Kim Hyun-Kyoung, Park Won Cheol, Lee Kwang Man, Hwang Hai-Li, Park Seong-Yeol, Sorn Sungbin, Chandra Vishal, Kim Kwang Gi, Yoon Woong-Bae, Bae Joon Seol, Shin Hyoung Doo, Shin Jong-Yeon, Seoh Ju-Young, Kim Jong-Il, Hong Kyeong-Man
Research Institute, National Cancer Center, Ilsandong-gu, Goyang, Korea; Department of Microbiology, Ewha Womans University School of Medicine, Seoul, Korea.
Department of Surgery, Wonkwang University School of Medicine, Iksan, Korea.
PLoS One. 2014 Jun 17;9(6):e100089. doi: 10.1371/journal.pone.0100089. eCollection 2014.
The concept of the utilization of rearranged ends for development of personalized biomarkers has attracted much attention owing to its clinical applicability. Although targeted next-generation sequencing (NGS) for recurrent rearrangements has been successful in hematologic malignancies, its application to solid tumors is problematic due to the paucity of recurrent translocations. However, copy-number breakpoints (CNBs), which are abundant in solid tumors, can be utilized for identification of rearranged ends.
As a proof of concept, we performed targeted next-generation sequencing at copy-number breakpoints (TNGS-CNB) in nine colon cancer cases including seven primary cancers and two cell lines, COLO205 and SW620. For deduction of CNBs, we developed a novel competitive single-nucleotide polymorphism (cSNP) microarray method entailing CNB-region refinement by competitor DNA.
Using TNGS-CNB, 19 specific rearrangements out of 91 CNBs (20.9%) were identified, and two polymerase chain reaction (PCR)-amplifiable rearrangements were obtained in six cases (66.7%). And significantly, TNGS-CNB, with its high positive identification rate (82.6%) of PCR-amplifiable rearrangements at candidate sites (19/23), just from filtering of aligned sequences, requires little effort for validation.
Our results indicate that TNGS-CNB, with its utility for identification of rearrangements in solid tumors, can be successfully applied in the clinical laboratory for cancer-relapse and therapy-response monitoring.
利用重排末端开发个性化生物标志物的概念因其临床适用性而备受关注。尽管针对复发性重排的靶向新一代测序（NGS）在血液系统恶性肿瘤中已取得成功，但其在实体瘤中的应用因复发性易位的缺乏而存在问题。然而，实体瘤中丰富的拷贝数断点（CNB）可用于识别重排末端。
作为概念验证，我们对9例结肠癌病例（包括7例原发性癌以及2种细胞系COLO205和SW620）进行了拷贝数断点处的靶向新一代测序（TNGS-CNB）。为了推断CNB，我们开发了一种新型竞争性单核苷酸多态性（cSNP）微阵列方法，该方法通过竞争DNA对CNB区域进行优化。
使用TNGS-CNB，在91个CNB中鉴定出19个特异性重排（20.9%），6例（66.7%）获得了两个可通过聚合酶链反应（PCR）扩增的重排。并且，显著的是，仅通过比对序列筛选，TNGS-CNB在候选位点（19/23）对可通过PCR扩增的重排具有较高的阳性识别率（82.6%），几乎无需费力进行验证。
我们的结果表明，TNGS-CNB可用于识别实体瘤中的重排，能够成功应用于临床实验室进行癌症复发和治疗反应监测。
