當檢測實體瘤仍是癌症診斷中的常規步驟時，現代技術，比如新一代測序技術就已經可以幫助科學家們深入追蹤癌症的發展變化了，許多腫瘤都會有一些脫落的細胞，而這些脫落的小囊泡名為外核體，而其DNA的軌跡會進入血液和其它體液組織中；近來有研究發現，這些外核體碎片可以作為標誌物來幫助監測疾病的進展，甚至可以幫助科學家們在患者疾病症狀出現之前診斷癌症的發生。
利用常規的血液樣本就可以對腫瘤DNA進行檢測，比如刊登在國際雜誌JAMA Oncology上的一篇研究論文中，研究者們就對4000多名孕婦機體的血液樣本進行了檢測，用來鑒別胎兒染色體的異常情況，隨後研究者發現了三例母源性的癌症，即卵巢癌、濾泡型淋巴瘤以及霍傑金淋巴瘤，在大多數腫瘤中，盡管其惡性程度較低，但研究者仍然可以利用個體的血液來作為研究腫瘤生物學的替代品。
而諸如“液體活組織檢查”對於血液和血漿樣本而言並不是特殊的，在其它研究中，研究者將膀胱癌複發的風險同尿液中DNA甲基化的水平聯係了起來，研究者也在糞便樣本中檢測到了腸癌的DNA，同時還在頭頸癌患者的唾液中鑒別出了癌症相關的基因突變；此前研究者常利用分子檢測的手段監測惡性疾病和癌症轉移，而如今隨著精確工具使用的增加，研究者可以在血液中，甚至是疾病發展早期階段就可以鑒別出少量的癌細胞和DNA。
來自約翰霍普金斯醫學院的教授Bert Vogelstein表示，糞便和尿液可以幫助檢測結直腸癌和膀胱癌，但血液檢測至少從概念上來講可以檢測所有癌症，目前我們麵臨的挑戰是檢測微量的癌細胞DNA。
普通的證據
去年刊登在國際雜誌Science Translational Medicine上的一篇研究論文中，研究者對640名病人進行了研究，發現對血漿中循環的腫瘤DNA進行檢測可以幫助確定大約40%至70%的各類癌症，包括腦癌、前列腺癌和卵巢癌等。比如在惡性的結直腸癌中，循環的腫瘤DNA常被用於確定87%的患者機體KRAS基因的特殊突變。
完整的癌細胞通常會溜入血流中，“誘捕”這些循環腫瘤細胞（CTCs）的早期嚐試依賴於表麵抗原和其它標誌物的識別，但CTCs會依賴於腫瘤的類型、疾病階段及其它因子來穿上不同的分子外衣；今年年初，哈佛醫學院的研究者Mehmet Toner在國際雜誌Nature Methods刊文表示，微流體設備可以利用物理學的方法來誘捕CTCs，而該方法並不依賴於腫瘤特異性的標誌物，Toner告訴The Scientist說道，完整無損的細胞具有巨大的研究價值，你可以仔細觀察DNA、RNA、信號分子、磷酸化模式以及表觀遺傳學，這些相比單一的生物標誌物要更為豐富，從長期角度而言，我們應當培養細胞來檢測藥物的敏感性，從而轉移到進行個體化醫學的開發。
除了DNA和整個細胞而言，近來又有研究指出，腫瘤細胞脫落的外核體或許可以充當癌症的生物標誌物，就在上個月，刊登在國際雜誌Nature上的一項研究報告中，來自得克薩斯大學MD癌症研究中心的科學家就對外核體進行了一項血清學測試，由於外核體攜帶有DNA、RNA和蛋白質，因此其可以被用於區分早期胰腺癌和晚期胰腺癌的患者。
但循環腫瘤細胞的痕跡依然是目前臨床應用的一大難題，改善分子檢測的敏感性和特異性對於建立臨床應用程序非常關鍵，研究者Vogelstein表示，目前我們並不知道局限性是否是技術上或生物學上的，我們發現大約40%至70%的腫瘤都可以被檢測到，但如果剩餘的早期階段的癌症並沒有分泌循環腫瘤DNA中的單一分子的話，那我們的技術再怎麼好也是無關緊要的。
目前很多基礎生物學都是不清楚的，研究者Tim Forshew教授說道，我們仍然並不能完全知道循環腫瘤DNA進入到血液中的機製，以及為何其會因不同的癌症而異，我們也並不能完全理解使得循環的腫瘤DNA可以在血液中被快速消除。盡管如此，利用簡單血液檢測來診斷癌症並且指導療法的可能性依然會讓很多製藥公司大發靈感來尋找新型的腫瘤血液檢測技術。
早期應用
研究者Forshew就職於一家提供循環腫瘤DNA診斷檢測技術的公司，他表示，從商業利益角度而言公司都比較關注循環腫瘤的標誌物，在他看來，類似這樣技術的重要應用就是研究並不容易進行活組織檢查的癌症的遺傳特性。許多其它公司，包括Epic科學、強生診斷、SRI國際等都能夠提供循環腫瘤DNA和細胞的檢測技術，然而這些檢測手段的臨床應用目前仍然受限於腫瘤轉移的監測。
科學家們進行的臨床研究通常是將血液中的腫瘤DNA同特殊的疾病參數相聯係起來，比如預測患者移除腫瘤術後疾病複發的風險，Vogelstein教授說道，這並不同於預後評估。近日，來自伊麗莎-霍爾醫學研究所的科學家們正在利用循環腫瘤DNA來評估結腸癌患者在術後因化療獲益的可能性，標準上而言，大部分患者在術後會接受輔助化學療法，但僅有4%至5%的患者會因此獲益，對於其餘患者而言，要麼手術比較充分，要麼就是化療後癌症依然複發的。
在去年美國臨床腫瘤學會會議上研究者公布了他們的初期研究結果，他們發現癌症複發風險和循環腫瘤DNA的水平密切相關，而如今科學家們又計劃進行一項大型的隨機試驗來評估利用循環腫瘤DNA來進行化療規範的實用性，通過血液檢測來知道化療，不僅可以改善癌症患者的生存，而且還可以降低接受化療患者的數量。
相比此前使用的前列腺特異抗原而言，腫瘤DNA、CTCs以及腫瘤衍生的外核體都可以作為較高價值的標誌物，這些新型標誌物可以為研究者提供一種絕佳的機會來管理癌症，最後研究者表示，此前我們總是晚疾病一步，而且我們試圖趕上疾病發生的節奏，而如今利用更多特異性且敏感性的工具我們就可以領先疾病一步診斷並且發現疾病，對於後期製定可用的治療手段或將帶來巨大的幫助。
參考文獻：
doi:10.1001/jamaoncol.2015.1883
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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 follicularlymphoma, 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.
doi:10.1126/scitranslmed.3007094.
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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 digitalpolymerasechain 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.
doi:10.1038/nmeth.3404
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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.
doi:10.1038/nature14581
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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.
