Xin-Xia Tian

Evaluation of ALK rearrangement in Chinese non-small cell lung cancer using FISH, immunohistochemistry, and real-time quantitative RT- PCR on paraffin-embedded tissues.

Patients with ALK gene rearrangements often manifest dramatic responses to crizotinib, an ALK inhibitor. Accurate identification of patients with ALK-positive non-small cell lung cancer (NSCLC) is essential for the clinical application of ALK-targeted therapy. However, assessing EML4-ALK rearrangement in NSCLC remains challenging in routine pathology practice. The aim of this study was to compare the diagnostic accuracy of FISH, immunohistochemistry (IHC), and real-time quantitative RT-PCR (QPCR) methodologies for detection of EML4-ALK rearrangement in NSCLC and to appraise immunohistochemistry as a pre-screening tool. In this study, a total of 473 paraffin-embedded NSCLC samples from surgical resections and biopsies were analyzed by IHC with ALK antibody. ALK rearrangement was further confirmed by FISH and QPCR. ALK protein expression was detected in twenty patients (20/473, 4.2%). Of the 20 ALK-positive cases by IHC, 15 cases were further confirmed as ALK rearrangement by FISH, and 5 cases were not interpretable. Also, we evaluated 13 out of the 20 IHC-positive tissues by QPCR in additional to FISH, and found that 9 cases were positive and 2 cases were equivocal, whereas 2 cases were negative although they were positive by both IHC and FISH. The ALK status was concordant in 5 out of 8 cases that were interpretable by three methods. Additionally, none of the 110 IHC-negative cases with adenocarcinoma histology showed ALK rearrangements by FISH. Histologically, almost all the ALK-rearranged cases were adenocarcinoma, except that one case was sarcomatoid carcinoma. A solid signet-ring cell pattern or mucinous cribriform pattern was presented at least focally in all ALK-positive tumors. In conclusion, our findings suggested that ALK rearrangement was associated with ALK protein expression. The conventional IHC assay is a valuable tool for the pre-screening of patients with ALK rearrangement in clinical practice and a combination of FISH and QPCR is required for further confirmation.

Genetic polymorphisms in AURKA and BRCA1 are associated with breast cancer susceptibility in a Chinese Han population

Centrosome defects can result in aneuploidy and genomic instability, and have important implications for breast cancer development. The Aurora‐A and BRCA1 proteins interact and both are strongly involved in centrosome regulation. Genetic variants in these two genes may have an effect on breast cancer development. Here, we report a comprehensive single nucleotide polymorphism (SNP) and haplotype‐tagging association study on these two genes in 1334 breast cancer cases and 1568 unaffected controls among the Chinese Han population. Apart from a missense SNP, rs2273535 (Phe31Ile), and a probable risk SNP, rs2064863, six htSNPs were analysed in three high‐LD blocks of AURKA spanning from 10 kb upstream to 2 kb downstream of AURKA. For BRCA1, six htSNPs were analysed in a large high‐LD region covering 98 kb (10 kb was extended to each end of BRCA1). The results showed that four SNPs in AURKA (data in recessive model, rs2273535: OR = 2.19, 95% CI = 1.03–4.66, p = 0.0422; rs2298016: OR = 0.38, 95% CI = 0.18–0.82, p = 0.0141; rs6024836: OR = 1.54, 95% CI = 1.18–2.00, p = 0.0014; rs10485805: OR = 0.68, 95% CI = 0.47–0.98, p = 0.0380) and one SNP in BRCA1 (rs3737559, dominant model OR = 1.35, 95% CI = 1.11–1.64, p = 0.0030) were associated with breast cancer susceptibility. After correction for multiple comparisons (FDR = 0.05), only rs6024836 and rs3737559 remained significant. Two haplotypes (CC of block 2, OR = 20.74, 95% CI = 4.35–98.88, p = 0.0001; GG of block 3, OR = 1.32, 95% CI = 1.12–1.56, p = 0.0010) and one diplotype (AG‐GG of block 3, OR = 1.63, 95% CI = 1.18–2.26, p = 0.0031) within AURKA showed strong associations with breast cancer risk. One haplotype of BRCA1 (CTGTTG, OR = 1.30, 95% CI = 1.06–1.59, p = 0.0118) was also associated with breast cancer risk. However, women harbouring both at‐risk genotypes of Aurora‐A and BRCA1 were at a slightly increased risk compared with those harbouring either at‐risk variant alone. Common genetic variants in the AURKA and BRCA1 genes may contribute to breast cancer development. Copyright

Quantification of alternative splicing variants of human telomerase reverse transcriptase and correlations with telomerase activity in lung cancer.

Telomerase plays important roles in the development and progression of malignant tumors, and its activity is primarily determined by transcriptional regulation of human telomerase reverse transcriptase (hTERT). Several mRNA alternative splicing variants (ASVs) for hTERT have been identified, but it remains unclear whether telomerase activity is directly associated with hTERT splicing transcripts. In this study, we developed novel real-time PCR protocols using molecular beacons and applied to lung carcinoma cell lines and cancerous tissues for quantification of telomerase activity and three essential hTERT deletion transcripts respectively. The results showed that lung carcinoma cell lines consistently demonstrated telomerase activity (14.22–31.43 TPG units per 100 cells) and various hTERT alternative splicing transcripts. For 165 lung cancer cases, telomerase activity showed significant correlation with tumor differentiation (poorly->moderately->well-differentiated, P<0.01) and with histotypes (combined small cell and squamous cell carcinoma>squamous cell carcinoma>adenosquamous carcinoma>adenocarcinoma, P<0.05). Although the overall hTERT transcripts were detected in all the samples, they were not associated with telomerase activity (r = 0.092, P = 0.24). Telomerase activity was significantly correlated with the transcriptional constituent ratio of α-deletion (r = -0.267, P = 0.026), β-deletion (r = -0.693, P = 0.0001) and γ-deletion (r = –0.614, P = 0.001). The positive rate and average constituent ratio of β-deletion transcripts (92.12%, 0.23) were higher than those of α-deletion (41.82%, 0.12) or γ-deletion (16.36%, 0.18) transcripts. The combined small-cell and squamous cell carcinomas expressed less deletion transcripts, especially β-deletion, than other histotypes, which might explain their higher telomerase activity. In conclusion, the molecular beacon-based real-time PCR protocols are rapid, sensitive and specific methods to quantify telomerase activity and hTERT ASVs. Telomerase activity may serve as a reliable and effective molecular marker to assist the evaluation of histological subtype and differentiation of lung carcinomas. Further studies on hTERT deletion splicing transcripts, rather than the overall hTERT transcripts, may improve our understanding of telomerase regulation.

Association of Germline Variation in CCNE1 and CDK2 with Breast Cancer Risk, Progression and Survival among Chinese Han Women

Background Somatic alterations of cyclin-dependent kinase 2 (CDK2)-cyclin E complex have been shown to contribute to breast cancer (BC) development and progression. This study aimed to explore the effects of single nucleotide polymorphisms (SNPs) in CDK2 and CCNE1 (a gene encoding G1/S specific cyclin E1 protein, formerly called cyclin E) on BC risk, progression and survival in a Chinese Han population. Methodology/Principal Findings We herein genotyped 6 haplotype-tagging SNPs (htSNPs) of CCNE1 and 2 htSNPs of CDK2 in 1207 BC cases and 1207 age-matched controls among Chinese Han women, and then reconstructed haplotype blocks according to our genotyping data and linkage disequilibrium status of these htSNPs. For CCNE1, the minor allele homozygotes of three htSNPs were associated with BC risk (rs3218035: adjusted odds ratio [aOR] = 3.35, 95% confidence interval [CI] = 1.69–6.67; rs3218038: aOR = 1.81, 95% CI = 1.22–2.70; rs3218042: aOR = 2.64, 95% CI = 1.31–5.34), and these three loci showed a dose-dependent manner in increasing BC risk (P trend = 0.0001). Moreover, the 5-SNP haplotype CCGTC, which carried none of minor alleles of the 3 at-risk SNPs, was associated with a favorable event-free survival (hazard ratio [HR] = 0.53, 95% CI = 0.32–0.90). Stratified analysis suggested that the minor-allele homozygote carriers of rs3218038 had a worse event-free survival among patients with aggressive tumours (in tumour size>2 cm group: HR = 2.06, 95% CI = 1.06–3.99; in positive lymph node metastasis group: HR = 2.41, 95% CI = 1.15–5.03; in stage II–IV group: HR = 2.03, 95% CI = 1.09–3.79). For CDK2, no significant association was found. Conclusions/Significance This study indicates that genetic variants in CCNE1 may contribute to BC risk and survival in Chinese Han population. They may become molecular markers for individual evaluation of BC susceptibility and prognosis. Nevertheless, further validation studies are needed.

Association Study of Germline Variants in CCNB1 and CDK1 with Breast Cancer Susceptibility, Progression, and Survival among Chinese Han Women

The CCNB1 and CDK1 genes encode the proteins of CyclinB1 and CDK1 respectively, which interact with each other and are involved in cell cycle regulation, centrosome duplication and chromosome segregation. This study aimed to investigate whether the genetic variants in these two genes may affect breast cancer (BC) susceptibility, progression, and survival in Chinese Han population using haplotype-based analysis. A total of ten tSNPs spanning from 2kb upstream to 2kb downstream of these genes were genotyped in 1204 cases and 1204 age-matched cancer-free controls. The haplotype blocks were determined according to our genotyping data and linkage disequilibrium (LD) status of these SNPs. For CCNB1, rs2069429 was significantly associated with increased BC susceptibility under recessive model (OR=2.352, 95%CI=1.480-3.737), so was the diplotype TAGT/TAGT (OR=1.947 95%CI=1.154-3.284, P=0.013). In addition, rs164390 was associated with Her2-negative BC. For CDK1, rs2448343 and rs1871446 were significantly associated with decreased BC risk under dominant models, so was the haplotype ATATT. These two SNPs also showed a dose-dependent effect on BC susceptibility. Using stratified association analysis, we found that women with the heterozygotes or minor allele homozygotes of rs2448343 had much less BC susceptibility among women with BMI<23. In CDK1, three closely located SNPs, rs2448343, rs3213048 and rs3213067, were significantly associated with tumor’s PR status: the heterozygotes of rs2448343 were associated with PR-positive tumors, while the minor allele homozygotes of rs3213048 and heterozygotes of rs3213067 were associated with PR-negative BC tumors. In survival analysis, rs1871446 was associated with unfavorable event-free survival under recessive model, so was the CDK1 diplotype ATATG/ATATG, which carried the minor allele homozygote of rs1871446. Our study indicates that genetic polymorphisms of CCNB1 and CDK1 are related to BC susceptibility, progression, and survival in Chinese Han women. Further studies need to be performed in other populations as an independent replication to verify these results.

Genetic polymorphisms in AURKA, BRCA1, CCNE1 and CDK2 are associated with ovarian cancer susceptibility among Chinese Han women

INTRODUCTION Centrosome aberrations and cell-cycle deregulation have important implications for ovarian cancer development. The AURKA, BRCA1, CCNE1 and CDK2 genes play pivotal roles in centrosome duplication and cell-cycle regulation. METHODS Using a haplotype-based analysis, this study aimed to investigate whether genetic polymorphisms in these four genes may contribute to ovarian cancer susceptibility. A total of 22 single nucleotide polymorphisms (SNPs) in these four genes were genotyped in 287 cases of ovarian serous cystadenocarcinomas and 618 age-matched cancer-free controls from the Chinese Han population, and then haplotype blocks were reconstructed according to our genotyping data and linkage disequilibrium (LD) status of these SNPs. RESULTS For AURKA, we found that haplotype GA [rs6064391 (T→G)+rs911162 (G→A)] was strongly associated with decreased ovarian cancer risk (adjusted OR=0.31, 95% CI=0.15-0.63, P=0.0012). For BRCA1, we found that haplotype CGTAG was associated with decreased ovarian cancer risk (adjusted OR=0.64, 95% CI=0.41-0.98, P=0.0417). Moreover, women harboring homozygous GA/CGTAG haplotypes showed the lowest risk (OR=0.12, 95% CI=0.02-0.94, P=0.0438). In CCNE1, the SNPs rs3218035 and rs3218042 were significantly associated with increased ovarian cancer risk (rs3218035: adjusted OR=5.20, 95% CI=1.85-14.52, P=0.0017; rs3218042: adjusted OR=4.98, 95% CI=1.75-14.19, P=0.0027). For CDK2, no significant association was found. CONCLUSIONS This study indicates that genetic polymorphisms of AURKA, BRCA1 and CCNE1 may affect ovarian cancer susceptibility in Chinese Han women.

Association of genetic polymorphisms in AURKA, BRCA1, CCNE1 and CDK2 with the risk of endometrial carcinoma and clinicopathological parameters among Chinese Han women

OBJECTIVE Centrosome aberrations and cell-cycle deregulations have important implications for the development of endometrial carcinoma. AURKA, BRCA1, CCNE1 and CDK2 genes play pivotal roles in centrosome duplication and cell-cycle regulation. This study aimed to investigate whether genetic polymorphisms in these four genes may contribute to endometrial carcinoma susceptibility and progression in Chinese Han women. STUDY DESIGN A total of twenty-two single nucleotide polymorphisms (SNPs) were selected according to the public HapMap database (HapMap Data Release #27; Chinese Beijing population), and genotyped using TaqMan Realtime PCR method in 530 endometrial adenocarcinoma cases and 825 age-matched controls from Chinese Han women. Then, haplotype blocks were reconstructed according to our genotyping data. RESULTS For AURKA, the rs911162 was associated with increased risk of endometrial carcinoma [in co-dominant model: adjusted odds ratio (aOR) = 4.92, 95% CI = 1.24-19.55, P = 0.0235]. The haplotype GA (rs6064391 + rs911162) in block 1 of AURKA was also associated with increased risk of endometrial carcinoma (aOR = 1.25, 95% CI = 1.07-2.06, P = 0.0189). For BRCA1, the minor allele homozygotes of rs8067269 could decrease the risk of endometrial carcinoma (in co-dominant models: aOR = 0.52, 95% CI = 0.34-0.80, P = 0.0032), so was the haplotype CTCAG (rs8176323 + rs8176199 + rs3737559 + rs8067269 + rs2070833) (aOR = 0.69, 95% CI = 0.50-0.95, P = 0.0234). Moreover, we found several associations between genetic variations in CCNE1, BRCA1 and AURKA and clinicopathological parameters. CONCLUSIONS This study indicates that genetic polymorphisms of AURKA, BRCA1 and CCNE1 may contribute to endometrial carcinoma susceptibility or progression in Chinese Han women.