change the way cervical cancer screening is conducted. One could entertain the possibility that HPV screening is the primary test, and that those Pap smears positive for 3q would then sequentially be tested for the presence of 3q gain. In collaboration with Pamela Paris (UCSF) we will also study prostate tissue sections for copy number changes of a high-risk diagnostic probe cocktail which was defined after aCGH analyses of prostate carcinomas associated with a high progression risk. The gene expression signature of genomic instability in breast cancer is an independent predictor of clinical outcome Recently, expression profiling of breast carcinomas has revealed gene signatures that predict clinical outcome, and discerned prognostically relevant breast cancer subtypes. Measurement of the degree of genomic instability provides a very similar stratification of prognostic groups. We therefore hypothesized that these features are linked. We used gene expression profiling of 48 breast cancer specimens that profoundly differed in their degree of genomic instability and identified a set of 12 genes that defines the two groups. The biological and prognostic significance of this gene set was established through survival prediction in published datasets from patients with breast cancer. Of note, the gene expression signatures that define specific prognostic subtypes in other breast cancer datasets, such as luminal A and B, basal, normal-like, and ERBB2+, and prognostic signatures including MammaPrint and Oncotype DX, predicted genomic instability in our samples. This remarkable congruence suggests a biological interdependence of poor-prognosis gene signatures, breast cancer subtypes, genomic instability, and clinical outcome and that the degree of genomic instability per se is the dominant breast cancer inherent prognostic trait.

对人肿瘤的宫颈细胞学（PAP-SMEARS）CGH分析表明，染色体畸变导致针对多种组织类型的基因组失衡。此外，这些变化定义了上皮肿瘤进展中的离散步骤。超过95％的宫颈癌携带额外的染色体副本，从而导致人端粒酶基因TERC的基因组扩增。因此，应用这些反复出现和特定染色体畸变的可视化以补充和增强人类癌症及其前体病变的细胞学诊断。这可以使用与荧光标记的DNA探针的相间遗传学来实现，这些探针直接识别直接在相间细胞中的特定染色体靶区域。该基因测试在常规收集的细胞学标本中的应用证明，高级上皮内病变（由CIN2和CIN3组成的HSIL（HSIL）可以与正常样品和低度上皮内病变（LSIL或CIN1）识别出具有正常样本（LSIL或CIN1）敏感性和特异性超过90％，与细胞学评估无关。为了探索3Q的增益和HTERC的基因组扩增是否增加了从LSIL到HSIL和侵入性癌的进展倾向，我们将探针设置为一系列先前染色的Pap-Smears。这些样品包括（i）CIN1和CIN2病变，这些病变逐渐发展为CIN3，（ii）自发回归的CIN1和CIN2病变，以及（iii）随后患上CIN3或宫颈癌的女性的正常子宫颈钉。我们的数据表明，HTERC的基因组扩增是从CIN1和CIN2到CIN3的过渡所必需的，并且可以预测进展。值得注意的是，在短期潜伏期后，从被诊断为CIN3或浸润性宫颈癌的女性的30％的细胞学正常子宫颈膜中发现了HTERC扩增。我们得出的结论是，常规收集的子宫颈膜中HTERC基因组扩增的检测可以帮助识别具有高进展风险的低级病变，并减少了假阴性个体细胞学筛查。在一项独立的研究中，我们很好奇人端粒酶基因的扩增是否也可以用于诊断另一种类型的宫颈癌，即宫颈腺癌。将来自12个原代宫颈腺癌的档案材料制备的相间核与针对染色体3和7的cint粒子和TERC基因的三色探针集杂交。这些发现与HPV感染相关。我们观察到高比例的核，在HPV阳性和HPV阴性肿瘤中TERC的拷贝数增加。 HPV阳性（平均3.7）和HPV阴性（平均5.0）肿瘤中TERC信号的绝对数量增加。人端粒酶基因TERC的扩增是宫颈腺癌的一致像差，并且与HPV基因组的存在无关。因此，我们的探针集的应用可以为评估子宫颈抹片检查的腺细胞提供客观的基因检测，从而为宫颈腺癌的诊断提供了客观的基因检测。基于这些结果，我们设计了一项大规模验证研究，其中PAP涂片中的TERC扩增与阴道镜检查和活检后的组织学诊断相关。在这项研究中，我们基于我们先前的结果，使用了Abbott/Vession的四色鱼测定法，其中包括用于检测HPV的生物素化DNA探针（通过该公司通过MTA提供给我们）。与瑞典斯德哥尔摩的Karolinska医院合作，我们从妇女那里收集了78个基于液体的细胞学（LBC）样本，因为妇女提到了Karolinska医院，这是由于可疑的PAP涂片。在LBC抽样时，通过阴道镜检查了所有患者，此时进行了活检。对于大多数患者，循环电外科切除手术（LEEP）和后续的PAP涂片后，随后进行了组织学诊断。还通过多个HPV测试（使用Norchip测定法，Quantovir HPV测试，HPV mRNA检测）分析LBC样品，并使用免疫细胞化学的p16表达水平进行分析。组织学诊断显示14例良性病例，17个样品被评为上皮内肿瘤（CIN）1级1、22 CIN2、23 CIN3和两个癌。正如预期的那样，TERC阳性病例的数量随发育异常的严重程度增加。然后，我们使用接收器操作特征（ROC）曲线进行了比较，该曲线对CIN2，CIN3和CANCER的良性病变和CIN1的识别提高了最高灵敏度和特异性。我们表明，TERC基因组扩增的检测显然优于其他任何测试，包括检测HPV，作为对常规收集的LBC样品中高级宫颈发育不良（CIN2和CIN3）的诊断测试。与组织学分类的相关性是诊断的标准。 为了进一步验证，我们参加了通过NCIS早期检测研究网络（EDRN）和疾病控制与预防中心（CDC）组织的前瞻性试验。目的是最终确定在子宫颈抹片检查中3Q染色体3Q基因组扩增的检测是否会改变进行宫颈癌筛查的方式。人们可以接受HPV筛选是主要测试的可能性，而这些PAP涂片为3Q的阳性将依次测试3Q增益的存在。与巴黎Pamela（UCSF）合作，我们还将研究前列腺组织切片，以进行高危诊断探针鸡尾酒的拷贝数变化，该鸡尾酒是在ACGH分析与高进展风险相关的前列腺癌后定义的。 乳腺癌基因组不稳定性的基因表达特征最近是临床结果的独立预测指标，乳腺癌的表达分析揭示了预测临床结果的基因特征，并辨别了预后相关的乳腺癌亚型。基因组不稳定性程度的测量提供了预后组非常相似的分层。因此，我们假设这些功能是链接的。我们使用了48种乳腺癌标本的基因表达分析，这些标本在其基因组不稳定性的程度上明显不同，并确定了一组定义这两组的基因。该基因集的生物学和预后意义是通过乳腺癌患者发表的数据集的生存预测确定的。值得注意的是，在其他乳腺癌数据集中定义了特定预后亚型的基因表达签名，例如腔A和B，基础，正常，正常和ERBB2+，以及包括乳腺和乳腺型和Oncotype DX在内的预后特征，预测了我们样品中的基因组不稳定。这种显着的一致性表明，较差的基因特征，乳腺癌亚型，基因组不稳定性和临床结果的生物学相互依存关系，基因组不稳定性本质上是固有的乳腺癌固有的预后性状。