Copy Number Gains at 8q24 and 20q11-q13 in Gastric Cancer Are More Common in Intestinal-Type than Diffuse-Type

<div><p>The present study was aimed at discovering DNA copy number alterations (CNAs) involved in the carcinogenesis of stomach and at understanding their clinicopathological significances in the Korean population. DNA copy numbers were analyzed using Agilent 244K or 400K array comparative genomic hybridization (aCGH) in fresh-frozen tumor and matched normal tissues from 40 gastric cancer patients. Some of the detected CNA regions were validated using multiplex ligation-dependent probe amplification (MLPA) in six of the 40 patients and customized Agilent 60K aCGH in an independent set of 48 gastric cancers. The mRNA levels of genes at common CNA regions were analyzed using quantitative real-time PCR. Copy number gains were more common than losses across the entire genome in tumor tissues compared to matched normal tissues. The mean number of alterations per case was 64 for gains and 40 for losses, and the median aberration length was 44016 bp for gains and 4732 bp for losses. Copy number gains were frequently detected at 7p22.1 (20%), 8q24.21 (27%–30%), 8q24.3 (22%–48%), 13q34 (20%–31%), and 20q11-q13 (25%–30%), and losses at 3p14.2 (43%), 4q35.2 (27%), 6q26 (23%), and 17p13.3 (20%–23%). CNAs at 7p22.1, 13q34, and 17p13.3 have not been reported in other populations. Most of the copy number losses were associated with down-regulation of mRNA levels, but the correlation between copy number gains and mRNA expression levels varied in a gene-dependent manner. In addition, copy number gains tended to occur more commonly in intestinal-type cancers than in diffuse-type cancers. In conclusion, the present study suggests that copy number gains at 8q24 and 20q11-q13 and losses at 3p14.2 may be common events in gastric cancer but CNAs at 7p22.1, 13q34, and 17p13.3 may be Korean-specific.</p></div

Correlations of CNAs levels with patient’s age at diagnosis and other CNAs in different chromosomal regions.

<p>(A) Correlations of CNAs levels with patient’s age were analyzed using Pearson’s correlation coefficient. Multiple tests were corrected using the Bonferroni correction. The Bonferroni-corrected <i>P</i>-values were calculated by multiplying the observed (uncorrected) <i>P</i>-values by the number of tested genes. Violet colors indicate Bonferroni-adjusted <i>P-</i>value < 0.05. (B) Unsupervised hierarchical clustering analysis of 15 genes in regions with recurrent (>20%) CNAs was performed to investigate correlations among CNAs levels of 11 genes showing copy number gains on 8q24.21, 8q24.3, 20q11.21, and 20q13.12, and 4 genes showing copy number losses. The numbers on the right side of the figure indicate patient identification number. The color scales indicate log2 intensity ratios of CNAs at individual gene. Value “zero (= log(2/2)” indicate a copy number of 2. Green and red colors represent copy number gain and loss, respectively.</p

Copy number alterations of gastric cancer using aCGH.

<p>(A) Overall aberration frequency in gastric cancers is shown. The horizontal line represents chromosomes in order from 1 to 22 and X. The vertical line represents the frequency of gains and losses in all cases. (B) The number of aberrations in each case is shown. Magenta bars indicate gains and viridian bars indicate losses. (C) Aberration length—density plot is shown. (D) Common CNAs in 40 gastric cancer samples are shown. Within each chromosome, aberrations are expressed in order from the p telomere to q telomere. Reds on the right of the chromosomes indicate gains, and greens on the left of the chromosomes indicate losses. (E) Common aberration length with p-value plot is shown. (F) Common aberrations in chromosome 8 (upper panel) and aberrations around <i>MYC</i> gene at 8q24.21 (lower panel) are shown. Vertical lines indicate log₂-based intensity ratio values, and each colored horizontal line represents a copy number alteration. Horizontal lines above the 0.25 of log₂-based intensity ratio indicate samples with copy number gains. The large vertical blue bar in the lower panel indicates the center of the currently analyzed region.</p

CNAs according to Lauren’s classification.

<p>(A-B) Correlations of log2 ratio of DNA copy number and mRNA fold change (FC) of <i>FHIT</i> (A) and <i>BOP1</i> (B) in tumor tissues compared to matched normal tissues were analyzed using Pearson’s correlation coefficients. Different color circles indicate different samples. (C-E) The differences in prevalence of copy number alterations of multiple genes were compared between diffuse-type cancers and intestinal-type cancers using Pearson’s chi-square test. Copy number gains of <i>MYC</i> (<i>P</i> = 0.03), <i>BOP1</i> (<i>P</i> = 0.03), and <i>CD40</i> (<i>P</i> = 0.01) occurred at a high prevalence in intestinal-type cancers compared to diffuse-type cancers. In general, copy number gains tend to occur more frequently in intestinal-type cancers than in diffuse-type cancers.</p

Genetic Aberrations in Imatinib-Resistant Dermatofibrosarcoma Protuberans Revealed by Whole Genome Sequencing

<div><p>Dermatofibrosarcoma protuberans (DFSP) is a very rare soft tissue sarcoma. DFSP often reveals a specific chromosome translocation, t(17;22)(q22;q13), which results in the fusion of collagen 1 alpha 1 (<i>COL1A1</i>) gene and platelet-derived growth factor-B (<i>PDGFB</i>) gene. The COL1A1-PDGFB fusion protein activates the PDGFB receptor and resultant constitutive activation of PDGFR receptor is essential in the pathogenesis of DFSP. Thus, blocking PDGFR receptor activation with imatinib has shown promising activity in the treatment of advanced and metastatic DFSP. Despite the success with targeted agents in cancers, acquired drug resistance eventually occurs. Here, we tried to identify potential drug resistance mechanisms against imatinib in a 46-year old female with DFSP who initially responded well to imatinib but suffered rapid disease progression. We performed whole-genome sequencing of both pre-treatment and post-treatment tumor tissue to identify the mutational events associated with imatinib resistance. No significant copy number alterations, insertion, and deletions were identified during imatinib treatment. Of note, we identified newly emerged 8 non-synonymous somatic mutations of the genes (<i>ACAP2</i>, <i>CARD10</i>, <i>KIAA0556</i>, <i>PAAQR7</i>, <i>PPP1R39</i>, <i>SAFB2</i>, <i>STARD9</i>, and <i>ZFYVE9</i>) in the imatinib-resistant tumor tissue. This study revealed diverse possible candidate mechanisms by which imatinib resistance to PDGFRB inhibition may arise in DFSP, and highlights the usefulness of whole-genome sequencing in identifying drug resistance mechanisms and in pursuing genome-directed, personalized anti-cancer therapy.</p></div

Minimal common regions^a of recurrent (>20%) copy number gains or losses.

<p>^a MCRs (Minimal Common Regions) were identified through the analysis of three arrays (aCGH-244K, aCGH-400K, aCGH-60K). MCR was defined as a 100 percent overlapping common region that was observed in all three arrays.</p><p>^b When CNAs occurred within whole DNA sequences of a gene, the gene is depicted as bold.</p><p>Minimal common regions<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137657#t001fn001" target="_blank">^a</a> of recurrent (>20%) copy number gains or losses.</p

A schematic diagram for identifying a minimal common region.

<p>The Aberration Detection Method 2 (ADM-2) algorithm with a sensitivity threshold of 6.0 was used to identify the CNAs in gastric cancer and to determine the frequencies of CNAs in each sample. The MCR (Minimal Common Region) of copy number gains or losses was identified through the analysis of CNAs in the three kinds of aCGHs (244K, 400K, and 60K).</p

The association of the expression of selected genes with copy number alterations.

<p>* The significance of mRNA fold change (FC) of individual genes was statistically analyzed by one-sample t-test.</p><p>The association of the expression of selected genes with copy number alterations.</p

Sequencing data analysis.

<p>The value of x-axis denotes the allelle frequency of imatinib-sensitive tumor sample and the value of y-axis denotes the allelle frequency of imatinib-resistant tumor sample of the respective 46 somatic mutations.</p

Newly identified somatic mutations in imatinib-resistant DFSP.

<p>Newly identified somatic mutations in imatinib-resistant DFSP.</p