Kunio Kitada

The complicated copy number alterations in chromosome 7 of a lung cancer cell line is explained by a model based on repeated breakage-fusion-bridge cycles

The drug-resistant lung cancer cell line PTX250, which has been previously established by exposure to an anti-cancer drug paclitaxel, has an increased copy number in the MDR1/ABCB1 locus region. In addition, the flanking regions also exhibit aberrant copy numbers, making the copy number profile of chromosome 7 complicated. In this study, we tested whether the breakage-fusion-bridge (BFB) cycle model can explain such copy number alterations. An analysis using fluorescence in situ hybridization (FISH) with a painting probe demonstrated that the aberrant chromosome, designated chromosome 7(amp), was derived from an intact chromosome 7. Using high-density comparative genomic hybridization arrays, we examined the copy number profile in detail and divided chromosome 7(amp) into seven segments. Based on copy numbers of each segment, which were determined using interphase- and metaphase-FISH analysis, we constructed a formation model for the complicated copy number alteration. Six-time BFB cycles and the cycle-termination by healing of broken ends were presupposed in the model. Locations and orientations of the segments observed in chromosome 7(amp) agreed well with those predicted from the model. Telomere addition was also cytogenetically confirmed. In all, it could be concluded that the complicated copy number alteration found in chromosome 7(amp) is generated from the intact chromosome 7 by the repeated BFB cycles.

Chromosome-specific segmentation revealed by structural analysis of individually isolated chromosomes.

Analysis of structural rearrangements at the individual chromosomal level is still technologically challenging. Here we optimized a chromosome isolation method using fluorescent marker‐assisted laser‐capture and laser‐beam microdissection and applied it to structural analysis of two aberrant chromosomes found in a lung cancer cell line. A high‐density array‐comparative genomic hybridization (array‐CGH) analysis of DNA samples prepared from each of the chromosomes revealed that these two chromosomes contained 296 and 263 segments, respectively, ranging from 1.5 kb to 784.3 kb in size, derived from different portions of chromosome 8. Among these segments, 242 were common in both aberrant chromosomes, but 75 were found to be chromosome‐specific. Sequences of 263 junction sites connecting the ends of segments were determined using a PCR/Sanger‐sequencing procedure. Overlapping microhomologies were found at 169 junction sites. Junction partners came from various portions of chromosome 8 and no biased pattern in the positional distribution of junction partners was detected. These structural characteristics suggested the occurrence of random fragmentation of the entire chromosome 8 followed by random rejoining of these fragments. Based on that, we proposed a model to explain how these aberrant chromosomes are formed. Through these structural analyses, it was demonstrated that the optimized chromosome isolation method described here can provide high‐quality chromosomal DNA for high resolution array‐CGH analysis and probably for massively parallel sequencing analysis.

Comparison of murine gene expression profiles between spontaneous and radiation-induced myelogenous leukemias: Stochastic and probabilistic expression variances in the former vs radiation-specific expression commonalities in the latter

OBJECTIVE To elucidate the common characteristics of murine radiation-induced myelogenous leukemias, global gene-chip expression profiles were compared with age-matched steady-state bone marrow tissue profiles and spontaneous myelogenous leukemia profiles. MATERIALS AND METHODS Six each of C3H/He mice-derived radiation-induced and spontaneously developed myelogenous leukemias were analyzed. Bone marrow cells from five each of 2- and 21-month-old mice were used to subtract nonleukemic information in the analysis. mRNAs from individual mice were analyzed separately using 45,101 gene chips followed by computational biological analysis. RESULTS First, principal component analysis (PCA) was performed to discriminate the gene expression profiles of individual mice with radiation-induced myelogenous leukemia from those of bone marrow cells from 2- or 21-month-old mice. Discriminant union genes for individual leukemias were then selected, which finally yielded 242 genes, among which six are radiation-related genes including Hus-1, Edf1a2, andVegf-c; 16 are apoptosis/cell-death-related genes, 13 are cell-cycle/cell-growth-related genes, and 50 are suppressor/promoter genes. PCA of these 242 genes consistently enabled the discrimination of the radiation-induced leukemias from the spontaneous leukemias. Second, the other components of the same PCA provided four different eigenvector clusters in an unsupervised manner representing four histopathological findings, with which the differential diagnosis in molecular taxonomy was significant as determined by analysis of variance of the global gene expression profiles. CONCLUSION Discriminant union genes in radiation-induced myelogenous leukemias against spontaneous myelogenous leukemias and age-matched nonleukemic bone marrow profilings generated by unsupervised computational analysis essentially represent probabilistic biomarkers for radiation-induced myelogenous leukemias, which may contribute to developing a model for risk of secondary carcinogenesis in patients treated by whole-body irradiation.

A Novel Mechanism of EML4-ALK Rearrangement Mediated by Chromothripsis in a Patient-Derived Cell Line

Introduction: EML4-ALK is a driver oncogene in non–small-cell lung cancer (NSCLC) and has been developed into a promising molecular target for antitumor agents. Although EML4-ALK is reported to be formed by inversion of chromosome 2, other mechanisms of this gene fusion remain unknown. This study aimed to examine the mechanism of EML4-ALK rearrangement using a novel cell line with the EML4-ALK fusion gene. Methods: An EML4-ALK-positive cell line, termed JFCR-LC649, was established from pleomorphic carcinoma, a rare subtype of NSCLC. We investigated the chromosomal aberrations using fluorescence in situ hybridization and comparative genomic hybridization (CGH). Alectinib/CH5424802, a selective ALK inhibitor, was evaluated in the antitumor activity against JFCR-LC649 in vitro and in vivo xenograft model. Results: We established an EML4-ALK-positive cell line, termed JFCR-LC649, derived from a patient with NSCLC and revealed that the JFCR-LC649 cells harbor variant 3 of the EML4-ALK fusion with twofold copy number gain. Interestingly, comparative genomic hybridization and metaphase-fluorescence in situ hybridization analysis showed that in addition to two normal chromosome 2, JFCR-LC649 cells contained two aberrant chromosome 2 that were fragmented and scattered. These observations provided the first evidence that EML4-ALK fusion in JFCR-LC649 cells was formed in chromosome 2 by a distinct mechanism of genomic rearrangement, termed chromothripsis. Furthermore, a selective ALK inhibitor alectinib/CH5424802 suppressed tumor growth of the JFCR-LC649 cells through inhibition of phospho-ALK in vitro and in vivo in a xenograft model. Conclusion: Our results suggested that chromothripsis may be a mechanism of oncogenic rearrangement of EML4-ALK. In addition, alectinib was effective against EML4-ALK-positive tumors with ALK copy number gain mediated by chromothripsis.

Amplification of the ABCB1 region accompanied by a short sequence of 200bp from chromosome 2 in lung cancer cells.

Lung cancer sublines No15-80-1 and No15-80-6 were selected by treatment of cell line NCI-H460 with paclitaxel at stepwise increasing concentrations from 50 nmol/L to 800 nmol/L. The two sublines exhibited amplifications of the ABCB1 region (previously MDR1) with different copy number profiles, but shared a common amplification pattern, which has been observed in amplification mediated by the breakage-fusion-bridge (BFB) cycle. Sequence analysis of the distal ends of the amplified regions, which were probably generated in a break-and-fusion of the initial round of the BFB cycle, revealed a head-to-head fused sequence of chromosome 7. The sequence was identical in the two sublines. A short sequence of 200bp derived from chromosome 2 was incorporated, suggesting translocation between chromosomes 2 and 7. The copy number of the short sequence was comparable to that of the neighboring sequence, suggesting coamplification. The timing of the occurrence of the putative translocation and the initiation of BFB-cycle-driven amplification during the stepwise selection were determined by using the unique junction sequences specific to these events as indicators. The results demonstrated that the translocation occurred at the step of 100 nmol/L treatment and the BFB cycle initiated in the step of 400 nmol/L-treatment. It is likely that the translocation, preceding amplification by several selection steps, activated ABCB1 gene expression. The diversity in amplification profiles between the two sublines was generated by the separately operating BFB cycles, after an initial break-and-fusion that probably occurred in a single cell.

Abstract 2192: A novel mechanism of EML4-ALK rearrangement in a patient-derived cell line and its tumor growth inhibition by an ALK inhibitor CH5424802.

Purpose: EML4-ALK is a driver oncogene in non-small cell lung cancer (NSCLC) and has been developed into a promising molecular target for antitumor agents. Although EML4-ALK is reported to be formed by inversion of chromosome 2, other mechanisms of gene fusion remain unknown. Here we characterize the EML4-ALK fusion gene of a new cell line derived from a patient of NSCLC. In addition, the efficacy of ALK inhibitors against this new cell line was examined. Experimental Design: An EML4-ALK-positive cell line, termed LC649, was established from pleomorphic carcinoma, a rare subtype of NSCLC. We investigated the chromosomal aberrations using fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). CH5424802, a selective ALK inhibitor, was evaluated in the antitumor activity against LC649 in vitro and in vivo xenograft model. Results: Our initial characterization revealed that LC649 harbors variant 3 of EML4-ALK fusion with 2-fold copy number gain. Interestingly, CGH and metaphase-FISH analysis showed that LC649 contained, in addition to two normal chromosome 2, two aberrant chromosome 2 that were fragmented and scattered. These observations provided the first evidence that EML4-ALK fusion in LC649 cells was formed in chromosome 2 by a distinct mechanism of genomic rearrangement, termed “chromothripsis”. Additionally, CH5424802 inhibited phospho-ALK and suppressed tumor growth of the LC649 cells in vitro and in vivo in a xenograft model. Conclusions: Our results suggested that chromothripsis may be a mechanism of oncogenic rearrangement of EML4-ALK. CH5424802 was effective against EML4-ALK positive tumors with ALK copy number gain mediated by chromothripsis. Citation Format: Tatsushi Kodama, Noriko Motoi, Hironori Ninomiya, Hiroshi Sakamoto, Kunio Kitada, Toshiyuki Tsukaguchi, Yasuko Sato, Nobuya Ishii, Yasuhito Terui, Kiyohiko Hatake, Yuichi Ishikawa. A novel mechanism of EML4-ALK rearrangement in a patient-derived cell line and its tumor growth inhibition by an ALK inhibitor CH5424802. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2192. doi:10.1158/1538-7445.AM2013-2192