Immunopheonotyping of circulating tumor cells

描述（由申请人提供）：转移是大多数癌症相关死亡的原因。转移的一个限制步骤是进入循环，因此循环肿瘤细胞（ctc）应该存在于所有转移性肿瘤患者中，这可能具有诊断和预后价值。原发性未知癌（CUP）仅占3-5%，对临床医生的诊断和治疗提出了挑战。组织活检是诊断的标准，但需要改进方法，包括依靠CTC分析的非侵入性血液检查。我们的总体目标是利用CTC来深入了解全身癌症进展和治疗方法，包括将CTC血液检查作为诊断、预后和治疗的工具。我们假设CTC人群的实时单细胞免疫表型分析将为组织起源的诊断提供信息，揭示诸如干细胞样特性的预后标记，并预测肿瘤的侵袭性。我们一直在开发一种使用多重q点共轭抗体的后veridex CTC分析方法，目的是检测CTC群体中每个单细胞中的多个标记物，以帮助诊断包括起源组织在内的CUP。我们开发了一种免疫荧光（IF）方案，其中包含一组最小的标记物来预测原发部位，并提供常见转移性肿瘤的预后信息：黑色素瘤、肺癌、结直肠癌、乳腺癌或前列腺癌。通过Veridex CellSearch有两种试剂盒可用于分离ctc：黑色素瘤试剂盒（使用黑色素瘤抗原）和上皮或癌试剂盒（使用EpCAM）。对病人的血液进行初步检测

DESCRIPTION (provided by applicant): Metastasis is responsible for the majority of cancer-related deaths. A limiting step in metastasis is access to circulation, thus circulating tumor cell (CTCs) should be present in all patients with metastatic tumors, which may have both diagnostic and prognostic value. Accounting for only 3-5%, carcinoma of unknown primary (CUP) presents a challenge for clinicians in diagnosis and therapy. Tissue biopsy is the standard for diagnosis but there is need for improved methodology including a non-invasive blood test that relies on CTC analysis. Our overall objective is to utilize CTCs to provide insight into systemic cancer progression and approaches to therapy including a CTC blood test as a tool for diagnosis, prognosis and therapy. We hypothesize that real- time, single-cell immunophenotype profiling of CTC populations will inform diagnosis of tissue of origin, reveal prognostic markers such as stem-like properties and predict tumor aggressiveness. We have been developing a post-Veridex CTC analysis method using multiplexed Q-dot conjugated antibodies with the goal of detection of multiple markers in each single cell within a CTC population to aid in CUP diagnosis including tissue of origin. We have developed an immunofluorescence (IF) protocol with a minimal set of markers to predict the primary sites and provide prognostic information for common metastatic tumors: melanoma, lung, colorectal, and breast or prostate cancer. Two kits are available to isolate CTCs through Veridex CellSearch: melanoma kit (using melanoma antigen), and epithelial or carcinoma kit (using EpCAM). A patient's blood is initially tested with both kits, and positive CTCs from the melanoma kit supports a diagnosis of melanoma, whereas positive CTCs from the epithelial kit supports carcinoma. For carcinomas, we use cytokeratin 7 (CK7), cytokeratin 20 (CK20), thyroid transcription factor 1 (TTF-1), estrogen receptor (ER) or prostate-specific antigen (PSA) as the minimal set of markers. We are developing a flexible micro spring array (FMSA) size-based CTC enrichment protocol in parallel. IF is optimized in cultured tumor cells, first with individual antibodies, then quantum dots (Q-dot) conjugated multiplex quadra-antibody set. In addition, we have isolated viable patient-derived CTCs and maintained them in culture for a few weeks. Specific Aim #1 Develop a multiplex-marker based algorithm system for predicting primary sites in carcinoma of unknown primary. Specific Aim #2 Utilize the FMSA device and other approaches for isolating live CTCs to study the biological properties of these cells in the context of metastatic spread. Successful application of this protocol to CTCs in patient samples is expected to provide a noninvasive, relatively quick and inexpensive blood test that can be both diagnostic and prognostic, which may be a valuable adjunct to routine biopsy. Circulating tumor cells may provide insight into the mechanism of systemic cancer progression, the characteristics of metastatic founder cells and changes within these cells as cancer progresses, thereby impacting the efficacy and evaluation of cancer therapy.