Ravat Panvichian

Impact of CYP2D6 polymorphisms on tamoxifen responses of women with breast cancer: a microarray-based study in Thailand.

This study was designed to investigate the frequency of CYP2D6 polymorphisms and evaluate the association between genetic polymorphisms of CYP2D6 and tamoxifen therapeutic outcome in Thai breast cancer patients. We recruited 48 breast cancer patients who received adjuvant tamoxifen for evaluating CYP2D6 genetic polymorphisms using microarray-based technology. Associations between genotypes-phenotypes and disease free survival were analyzed. Median follow up time was 5.6 years. The mean age of the subjects was 50 years. The 3 common allelic frequencies were 43.8% (*10), 36.5 (*1) and 10.4% (*2) which are related to extensive metabolizer (EM) and intermediate metabolizer (IM) with 70.8% and 29.2 %, respectively. No association between CYP2D6 genotypes and DFS was demonstrated. Nevertheless, exploratory analysis showed statistically significant shorter DFS in the IM group of post-menopause patients (HR, 6.85; 95% CI, 1.48 -31.69; P = 0.005). Furthermore, we observed statistically significant shorter DFS of homozygous CYP2D6*10 when compared with heterozygous CYP2D6*10 and other genotypes (P=0.005). CYP2D6*10 was the most common genotype in our subjects. Post-menopause patients with homozygous CYP2D6*10 and IM have shorter DFS. To confirm this relationship, larger samples and comprehensively designed trials in Thailand are required.

CYP2D6 polymorphisms influence the efficacy of adjuvant tamoxifen in Thai breast cancer patients.

Aim: We evaluated single nucleotide polymorphisms (SNPs) of CYP2D6 to identify those that influence the efficiency of tamoxifen in adjuvant treatment of breast cancer through a matched case–control study. Methods: Peripheral blood DNA was collected from 20 patients with disease recurrence during adjuvant tamoxifen treatment and from 19 patients who had completed 5 years of tamoxifen therapy without recurrence of breast cancer. CYP2D6*4 (1846G > A; rs3892097), CYP2D6*10 (100C > T, rs1065852), and CYP2D6*5 (deletion) were genotyped. The correlation between disease-free survival (DFS) and genotype and clinical outcome were assessed using Kaplan–Meier analysis and a log-rank test. Results: We found the allelic frequency of CYP2D6*10 during this study. Patients with the CYP2D6*10 homozygous variant T/T genotype had a significantly shorter median of DFS than those with C/T (P = 0.036), but DFS was not significantly different from that of patients with the C/C genotype (P = 0.316). One patient who was a carrier both of CYP2D6 G/A (1846G > A) and T/T (100C > T) had DFS of 22.7 months. Conclusions: This study demonstrated that CYP2D6*10/*10 was significantly associated with shorter DFS in Thai breast cancer patients receiving tamoxifen. This was a pilot study investigating the correlation of CYP2D6 polymorphisms and their influence on clinical outcomes in Thai estrogen receptor-positive breast cancer patients.

TOP2A Amplification and Overexpression in Hepatocellular Carcinoma Tissues

Hepatocellular carcinoma (HCC) is the leading cause of cancer death in men worldwide owing to limited insights into pathogenesis and unsatisfactory efficacy of current therapies. HER2 and TOP2A genes are coamplified in breast and some other cancers. In this study, we investigated gene aberrations of HER2 and TOP2A and protein expressions of HER2, TOP2A, Ki-67, and p53 in tumor and matched nontumor tissues, as well as their associations with clinicopathological features. Gene aberrations were evaluated by FISH and protein expressions by IHC. Neither HER2 overexpression nor HER2 gene amplification was observed in both tumor tissues and matched nontumor tissues. By contrast, TOP2A overexpression was detected in 72.5% of tumor tissues but not detected in matched nontumor tissues. However, TOP2A gene amplification was not observed in both tumor and matched nontumor tissues. TOP2A overexpression was significantly associated with HCC tumor tissues (P < 0.001), hepatitis B surface antigen (HBsAg) in the serum (P = 0.004), and Ki-67 (P = 0.038) but not with age, tumor size, alpha-fetoprotein, TP53, and copy number of TOP2A gene and chromosome 17 centromere. In conclusion, TOP2A overexpression in HCC was not secondary to gene amplification. In addition, neither HER2 amplification nor overexpression could be used as prognostic and predictive marker in HCC.

Missense Mutations in Exons 18–24 of EGFR in Hepatocellular Carcinoma Tissues

Epidermal growth factor receptor (EGFR), a transmembrane tyrosine kinase receptor, plays important roles in various cancers. In nonsmall cell lung cancer (NSCLC), EGFR mutations cluster around the ATP-binding pocket (exons 18–21) and some of these mutations activate the kinase and induce an increased sensitivity to EGFR-tyrosine kinase inhibitors. Nevertheless, data of EGFR mutations in HCC are limited. In this study, we investigated EGFR expression by immunohistochemistry and EGFR mutations (exons 18–24) by PCR cloning and sequencing. EGFR overexpression in HCC and matched nontumor tissues were detected in 13/40 (32.5%) and 10/35 (28.6%), respectively. Moreover, missense and silent mutations were detected in 13/33 (39.4%) and 11/33 (33.3%) of HCC tissues, respectively. The thirteen different missense mutations were p.L730P, p.V742I, p.K757E, p.I780T, p.N808S, p.R831C, p.V851A, p.V897A, p.S912P, p.P937L, p.T940A, p.M947V, and p.M947T. We also found already known SNP, p.Q787Q (CAG>CAA), in 13/33 (39.4%) of HCC tissues. However, no significant association was detected between EGFR mutations and EGFR overexpression, tissue, age, sex, tumor size, AFP, HBsAg, TP53, and Ki-67. Further investigation is warranted to validate the frequency and activity of these missense mutations, as well as their roles in HCC tumorigenesis and in EGFR-targeted therapy.

Pharmacogenetic Study of 5-Fluorouracil-Related Severe Toxicity in Thai Cancer Patients: A Novel SNP Detection

Objective: The objective of this study is to determine the extent of (DPYD) Dihydropyrimidine Dehydrogenase Gene] polymorphisms of Thai cancer patients who received 5-FU based chemotherapy regimens. Methods: The study was conducted a pharmacogenetic analysis to determine the polymorphisms of DPYD gene in 116. Thai cancer patients. 76 patients developed severe (grade 3-4) toxicities after receiving the first or second cycle of 5-FU based chemotherapy. The other subject group consisted of 40 patients without severe toxicity. The DNA sequencing of every amplicon was done to identify 11 mutations as reported in Asian population. The actual change of absolute neutrophil count (ANC), hematocrit, platelet and percentage of neutrophil were compared. Results: We detected 13 SNPs of which 6 SNPs were found in exons; 967G>A, 1011A>T, 1236G>A, 1774C>T, 1896T>C and 1627A>G. The other 7 SNPs were found in intron but only IVS14+1G>A is the intron splice site. We found homozygous GG of 1627A>G in 4 patients who had severe toxicities. Statistically significant difference in actual ANC change and percentage of neutrophil change in homozygous GG [P = .011 and .009] were found. The median nadir ANC of homozygous GG is 399.6 cells/mm3. This SNP has cause the amino acid change from isoleucine to valine. Novel heterozygous SNPs (967G>A, 1774C>T) that cause the amino acid change were found in two patients with severe toxicities. Conclusions: 1627A>G, 967G>A, 1774C>T and IVS14+G>A might be the cause of (DPD) Dihydro Pyrimidine Dehydrogenase deficiency in Thai patients. The further study needs to establish the functional DPD protein in this population. Ten novel SNPs were discovered in our study.

HER2 expression in breast cancer with nonamplified HER2 and gains of chromosome 17 centromere.

Gains of chromosome 17 centromere (CEP17) may be accompanied by gains of chromosome 17q. To evaluate the effect of CEP17 gains (CEP17>3 copies per tumor nucleus) on the expression of the HER2 gene, which is located on chromosome 17q12-21.32, we analyzed HER2 amplification and expression in breast carcinomas with and without CEP17 gains. We isolated tumor nuclei from frozen tissues of 37 breast carcinomas for analysis of the HER2 gene and CEP17 by fluorescence in situ hybridization. HER2 expression was detected by immunohistochemistry (IHC) performed on formalin-fixed, paraffin-embedded sections of the corresponding tumors. Tumors with amplified HER2 as determined by both HER2 copy number and HER2/CEP17 ratio were detected in 29.7% (11/37). CEP17 gains were significantly associated with HER2 amplification (P=0.005) but not associated with estrogen receptor status, tumor grade, and lymph node status (P>0.05). In contrast, HER2 amplification was significantly associated with estrogen receptor negativity (P=0.020) but not with tumor grade and lymph node status (P>0.05). IHC analysis was performed in 7 HER2-amplified tumors and all of these were IHC 3+, which were used as positive controls. Among HER2–non-amplified tumors with CEP17 gains, only 1 tumor (1/8, 12.5%) was IHC 3+. However, none of the HER2–non-amplified tumors without CEP17 gains was IHC 3+. In HER2–non-amplified tumors, there was no significant association between HER2 protein expression as detected by IHC and CEP17 or HER2 copy number (P=0.999, P=0.785, respectively). These findings indicate that in the absence of HER2 amplification, CEP17 gains do not have a significant effect on HER2 protein expression.

Droplet digital PCR using HER2/EIF2C1 ratio for detection of HER2 amplification in breast cancer tissues

Breast cancers with amplification and overexpression of human epithelial growth factor receptor 2 (HER2) are associated with poor prognosis, and targeted for anti-HER2 therapy. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are currently the recommended methods to asses HER2 overexpression/amplification. Droplet digital PCR (ddPCR), a highly accurate method to quantify DNA copy number, is potentially a robust alternative for HER2 diagnostics. In the FISH assay and most of previous ddPCR reports, chromosome 17 centromere (CEP17) has been used as the reference control to determine HER2/CEP17 ratio. Nevertheless, miss-classification could occur when HER2 is co-amplified with CEP17. To avoid this inherent defect, in the present study, we employed ddPCR assay using the human eukaryotic translation initiation factor 2C1 (EIF2C1) gene located at chromosome 1p34.3 as the reference control to quantify HER2 copy number in 31 frozen breast cancer tissues. HER2 status of these samples had been determined by FISH and classified as HER2-amplified and HER2-non-amplified breast cancers. The results showed that HER2 determined by ddPCR using HER2/EIF2C1 ratio was in good concordance with HER2 determined by FISH using HER2/CEP17 ratio, the concordance rate 87.1% (27/31), Kappa  = 0.719. The sensitivity and specificity of ddPCR assay was 90% (9/10) and 85.7% (18/21), respectively. The median HER2/EIF2C1 copy number ratio in HER2-amplified cancers (6.55, range 1.3–17.3) was significantly higher than in HER2-non-amplified cancers (1.05, range 0.6–3.6, p < 0.001). This study demonstrated that ddPCR using HER2/EIF2C1 ratio could accurately assess HER2 status in frozen breast cancer tissues. Thus, our findings warrant further studies into breast cancer with HER2-equivocal by IHC/FISH.