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Phys Biol:HD-CTC血液测试——检测癌症的新活组织检查

来源:生物谷 2012-02-04 10:28

斯克里普斯研究所的科学家们和癌症医生们的共同研究,已经成功地证明了一个从癌症患者固体肿瘤中分离细胞来检测和分析循环肿瘤细胞(CTCs)先进测试的作用在5篇新论文中报道的这项研究结果表明,高度敏感的血液分析提供了不久便可与从某些类型手术活检中所得信息相媲美的信息。

"这是新一代技术",斯克里普斯研究所副教授Peter Kuhn博士说,他是这项新研究的高级研究员和此项高分辨率血液测试的主要发明人。"它大大提高我们监测、预测和理解包括转移的癌症发展的能力,这是癌症患者死亡的主要原因。"

该研究发表在2012年2月3日的期刊《物理生物学》(Physical Biology)。

称为HD-CTC的新测试,以一种从普通红色和白色血细胞中区分可能CTCs的方式来指示病人血液样本中的细胞。然后,用一个数字显微镜和图像处理算法来分离那些大小与形状("形态学")不象正常细胞的可疑细胞。正如手术活检一样,病理学家可以检查可疑细胞的影像来排除假阳性并记录它们的形态。

Kuhn强调,这基本设置能用不同的细胞标记和图像处理技术很容易地修改。

五项新研究,五步向前

为测试新技术,斯克里普斯研究所Kuhn实验室的成员,与加利福尼亚拉霍亚加州斯克里普斯健康研究所、圣地亚哥加利福尼亚大学摩尔斯癌症中心、蒙大纳比林斯诊所、圣地亚哥加利福尼亚大学医学肿瘤学分部、洛杉机南加州大学应用分子医学中心和荷兰阿姆斯特丹Antoni van Leeuwenhoek医院荷兰癌症研究所的病理学家和肿瘤学家一起组成研究小组。

因合作引起的五个新研究,不仅表明为许多不同癌症类型而做的新测试的准确性和有效性,也开始探索诊断和监测患者的技术的实用性和在实验室改善癌症研究。而CTCs的其他测试也通常在可疑CTCs被浓缩中使用"富集"步骤--这些方法不注意地排除了某些类型的CTCs--新研究表明HD-CTC就如一个无细胞遗漏过程一样起作用,使能够有一个更完整的分析。

也引人注目的是图像质量。"高清晰度方法提供了这些难以捉摸细胞的详细描写,这些细胞在散布身体的行动中被抓获", 斯克里普斯健康研究所和加利福尼亚大学圣地亚哥医学分校的诊断病理学家Kelly Bethel医学博士,他是Kuhn研究小队的高级临床研究员,"这是前所未有的--之前我们一直没能象这样常规地和高清度地看到他们。"

在第一个研究中,为证明该测试对不同癌症类型的敏感性与精确度,研究小组使用HD-CTC对83个晚期癌症病人进行了检查。科学家们发现,该测试在80%转移性前列腺癌患者中检测出每毫升血液中5个或更多个CTCs,其中70%患转移性乳腺癌, 50%患转移性胰腺癌,没有健康的受试者。目前的CTCs测试金标准,它也称为细胞搜索(CellSearch),在这些样本中检测肿瘤细胞的敏感性明显低一些。

大多数CTC计数超过检测阈值的患者也显示出CTCs的小聚集物,这就是癌症生物学家所说的"微型肿瘤栓子"( microtumor emboli)。这些被怀疑是早期转移性肿瘤,也就是常常杀死晚期癌症病人的血凝块触发子。在第二项研究中,科学家指出,HD-CTC能在71例晚期前列腺癌、肺癌、胰腺癌和乳腺癌患者中检测出43%的这些聚集物,在15名健康受试者中没有检测出一例。"这告诉我们,HD-CTC可能有助于研究癌症转移和相关血凝块的起源,也有助于预测他们", Kuhn说。

在第三项研究中,研究小组使用HD-CTC来比较前列腺癌患者的循环肿瘤细胞和前列腺癌细胞系,此细胞系是研究人员在实验室经常使用的前列腺癌生物学的方便模型。研究小组发现,在细胞形态及被HD-CTC荧光标签标记的方式上,这2类细胞存在着明显差异。"这强调必需要研究来自患者的癌细胞,而不只是模型癌细胞,这些模型癌细胞在某些方面可能完全不同于事情真象", Kuhn说。

在第四项研究中,研究人员对28例晚期非小细胞肺癌患者进行了长达一年的HD-CTC测试。研究小组在68%样本中能检测出CTCs,并发现检测出的CTCs数量往往如其他检测表明的癌症进展一样不断上升。

在这一系列的第五即最后的研究中,该研究小组在78例刚被诊断为非小细胞肺癌各个阶段的病人中使用HD-CTC。"我们演示了,我们能灵敏地检测出CT Cs甚至患者是在癌症的早期阶段", Kuhn说。

这一研究结果指出了使用HD-CTC血液测试的可能性,即不仅能评估已经确诊的癌症,也能有助于检测还不知道自己已患癌症的人。"如果HD-CTC在癌症诊断后的那天起作用,正如我们已经证明的,那么就很容易设象它在诊断前也起作用" Kuhn说。

Kuhn和他的同事们现在打算研究HD-CTC作为一个潜在筛选测试的使用并进一步开发为临床监测和癌症研究之用。Kuhn已经成立了一个总部设在圣地亚哥的生物技术公司--Epic科技公司--来开发HD-CTC为个性化癌症治疗中的商业化附带诊断产品。(生物谷bioon.com)

High-- imaging of circulating tumor cells and associated cellular events in non-small cell lung cancer patients: a longitudinal analysis

Jorge Nieva, MarcoWendel, Madelyn Luttgen, Dena Marrinucci, Lyudmila Bazhenova, Anand Kolatkar, Roger Santala, BrockWhittenberger, James Burke, Melissa Torrey, Kelly Bethel, and Peter Kuhn

Abstract     Sampling circulating tumor cells (CTCs) from peripheral blood is ideally accomplished using assays that detect high numbers of cells and preserve them for downstream characterization. We sought to evaluate a method using enrichment free fluorescent labeling of CTCs followed by automated digital microscopy in patients with non-small cell lung cancer. Twenty-eight patients with non-small cell lung cancer and hematogenously seeded metastasis were analyzed with multiple blood draws. We detected CTCs in 68% of analyzed samples and found a propensity for increased CTC detection as the disease progressed in individual patients. CTCs were present at a median concentration of 1.6 CTCs ml?1 of analyzed blood in the patient population. Higher numbers of detected CTCs were associated with an unfavorable prognosis.

Fluid biopsy in patients with metastatic prostate, pancreatic and breast cancers

Dena Marrinucci1, Kelly Bethel, Anand Kolatkar, Madelyn Luttgen, Michael Malchiodi,, Franziska Baehring, Katharina Voigt, Daniel Lazar, Jorge Nieva, Lyudmilda Bazhenova, Andrew H Ko, W Michael Korn, Ethan Schram, Michael Coward, Xing Yang, Thomas Metzner, Rachelle Lamy, Meghana Honnatti, Craig Yoshioka, Joshua Kunken, Yelena Petrova, Devin Sok, David Nelson, and Peter Kuhn

Abstract        Hematologic spread of carcinoma results in incurable metastasis; yet, the basic characteristics and travel mechanisms of cancer cells in the bloodstream are unknown. We have established a fluid phase biopsy approach that identifies circulating tumor cells (CTCs) without using surface protein-based enrichment and presents them in sufficiently high definition (HD) to satisfy diagnostic pathology image quality requirements. This 'HD-CTC' assay finds >5 HD-CTCs mL?1 of blood in 80% of patients with metastatic prostate cancer (n = 20), in 70% of patients with metastatic breast cancer (n = 30), in 50% of patients with metastatic pancreatic cancer (n = 18), and in 0% of normal controls (n = 15). Additionally, it finds HD-CTC clusters ranging from 2 HD-CTCs to greater than 30 HD-CTCs in the majority of these cancer patients. This initial validation of an enrichment-free assay demonstrates our ability to identify significant numbers of HD-CTCs in a majority of patients with prostate, breast and pancreatic cancers.

Cytometric comparisons between circulating tumor cells from prostate cancer patients and the prostate-tumor-derived LNCaP cell line

Daniel C Lazar, Edward H Cho, Madelyn S Luttgen, Thomas J Metzner, Maria Loressa Uson, Melissa Torrey, Mitchell E Gross, and Peter Kuhn

Abstract       Many important experiments in cancer research are initiated with cell line data analysis due to the ease of accessibility and utilization. Recently, the ability to capture and characterize circulating tumor cells (CTCs) has become more prevalent in the research setting. This ability to detect, isolate and analyze CTCs allows us to directly compare specific protein expression levels found in patient CTCs to cell lines. In this study, we use immunocytochemistry to compare the protein expression levels of total cytokeratin (CK) and androgen receptor (AR) in CTCs and cell lines from patients with prostate cancer to determine what translational insights might be gained through the use of cell line data. A non-enrichment CTC detection assay enables us to compare cytometric features and relative expression levels of CK and AR by indirect immunofluorescence from prostate cancer patients against the prostate cancer cell line LNCaP. We measured physical characteristics of these two groups and observed significant differences in cell size, fluorescence intensity and nuclear to cytoplasmic ratio. We hope that these experiments will initiate a foundation to allow cell line data to be compared against characteristics of primary cells from patients.

Characterization of circulating tumor cell aggregates identified in patients with epithelial tumors

Edward H Cho, MarcoWendel, Madelyn Luttgen1, Craig Yoshioka, Dena Marrinucci1, Daniel Lazar, Ethan Schram, Jorge Nieva, Lyudmila Bazhenova, Alison Morgan, Andrew H Ko, W Michael Korn, Anand Kolatkar, Kelly Bethel, and Peter Kuhn

Abstract        Circulating tumor cells (CTCs) have been implicated as a population of cells that may seed metastasis and venous thromboembolism (VTE), two major causes of mortality in cancer patients. Thus far, existing CTC detection technologies have been unable to reproducibly detect CTC aggregates in order to address what contribution CTC aggregates may make to metastasis or VTE. We report here an enrichment-free immunofluorescence detection method that can reproducibly detect and enumerate homotypic CTC aggregates in patient samples. We identified CTC aggregates in 43% of 86 patient samples. The fraction of CTC aggregation was investigated in blood draws from 24 breast, 14 non-small cell lung, 18 pancreatic, 15 prostate stage IV cancer patients and 15 normal blood donors. Both single CTCs and CTC aggregates were measured to determine whether differences exist in the physical characteristics of these two populations. Cells contained in CTC aggregates had less area and length, on average, than single CTCs. Nuclear to cytoplasmic ratios between single CTCs and CTC aggregates were similar. This detection method may assist future studies in determining which population of cells is more physically likely to contribute to metastasis and VTE.

Fluid biopsy for Circulating Tumor Cell identification in Patients with early and late stage Non-Small Cell Lung Cancer; a glimpse into lung cancer biology

Marco Wendel, Lyudmila Bazhenova, Rogier Boshuizen, Anand Kolatkar, Meghana Honnatti, Edward H. Cho, Dena Marrinucci, Ajay Sandhu, Anthony Perricone, Patricia Thistlethwaite, Kelly Bethel, Jorge Nieva, Michel van den Heuvel, and Peter Kuhn

Abstract    Circulating tumor cell (CTC) counts are an established prognostic marker in metastatic prostate, breast and colorectal cancer, and recent data suggest a similar role in late stage non-small cell lung cancer (NSCLC). However, due to sensitivity constraints in current enrichment-based CTC detection technologies, there are few published data about CTC prevalence rates and morphologic heterogeneity in early-stage NSCLC, or the correlation of CTCs with disease progression and their usability for clinical staging. We investigated CTC counts, morphology and aggregation in early stage, locally advanced and metastatic NSCLC patients by using a fluid-phase biopsy approach that identifies CTCs without relying on surface-receptor-based enrichment and presents them in sufficiently high definition (HD) to satisfy diagnostic pathology image quality requirements. HD-CTCs were analyzed in blood samples from 78 chemotherapy-na?ve NSCLC patients. 73% of the total population had a positive HD-CTC count (>0 CTC in 1 mL of blood) with a median of 4.4 HD-CTCs mL?1 (range 0-515.6) and a mean of 44.7 (±95.2) HD-CTCs mL?1. No significant difference in the medians of HD-CTC counts was detected between stage IV (n = 31, range 0-178.2), stage III (n = 34, range 0-515.6) and stages I/II (n = 13, range 0-442.3). Furthermore, HD-CTCs exhibited a uniformity in terms of molecular and physical characteristics such as fluorescent cytokeratin intensity, nuclear size, frequency of apoptosis and aggregate formation across the spectrum of staging. Our results demonstrate that despite stringent morphologic inclusion criteria for the definition of HD-CTCs, the HD-CTC assay shows high sensitivity in the detection and characterization of both early- and late-stage lung cancer CTCs. Extensive studies are warranted to investigate the prognostic value of CTC profiling in early-stage lung cancer. This finding has implications for the design of extensive studies examining screening, therapy and surveillance in lung cancer patients.

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