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【盘点】基于血液的新型癌症检测技术进展
浏览次数:279   发布时间:2015-07-30  来源:生物谷   [返回列表

当检测实体瘤仍是癌症诊断中的常规步骤时,现代技术,比如新一代测序技术就已经可以帮助科学家们深入追踪癌症的发展变化了,许多肿瘤都会有一些脱落的细胞,而这些脱落的小囊泡名为外核体,而其DNA的轨迹会进入血液和其它体液组织中;近来有研究发现,这些外核体碎片可以作为标志物来帮助监测疾病的进展,甚至可以帮助科学家们在患者疾病症状出现之前诊断癌症的发生。

利用常规的血液样本就可以对肿瘤DNA进行检测,比如刊登在国际杂志JAMA Oncology上的一篇研究论文中,研究者们就对4000多名孕妇机体的血液样本进行了检测,用来鉴别胎儿染色体的异常情况,随后研究者发现了三例母源性的癌症,即卵巢癌、滤泡型淋巴瘤以及霍杰金淋巴瘤,在大多数肿瘤中,尽管其恶性程度较低,但研究者仍然可以利用个体的血液来作为研究肿瘤生物学的替代品。

而诸如“液体活组织检查”对于血液和血浆样本而言并不是特殊的,在其它研究中,研究者将膀胱癌复发的风险同尿液中DNA甲基化的水平联系了起来,研究者也在粪便样本中检测到了肠癌的DNA,同时还在头颈癌患者的唾液中鉴别出了癌症相关的基因突变;此前研究者常利用分子检测的手段监测恶性疾病和癌症转移,而如今随着精确工具使用的增加,研究者可以在血液中,甚至是疾病发展早期阶段就可以鉴别出少量的癌细胞和DNA。

来自约翰霍普金斯医学院的教授Bert Vogelstein表示,粪便和尿液可以帮助检测结直肠癌和膀胱癌,但血液检测至少从概念上来讲可以检测所有癌症,目前我们面临的挑战是检测微量的癌细胞DNA。

普通的证据

去年刊登在国际杂志Science Translational Medicine上的一篇研究论文中,研究者对640名病人进行了研究,发现对血浆中循环的肿瘤DNA进行检测可以帮助确定大约40%至70%的各类癌症,包括脑癌、前列腺癌和卵巢癌等。比如在恶性的结直肠癌中,循环的肿瘤DNA常被用于确定87%的患者机体KRAS基因的特殊突变。

完整的癌细胞通常会溜入血流中,“诱捕”这些循环肿瘤细胞(CTCs)的早期尝试依赖于表面抗原和其它标志物的识别,但CTCs会依赖于肿瘤的类型、疾病阶段及其它因子来穿上不同的分子外衣;今年年初,哈佛医学院的研究者Mehmet Toner在国际杂志Nature Methods刊文表示,微流体设备可以利用物理学的方法来诱捕CTCs,而该方法并不依赖于肿瘤特异性的标志物,Toner告诉The Scientist说道,完整无损的细胞具有巨大的研究价值,你可以仔细观察DNA、RNA、信号分子、磷酸化模式以及表观遗传学,这些相比单一的生物标志物要更为丰富,从长期角度而言,我们应当培养细胞来检测药物的敏感性,从而转移到进行个体化医学的开发。

除了DNA和整个细胞而言,近来又有研究指出,肿瘤细胞脱落的外核体或许可以充当癌症的生物标志物,就在上个月,刊登在国际杂志Nature上的一项研究报告中,来自得克萨斯大学MD癌症研究中心的科学家就对外核体进行了一项血清学测试,由于外核体携带有DNA、RNA和蛋白质,因此其可以被用于区分早期胰腺癌和晚期胰腺癌的患者。

但循环肿瘤细胞的痕迹依然是目前临床应用的一大难题,改善分子检测的敏感性和特异性对于建立临床应用程序非常关键,研究者Vogelstein表示,目前我们并不知道局限性是否是技术上或生物学上的,我们发现大约40%至70%的肿瘤都可以被检测到,但如果剩余的早期阶段的癌症并没有分泌循环肿瘤DNA中的单一分子的话,那我们的技术再怎么好也是无关紧要的。

目前很多基础生物学都是不清楚的,研究者Tim Forshew教授说道,我们仍然并不能完全知道循环肿瘤DNA进入到血液中的机制,以及为何其会因不同的癌症而异,我们也并不能完全理解使得循环的肿瘤DNA可以在血液中被快速消除。尽管如此,利用简单血液检测来诊断癌症并且指导疗法的可能性依然会让很多制药公司大发灵感来寻找新型的肿瘤血液检测技术。

早期应用

研究者Forshew就职于一家提供循环肿瘤DNA诊断检测技术的公司,他表示,从商业利益角度而言公司都比较关注循环肿瘤的标志物,在他看来,类似这样技术的重要应用就是研究并不容易进行活组织检查的癌症的遗传特性。许多其它公司,包括Epic科学、强生诊断、SRI国际等都能够提供循环肿瘤DNA和细胞的检测技术,然而这些检测手段的临床应用目前仍然受限于肿瘤转移的监测。

科学家们进行的临床研究通常是将血液中的肿瘤DNA同特殊的疾病参数相联系起来,比如预测患者移除肿瘤术后疾病复发的风险,Vogelstein教授说道,这并不同于预后评估。近日,来自伊丽莎-霍尔医学研究所的科学家们正在利用循环肿瘤DNA来评估结肠癌患者在术后因化疗获益的可能性,标准上而言,大部分患者在术后会接受辅助化学疗法,但仅有4%至5%的患者会因此获益,对于其余患者而言,要么手术比较充分,要么就是化疗后癌症依然复发的。

在去年美国临床肿瘤学会会议上研究者公布了他们的初期研究结果,他们发现癌症复发风险和循环肿瘤DNA的水平密切相关,而如今科学家们又计划进行一项大型的随机试验来评估利用循环肿瘤DNA来进行化疗规范的实用性,通过血液检测来知道化疗,不仅可以改善癌症患者的生存,而且还可以降低接受化疗患者的数量。

相比此前使用的前列腺特异抗原而言,肿瘤DNA、CTCs以及肿瘤衍生的外核体都可以作为较高价值的标志物,这些新型标志物可以为研究者提供一种绝佳的机会来管理癌症,最后研究者表示,此前我们总是晚疾病一步,而且我们试图赶上疾病发生的节奏,而如今利用更多特异性且敏感性的工具我们就可以领先疾病一步诊断并且发现疾病,对于后期制定可用的治疗手段或将带来巨大的帮助。(原文链接

参考文献:

 

Presymptomatic Identification of Cancers in Pregnant Women During Noninvasive Prenatal Testing

Frédéric Amant, MD, PhD1; Magali Verheecke, MD1; Iwona Wlodarska, PhD2; Luc Dehaspe, PhD2; Paul Brady, PhD2; Nathalie Brison, PhD2; Kris Van Den Bogaert, PhD2; Daan Dierickx, MD, PhD3; Vincent Vandecaveye, MD, PhD4; Thomas Tousseyn, MD, PhD5; Philippe Moerman, MD, PhD5; Adriaan Vanderstichele, MD2; Ignace Vergote, MD, PhD2; Patrick Neven, MD, PhD2; Patrick Berteloot, MD6; Katrien Putseys, MD7; Lode Danneels, MD8; Peter Vandenberghe, MD, PhD2,3; Eric Legius, MD, PhD2; Joris Robert Vermeesch, PhD2

 

Importance Noninvasive prenatal testing (NIPT) for fetal aneuploidy by scanning cell-free fetal DNA in maternal plasma is rapidly becoming a major prenatal genetic test. Similar to placental DNA, tumor DNA can be detected in the plasma, and analysis of cell-free tumor DNA can be used to characterize and monitor cancers. We show that plasma DNA profiling allows for presymptomatic detection of tumors in pregnant women undergoing routine NIPT. Observations During NIPT in over 4000 prospective pregnancies by parallel sequencing of maternal plasma cell-free DNA, 3 aberrant genome representation (GR) profiles were observed that could not be attributed to the maternal or fetal genomic constitution. A maternal cancer was suspected, and those 3 patients were referred for whole-body diffusion-weighted magnetic resonance imaging, which uncovered an ovarian carcinoma, a follicular lymphoma, and a Hodgkin lymphoma, each confirmed by subsequent pathologic and genetic investigations. The copy number variations in the subsequent tumor biopsies were concordant with the NIPT plasma GR profiles. Conclusions and Relevance We show that maternal plasma cell-free DNA sequencing for noninvasive prenatal testing also may enable accurate presymptomatic detection of maternal tumors and treatment during pregnancy.

 

Detection of circulating tumor DNA in early- and late-stage human malignancies.

Bettegowda C1, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, Bartlett BR, Wang H, Luber B, Alani RM, Antonarakis ES, Azad NS, Bardelli A, Brem H, Cameron JL, Lee CC, Fecher LA, Gallia GL, Gibbs P, Le D, Giuntoli RL, Goggins M, Hogarty MD, Holdhoff M, Hong SM, Jiao Y, Juhl HH, Kim JJ, Siravegna G, Laheru DA, Lauricella C, Lim M, Lipson EJ, Marie SK, Netto GJ, Oliner KS, Olivi A, Olsson L, Riggins GJ, Sartore-Bianchi A, Schmidt K, Shih lM, Oba-Shinjo SM, Siena S, Theodorescu D, Tie J, Harkins TT, Veronese S, Wang TL, Weingart JD, Wolfgang CL, Wood LD, Xing D, Hruban RH, Wu J, Allen PJ, Schmidt CM, Choti MA, Velculescu VE, Kinzler KW, Vogelstein B, Papadopoulos N, Diaz LA Jr.

 

The development of noninvasive methods to detect and monitor tumors continues to be a major challenge in oncology. We used digital polymerase chain reaction-based technologies to evaluate the ability of circulating tumor DNA (ctDNA) to detect tumors in 640 patients with various cancer types. We found that ctDNA was detectable in >75% of patients with advanced pancreatic, ovarian, colorectal, bladder, gastroesophageal, breast, melanoma, hepatocellular, and head and neck cancers, but in less than 50% of primary brain, renal, prostate, or thyroid cancers. In patients with localized tumors, ctDNA was detected in 73, 57, 48, and 50% of patients with colorectal cancer, gastroesophageal cancer, pancreatic cancer, and breast adenocarcinoma, respectively. ctDNA was often present in patients without detectable circulating tumor cells, suggesting that these two biomarkers are distinct entities. In a separate panel of 206 patients with metastatic colorectal cancers, we showed that the sensitivity of ctDNA for detection of clinically relevant KRAS gene mutations was 87.2% and its specificity was 99.2%. Finally, we assessed whether ctDNA could provide clues into the mechanisms underlying resistance to epidermal growth factor receptor blockade in 24 patients who objectively responded to therapy but subsequently relapsed. Twenty-three (96%) of these patients developed one or more mutations in genes involved in the mitogen-activated protein kinase pathway. Together, these data suggest that ctDNA is a broadly applicable, sensitive, and specific biomarker that can be used for a variety of clinical and research purposes in patients with multiple different types of cancer.

 

A microfluidic device for label-free, physical capture of circulating tumor cell clusters

A Fatih Sarioglu, Nicola Aceto, Nikola Kojic, Maria C Donaldson, Mahnaz Zeinali, Bashar Hamza, Amanda Engstrom, Huili Zhu, Tilak K Sundaresan, David T Miyamoto, Xi Luo, Aditya Bardia, Ben S Wittner, Sridhar Ramaswamy, Toshi Shioda, David T Ting, Shannon L Stott, Ravi Kapur, Shyamala Maheswaran, Daniel A Haber & Mehmet Toner

 

Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC clusters). Existing technologies for CTC enrichment are designed to isolate single CTCs, and although CTC clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here we developed a microchip technology (the Cluster-Chip) to capture CTC clusters independently of tumor-specific markers from unprocessed blood. CTC clusters are isolated through specialized bifurcating traps under low–shear stress conditions that preserve their integrity, and even two-cell clusters are captured efficiently. Using the Cluster-Chip, we identified CTC clusters in 30–40% of patients with metastatic breast or prostate cancer or with melanoma. RNA sequencing of CTC clusters confirmed their tumor origin and identified tissue-derived macrophages within the clusters. Efficient capture of CTC clusters will enable the detailed characterization of their biological properties and role in metastasis.

 

Glypican-1 identifies cancer exosomes and detects early pancreatic cancer

Sonia A. Melo, Linda B. Luecke, Christoph Kahlert, Agustin F. Fernandez, Seth T. Gammon, Judith Kaye, Valerie S. LeBleu, Elizabeth A. Mittendorf, Juergen Weitz, Nuh Rahbari, Christoph Reissfelder, Christian Pilarsky, Mario F. Fraga, David Piwnica-Worms & Raghu Kalluri

 

Exosomes are lipid-bilayer-enclosed extracellular vesicles that contain proteins and nucleic acids. They are secreted by all cells and circulate in the blood. Specific detection and isolation of cancer-cell-derived exosomes in the circulation is currently lacking. Using mass spectrometry analyses, we identify a cell surface proteoglycan, glypican-1 (GPC1), specifically enriched on cancer-cell-derived exosomes. GPC1+ circulating exosomes (crExos) were monitored and isolated using flow cytometry from the serum of patients and mice with cancer. GPC1+ crExos were detected in the serum of patients with pancreatic cancer with absolute specificity and sensitivity, distinguishing healthy subjects and patients with a benign pancreatic disease from patients with early- and late-stage pancreatic cancer. Levels of GPC1+ crExos correlate with tumour burden and the survival of pre- and post-surgical patients. GPC1+ crExos from patients and from mice with spontaneous pancreatic tumours carry specific KRAS mutations, and reliably detect pancreatic intraepithelial lesions in mice despite negative signals by magnetic resonance imaging. GPC1+ crExos may serve as a potential non-invasive diagnostic and screening tool to detect early stages of pancreatic cancer to facilitate possible curative surgical therapy.

 

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