SAN DIEGO (EGMN) – Scientists have developed a new way to track cancer using DNA-based blood tests to monitor individualized biomarkers after treatment of solid tumors, and thereby to detect cancer recurrence, an advance that could further the personalized management of cancer patients.

The method, known as PARE (Personalized Analysis of Rearranged Ends), “is based on next-generation mate-paired analysis of resected tumor DNA to identify individualized tumor-specific rearrangements,” researchers led by Dr. Victor Velculescu explained in a study scheduled to be published on Feb. 24 in Science Translational Medicine. “Such alterations are used to develop [polymerase chain reaction–based] quantitative analyses for personalized tumor monitoring of plasma samples or other bodily fluids.”

During a press briefing at the annual meeting of the American Association for the Advancement of Science, one of the study’s authors, Dr. Luis A. Diaz Jr., said that PARE “allows us to measure the amount of cancer DNA in any clinical specimen. As soon as a patient’s cancer is identified by biopsy, it can be scanned for gene rearrangements. These rearrangements will then be a template to act as a fingerprint for that individual cancer. This can be applied in a variety of clinical scenarios.”

Dr. Velculescu, codirector of the cancer biology program at Johns Hopkins Kimmel Cancer Center in Baltimore, and his associates used six sets of tissue samples obtained from four patients with colorectal cancer and two patients with breast cancer to catalog the number of gene sequences in each patient. They did this by first identifying regions where the number of DNA sequences was more or less than anticipated and where sections of different chromosomes fused together.

The researchers then analyzed these regions further to identify DNA sequences that displayed incorrect ordering, orientation, or spacing, and observed that an average of 9 rearrangements was found in each of the six samples (range, 4-17).

“The rearrangements represent the most dramatic form of genetic changes that can occur in the genome,” Dr. Velculescu said at the briefing. “If the genome were like a book with many chapters, the rearrangement would be like swapping of two chapters within book, so that both chapters would be out of order. If one could recognize rearrangements accurately, that could potentially be one of the best ways to distinguish cancer cells from normal cells.”

Next, they looked for the same changes as shed from tumors into the blood of patients. After amplifying DNA that was found in the blood of two of the colorectal cancer patients, they determined that the tests were robust enough to detect rearranged tumor DNA.

For example, the fraction of mutant DNA contained in the blood of one of the colorectal cancer patients was 37% prior to surgery for tumor removal, and it dropped to 14% after surgery. “The mutant DNA fraction decreased further after chemotherapy and subsequent removal of metastatic lesions from the right lobe of the liver,” the researchers reported. “However, the fraction of mutant tumor DNA did not reach zero (remaining at 0.3% at day 137), consistent with the fact that this patient had residual metastatic lesions in the remaining left lobe of the liver.”

The researchers noted certain limitations of the study, including the chance that some rearranged genetic sequences “could be lost during tumor progression,” and that the PARE assay currently costs about $5,000, making it expensive for general clinical use. “This cost is a consequence of the high physical coverage and the inefficiencies associated with stringent mapping of 25-bp sequence data to the human genome,” the researchers explained.

“As read quality and length continue to improve, less stringent mapping criteria and lower physical coverage will permit analyses similar to those in this study but with substantially less sequencing effort. Moreover, the cost of massively parallel sequencing, which has decreased substantially over the last 2 years, continues to spiral downwards.”

Despite such limitations, Dr. Velculescu and his associates maintain that the potential applications for PARE are “numerous,” including the identification of tumor-free surgical margins, the analysis of regional lymph nodes, and the measurement of circulating tumor DNA after surgery, radiation, or chemotherapy. “Short-term monitoring of circulating tumor DNA may be particularly useful in the testing of new drugs, as it could provide an earlier indication of efficacy than possible through conventional diagnostic methods such as computed tomography scanning,” they concluded.

At the briefing, Dr. Diaz, an oncologist at Johns Hopkins, said that PACE could be used to help clinicians determine who is cured or not cured after surgical resection. “Currently, as physicians we can’t tell a patient after breast, colon, or lung cancer surgery whether or not they’ve been cured,” he commented. “A fraction of these patients will be cured by surgery alone, but many will have residual disease. We hope that PARE will be able to discriminate between those individuals that are cured and those that are not cured by detecting residual disease at first surgery. This approach would thereby spare cured individuals from unnecessary and potentially toxic and harmful chemotherapy.”

Under a licensing agreement between the Johns Hopkins University and Genzyme Corp., three of the investigators, including Dr. Velculescu, are entitled to a share of royalties received by the university on sales of products related to research described in the study.

Copyright (c) 2009 Elsevier Global Medical News. All rights reserved. This material may not be published, broadcast, rewritten, or redistributed.

圣地亚哥(EGMN)——科学家现已研发了一种追踪癌症的新方法，在实体瘤治疗后，采用基于DNA的血液检查来监测个性化的生物标志物，从而发现癌症复发，这一进展将癌症患者的个性化管理又向前推进了一步。

 

计划将于2月24日发表在《科学——转化医学》(Science Translational Medicine)上的一篇文章中，Victor Velculescu博士领导的研究者解释道，这种名为PARE(重排终端个性化分析)的方法是“基于所切除肿瘤DNA的下一代配对分析，目的是确定个性化的肿瘤特异性重排。”研究者说：“这种改变是用于研发(基于多聚酶链反应)的定量分析，以实现血浆样本或其他体液的个性化肿瘤监测。”

 

在美国科学促进学会2010年年会上，该研究的作者之一Luis A. Diaz Jr.博士在一次新闻发布会上表示，PARE“使我们能够测定任何临床样本中癌症DNA的含量。一旦通过活检发现患者患有癌症，即可进行基因重排扫描。然后将得到的重排结果作为模板，视为该个体癌症的指纹图谱，可广泛用于各种临床情况。”

 

作为这项癌症生物学研究项目的负责人之一，美国巴尔的摩约翰霍普金斯凯门癌症中心的Velculescu博士及其助手采用取自4例结直肠癌患者和2例乳腺癌患者的6套组织样本，列出了每例患者基因序列的数量。研究者首先确定了DNA序列数量多于或少于预期数量，以及不同的染色体片段融合到了一起的区域。

 

然后，研究者对这些区域进行进一步分析以找出排列、定位或间距错误的DNA序列，观察发现在这6份样本中，每份平均出现了9个基因重排。

 

Velculescu博士在新闻发布会上说：“这些重排反映了基因组中出现的最明显的遗传学改变类型。如果将基因组比作一本由许多章节组成的书，那么重排便是书中的2个章节颠倒了，因此这2个章节的顺序都乱了。如果我们能够准确找出这些重排，这很可能是区分癌细胞与正常细胞的最佳方法之一。”

 

下一步，研究者在患者的血液中寻找与肿瘤改变相同的变化。在对2例结直肠癌患者血液中找到的DNA进行扩增后，研究者确定该血液检查足以检测出重排的肿瘤DNA。

 

例如，其中1例结直肠癌患者的血液中所含的DNA突变片段在行肿瘤摘除术之前占37%，术后即降至14%。研究者报告称：“在化疗以及之后开展的肝脏右叶转移灶摘除术后，DNA突变片段进一步减少。但突变的肿瘤DNA片段并未达到0 (第137天时仍有0.3%)，这与该患者肝脏左叶仍残余有转移灶的事实相符。”

 

同时，研究者也指出该研究存在一定的局限性，包括部分重排的基因序列有可能“在肿瘤进展过程中丢失”，以及目前PARE检测的费用大约在5,000美元左右，价格高昂难以普遍用于临床。研究者解释道：“检查费用高是因为所涉及的身体部位较广，且准确定位人体基因组的25-bp序列数据耗时很长。”

 

“随着读取质量和长度的不断改善，定位标准可能不那么严格，所涉及的身体部位也将缩小，但能够达到与本研究类似的分析效果，只是在基因测序上所花的精力将大大减少。而且，大规模平行测序的价格在过去2年内已大幅下降，还将继续呈螺旋式下降。”

 

虽然存在这些局限性，但Velculescu博士及其助手坚持认为PARE的潜在使用范围“非常广”，包括判定无肿瘤的手术定界、分析局部淋巴结以及测定手术、放疗或化疗后的循环肿瘤DNA。研究者总结道：“短期监测循环肿瘤DNA可能对检测新药尤其有用，因为相对于传统的诊断方法，如计算机断层扫描，PARE能够更早地显示药物的疗效。”

 

在新闻发布会上，约翰霍普金斯大学的肿瘤学专家Diaz博士说，PARE可用于帮助临床医生确定手术切除后患者是否得以治愈。Diaz博士评论道，“目前，在乳腺癌、结肠癌或肺癌手术后医生无法告知患者是否治愈。小部分患者可能单纯用手术就能治愈，但多数患者都有残余病灶。我们希望PARE能够通过检测初次手术后的残余病灶判别哪些患者治愈了，哪些患者没有治愈。那么已经治愈的患者便无需再接受不必要且有潜在毒性和有害性的化疗。”

 

依照约翰霍普金斯大学与健赞公司之间的许可协议，包括Velculescu博士在内的3名研究者有权收取翰霍普金斯大学因出售与该研究相关的产品所获使用费的提成。

 

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