来自加州大学San Diego分校Moores癌症中心的科学家们表示，他们找到了一种诊断癌症新技术，该技术可以在癌症发展的最早期也就是只有数个癌细胞的阶段就发现它们。而目前最好的方法也只能在肿瘤发展到包含约100万个细胞时作出诊断。
科学家在4月18日的在线《PLoS ONE》上的文章中描述了他们的一系列验证试验。他们可以在99.9%的正常DNA中寻找并放大微量导致癌症的DNA。目前的方法无法用于临床设备，因为它们需要相对较纯的癌细胞群，这对临床样品而言很困难。
Moores中心主任Dennis A. Carson说：“我们找到了在很早期发现各种由于DNA损伤引起的癌症的新技术。目前实验室正在与工程师合作，以研制用于临床的设备，这将广泛用于病人。”
Carson表示，尽管还需要数年时间进行临床测试，但是最终人们将通过简单的样本——例如血液、尿液等分析癌细胞的DNA标记。此外医生还可以简便且低成本的监测病人状态。一旦治疗起效，变异DNA将不存在，病人也就被治愈了。
这一被称为PAMP的技术基于一种酶反应，它发生在一片DNA被删除或者异常的和另一DNA结合时。而变异的具体位置并不重要，该方法将从正常DNA中探测出任何变异的片断，然后将这些变异分子放大。
当癌细胞变异时，通常会使两片应该分离的DNA结合起来。而酶反应就在此时起作用，这一技术能放大变异DNA，然后利用微阵列技术确定具体的变异。
原文链接：http://www.physorg.com/news96302531.html
译自：physorg.com
原始出处:
A Novel Approach for Determining Cancer Genomic Breakpoints in the Presence of Normal DNA
Yu-Tsueng Liu*, Dennis A. Carson
Moores UCSD Cancer Center, University of California San Diego, La Jolla, California, United States of America
CDKN2A (encodes p16INK4A and p14ARF) deletion, which results in both Rb and p53 inactivation, is the most common chromosomal anomaly in human cancers. To precisely map the deletion breakpoints is important to understanding the molecular mechanism of genomic rearrangement and may also be useful for clinical applications. However, current methods for determining the breakpoint are either of low resolution or require the isolation of relatively pure cancer cells, which can be difficult for clinical samples that are typically contaminated with various amounts of normal host cells. To overcome this hurdle, we have developed a novel approach, designated Primer Approximation Multiplex PCR (PAMP), for enriching breakpoint sequences followed by genomic tiling array hybridization to locate the breakpoints. In a series of proof-of-concept experiments, we were able to identify cancer-derived CDKN2A genomic breakpoints when more than 99.9% of wild type genome was present in a model system. This design can be scaled up with bioinformatics support and can be applied to validate other candidate cancer-associated loci that are revealed by other more systemic but lower throughput assays.
Introduction
Tumors evolve through the continuous accumulation and selection of randomly mutated genes. While sets of advantageous mutations are selected in tumors, neutral or even slightly detrimental mutations may also occur due to genomic instability and genetic drift. Recently, much effort has been expended to identify in primary human cancers point mutations in the exons of cancer-related genes. However, systemic mapping of genomic DNA rearrangements has lagged behind, due to technical difficulties in detecting smaller deletions, tumor heterogeneity, and the necessity to purify malignant from normal cells [1]. Historically, such work was done by time consuming and labor intensive genetics and molecular cloning on established cancer cell lines [2], [3], [4]. One of the most striking examples is the homozygous deletion of the CDKN2A (INK4A/ARF) tumor suppressor locus, which was discovered in this and other laboratories [3], [4], [5], [6], [7], [8]. The CDKN2A deletions occur early during tumor development [9], [10], [11]. The p16INK4a (one of the CDKN2A products [12]) protein constrains cell cycle progression by the Rb pathway and may be responsible for the decline in the replicative potential of stem cells during aging [13]. The p14ARF (the other alternative reading frame of CDKN2A [14]) gene product regulates the expression of MDM2, the turnover of p53, and thereby controls the cellular response to stress (reviewed in [6], [7], [8], [15], [16], [17]). Because the Rb and p53 pathways are central to cancer gate-keeping and caretaking [18], [19], strong selection pressures exist for the disruption of the entire CDKN2A gene segment on both chromosomes. Few other deletions are as well characterized, although it is expected that more will be found when more data from array based comparative genomic hybridization (array-CGH) are reported and also through The Cancer Genome Atlas (TCGA) project [20], [21], [22], [23], [24]. It will be important to validate the relevance of those genomic rearrangements to cancer development since many of the genomic structural changes may be simply due to genome instability in cancer. Large scale studies with clinical samples will be the most reliable confirmation.
While point mutations and very small insertions or deletions in genomic DNA can be detected by exon re-sequencing, it can be more difficult to detect gene dosage changes of larger genomic fragments, especially deletions [1]. Current established techniques for deletion mapping, including Southern blotting [25], fluorescent in situ hybridization (FISH) [26], quantitative PCR [26], [27], [28], [29], [30], and array-CGH [31] rely on the absence of a detectable wild type signal [1]. This is problematic when a significant number of normal cells are present in a tumor sample. Array-CGH has the potential to analyze alterations of DNA copy number on a genome-wide scale with relatively high resolution, depending on whether BACs, PCR products or oligonucleotides are used for the array elements. However, these techniques often fail where there is a heterogeneous cell population or samples of poor quality [31]. FISH is less vulnerable to the presence of heterogeneous cell populations, but has relatively low resolution and is difficult to scale up. Except for FISH, the other techniques mentioned are not practical for mapping genomic translocations and inversions. End-sequencing profiling was developed to address this issue but the approach was costly and hard to scale up [32]. Therefore, there is a need to develop a scalable approach for detecting such genomic structural changes in solid tumors where heterogeneous cell populations are present.
Here we report a novel approach, designated as Primer Approximation Multiplex PCR (PAMP), to enrich small amounts of deleted genomic DNA sequences in the presence of wild type DNA. The genomic locations of the enriched sequences are subsequently decoded by a genomic tiling array and confirmed by sequencing.
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