Yuichi Takatsuka

Significant Effect of Polymorphisms in CYP2D6 and ABCC2 on Clinical Outcomes of Adjuvant Tamoxifen Therapy for Breast Cancer Patients

PURPOSE The clinical efficacy of tamoxifen is suspected to be influenced by the activity of drug-metabolizing enzymes and transporters involved in the formation, metabolism, and elimination of its active forms. We investigated relationships of polymorphisms in transporter genes and CYP2D6 to clinical outcome of patients receiving tamoxifen. PATIENTS AND METHODS We studied 282 patients with hormone receptor-positive, invasive breast cancer receiving tamoxifen monotherapy, including 67 patients who have been previously reported. We investigated the effects of allelic variants of CYP2D6 and haplotype-tagging single nucleotide polymorphisms (tag-SNPs) of ABCB1, ABCC2, and ABCG2 on recurrence-free survival using the Kaplan-Meier method and Cox regression analysis. Plasma concentrations of tamoxifen metabolites were measured in 98 patients receiving tamoxifen 20 mg/d. RESULTS CYP2D6 variants were significantly associated with shorter recurrence-free survival (P = .000036; hazard ratio [HR] = 9.52; 95% CI, 2.79 to 32.45 in patients with two variant alleles v patients without variant alleles). Among 51 tag-SNPs in transporter genes, a significant association was found at rs3740065 in ABCC2 (P = .00017; HR = 10.64; 95% CI, 1.44 to 78.88 in patients with AA v GG genotypes). The number of risk alleles of CYP2D6 and ABCC2 showed cumulative effects on recurrence-free survival (P = .000000055). Patients carrying four risk alleles had 45.25-fold higher risk compared with patients with <or= one risk allele. CYP2D6 variants were associated with lower plasma levels of endoxifen and 4-hydroxytamoxifen (P = .0000043 and .00052), whereas no significant difference was found among ABCC2 genotype groups. CONCLUSION Our results suggest that polymorphisms in CYP2D6 and ABCC2 are important predictors for the prognosis of patients with breast cancer treated with tamoxifen.

A genome-wide association study identifies locus at 10q22 associated with clinical outcomes of adjuvant tamoxifen therapy for breast cancer patients in Japanese

Although many association studies of polymorphisms in candidate genes with the clinical outcomes of breast cancer patients receiving adjuvant tamoxifen therapy have been reported, genetic factors determining individual response to tamoxifen are not fully understood. To identify genetic polymorphisms associated with clinical outcomes of patients with tamoxifen treatment, we conducted a genome-wide association study (GWAS). We studied 462 Japanese patients with hormone receptor-positive, invasive breast cancer receiving adjuvant tamoxifen therapy. Of them, 240 patients were analyzed by genome-wide genotyping using the Illumina Human610-Quad BeadChips, and two independent sets of 105 and 117 cases were used for replication studies. In the GWAS, we detected significant associations with recurrence-free survival at 15 single-nucleotide polymorphisms (SNPs) on nine chromosomal loci (1p31, 1q41, 5q33, 7p11, 10q22, 12q13, 13q22, 18q12 and 19p13) that satisfied a genome-wide significant threshold (log-rank P= 2.87 × 10(-9)-9.41 × 10(-8)). Among them, rs10509373 in C10orf11 gene on 10q22 was significantly associated with recurrence-free survival in the replication study (log-rank P= 2.02 × 10(-4)) and a combined analysis indicated a strong association of this SNP with recurrence-free survival in breast cancer patients treated with tamoxifen (log-rank P= 1.26 × 10(-10)). Hazard ratio per C allele of rs10509373 was 4.51 [95% confidence interval (CI), 2.72-7.51; P= 6.29 × 10(-9)]. In a combined analysis of rs10509373 genotype with previously identified genetic makers, CYP2D6 and ABCC2, the number of risk alleles of these three genes had cumulative effects on recurrence-free survival among 345 patients receiving tamoxifen monotherapy (log-rank P= 2.28 × 10(-12)). In conclusion, we identified a novel locus associated with recurrence-free survival in Japanese breast cancer patients receiving adjuvant tamoxifen therapy.

Comparison of frozen section and touch imprint cytology for evaluation of sentinel lymph node metastasis in breast cancer

Background: Sentinel lymph node metastasis of breast cancer is evaluated by frozen section (FS) or touch imprint cytology (TIC). However, which of the two methods is superior remains controversial. Here we directly compared the sensitivity of these methods prospectively.Methods: The study included 208 SNs harvested from 107 consecutive patients with breast cancer who underwent sentinel lymph node biopsy. SNs were serially sectioned at 2-mm intervals, and two sections were subjected to intraoperative evaluation of FS with hematoxylin and eosin staining. TIC specimens were prepared from all cut surfaces and analyzed by Papanicolaou (TIC) and cytokeratin (TIC with immunohistochemistry; TIHC) immunohistochemistry.Results: Thirty-five SNs from 27 patients were positive by final histopathology. The sensitivity per sentinel lymph node of FS was 89%; it was 86% for TIC and 89% for TIHC. Among 173 negative SNs, the results of FS were concordant with final histopathology, but TIC and TIHC were positive in 1 and 5 histopathology-negative SNs, respectively. The sensitivity per patient of FS was 85%; it was 85% for TIC and 89% for TIHC. Among 80 patients with node-negative disease, the results of FS and TIC were concordant with final histopathology, whereas TIHC was positive in 3 patients (3.8% were upstaged). A slight improvement of sensitivity per patient was achieved by the combination of FS and TIC (to 89%) or FS and TIHC (to 93%).Conclusions: The sensitivity of FS was almost equivalent to that of TIC. TIHC had a better sensitivity than FS and TIC, but it upstaged a few node-negative patients.Background: Sentinel lymph node metastasis of breast cancer is evaluated by frozen section (FS) or touch imprint cytology (TIC). However, which of the two methods is superior remains controversial. Here we directly compared the sensitivity of these methods prospectively. Methods: The study included 208 SNs harvested from 107 consecutive patients with breast cancer who underwent sentinel lymph node biopsy. SNs were serially sectioned at 2-mm intervals, and two sections were subjected to intraoperative evaluation of FS with hematoxylin and eosin staining. TIC specimens were prepared from all cut surfaces and analyzed by Papanicolaou (TIC) and cytokeratin (TIC with immunohistochemistry; TIHC) immunohistochemistry. Results: Thirty-five SNs from 27 patients were positive by final histopathology. The sensitivity per sentinel lymph node of FS was 89%; it was 86% for TIC and 89% for TIHC. Among 173 negative SNs, the results of FS were concordant with final histopathology, but TIC and TIHC were positive in 1 and 5 histopathology-negative SNs, respectively. The sensitivity per patient of FS was 85%; it was 85% for TIC and 89% for TIHC. Among 80 patients with node-negative disease, the results of FS and TIC were concordant with final histopathology, whereas TIHC was positive in 3 patients (3.8% were upstaged). A slight improvement of sensitivity per patient was achieved by the combination of FS and TIC (to 89%) or FS and TIHC (to 93%). Conclusions: The sensitivity of FS was almost equivalent to that of TIC. TIHC had a better sensitivity than FS and TIC, but it upstaged a few node-negative patients.

Midkine expression in human breast cancers : Expression of truncated form

The expression of midkine (MK), a growth/differentiation factor,was assessed in 34 surgically resected specimens ofprimary breast cancer or mastopathy. Using reverse transcriptase-polymerasechain reaction (RT-PCR) analysis, all of the non-cancerousand cancerous tissues were found to express MKexcept for one breast cancer specimen. Northern blotanalysis revealed that MK mRNA was also expressedin the normal breast tissues examined. Immunohistochemical analysisof the MK protein was performed on alimited number of the specimens, showing that somecancerous tissues were immunoreactive with anti-MK antibodies. Furthermore,using RT-PCR analysis, expression of not only thewild-type but also a truncated form of MK,which was recently found in various human tumorcell lines, was detected in 6 of 26cancerous tissues but not in non-cancerous tissues.

Lessons for pharmacogenomics studies: association study between CYP2D6 genotype and tamoxifen response.

We earlier reported a significant association between the cytochrome P450 2D6 (CYP2D6) genotype and the clinical outcome in 282 Japanese breast cancer patients receiving tamoxifen monotherapy. Although many research groups have provided evidence indicating the CYP2D6 genotype as one of the strongest predictors of tamoxifen response, the results still remain controversial. We hypothesized that concomitant treatment was one of the causes of these controversial results. We then studied 167 breast cancer patients who received tamoxifen-combined therapy to evaluate the effects of concomitant treatment on the association analysis and observed no significant association between CYP2D6 genotype and recurrence-free survival (P=0.44, hazard ratio: 0.64, 95% confidential interval: 0.20-1.99 in patients with two variant alleles vs. patients without a variant allele). When we carried out two subgroup analyses for nodal status and tumor size, we observed a positive association between the CYP2D6 genotype and the clinical outcome only in patients who received tamoxifen monotherapy. This study explained a part of the discrepancies among the reported results.

Carcinoembryonic antigen estimation in nipple discharge as an adjunctive tool in the diagnosis of early breast cancer.

To assess the usefulness of carcinoembryonic antigen (CEA) estimation in nipple discharge for the detection of nonpalpable breast cancer, CEA activity in nipple discharge was measured by enzyme immunoassay using monoclonal antibody. The specificity of the antibody for breast cancer was assessed by an immunohistochemical method. Mean CEA levels in the nipple discharge from 18 patients with benign breast diseases (ten intraductal papilloma; eight fibrocystic disease) was 43 ng/ml (SD, 34 ng/ml), suggesting an upper reference limit of 100 ng/ml. Six of seven nonpalpable breast cancer patients had higher CEA levels than this tentative cutoff value, as did three of five patients with borderline lesions. The incidence of elevated CEA levels in nipple discharge correlated significantly with the incidence of intratumoral antigen expression. These results lead us to conclude that CEA measurement in nipple discharge may be a useful adjunct in the diagnosis of nonpalpable breast cancer.

Ca2+-dependent neutral protease (Calpain) activity in breast cancer tissue and estrogen receptor status

Cancer invasion and metastasis require action of tumor-associated proteases, which degrade the extracellular matrix. It has been reported that calpain, a calcium-activated neutral protease and a thiol protease regulated by Ca2+, proteolyzes estrogen receptor (ER) and that calpain may play an important role in the regulation of ER function. In the present study, the activities of calpain were measured in human normal breast tissues and breast cancer tissues stratified by estrogen receptor levels. There were no correlations between calpain activity and tumor size or lymph node involvement. Activities of calpain were significantly higher in breast cancer tissues compared with those of normal breast tissues, and were higher in the ER-positive tumors than in ER negative ones. These results indicate that calpain is related to mammary malignant transformation and is involved in the regulation of the ER function in breast cancer tissues.

Phase II Study of Weekly Paclitaxel for Docetaxel-Resistant Metastatic Breast Cancer in Japan

Abstract:  The purpose of the study was to evaluate the efficacy of weekly paclitaxel (PTX) against metastatic breast cancer (MBC) that was resistant to docetaxel (DTX) given every 3 weeks. A multicenter phase II study was performed. Women with MBC resistant to DTX were eligible for enrollment. DTX resistance was defined as no tumor response to DTX and stable disease, partial response, or complete response to DTX preceding disease progression. PTX 80 mg/m2 was administered over 1 hour once a week for 3 weeks per 4‐week cycle. Among 47 enrolled patients, 46 patients were assessable for response and toxicity. The overall objective response rate (complete responses [CRs] and partial responses [PRs]) was 17.4% and overall clinical benefit rate (CRs, PRs, and stable disease ≥24 weeks) was 26.1%. The median time to progression was 11 weeks. There were a few severe hematologic toxicities related to the therapy, with grade 4 neutropenia (4.3%) and thrombocytopenia (2.2%). Grade 3 anaphylaxis and grade 3 neuropathy were observed in one patient (2.2%) each. The median delivered dose intensity was 77.6 mg/m2/week, 97.1% of the planned dose intensity. Weekly PTX has activity in patients with MBC resistant to DTX every 3 weeks. 

A genome-wide association study identifies a genetic variant in the SIAH2 locus associated with hormonal receptor-positive breast cancer in Japanese.

In Japan, breast cancer is the most common cancer among women and the second leading cause of cancer death among women worldwide. To identify genetic variants associated with the disease susceptibility, we performed a genome-wide association study (GWAS) using a total of 1086 Japanese female patients with hormonal receptor-positive (HRP) breast cancer and 1816 female controls. We selected 33 single-nucleotide polymorphisms (SNPs) with suggestive associations in GWAS (P-value of <1 × 10−4) as well as 4 SNPs that were previously implicated their association with breast cancer for further replication by an independent set of 1653 cases and 2797 controls. We identified significant association of the disease with a SNP rs6788895 (Pcombined of 9.43 × 10−8 with odds ratio (OR) of 1.22) in the SIAH2 (intron of seven in absentia homolog 2) gene on chromosome 3q25.1 where the involvement in estrogen-dependent diseases was suggested. In addition, rs3750817 in intron 2 of the fibroblast growth factor receptor 2 gene, which was reported to be associated with breast cancer susceptibility, was significantly replicated with Pcombined of 8.47 × 10−8 with OR=1.22. Our results suggest a novel susceptibility locus on chromosome 3q25.1 for a HRP breast cancer.

Surgical margin status as a cause of local failure after breast conserving therapy

BackgroundTo elucidate the cause of in-breast recurrence after breast conserving surgery, we analyzed the characteristics of resected specimens histopathologically, especially the surgical margin status.Materials and Methods1) Pathological surgical margin positivity was reevaluated in terms of the distance from the resected surgical margin by pathologists from seven institutions in 486 cases with complete stepwise pathological examination.2) We reviewed pathological specimens including surgical margins from 30 patients with in-breast recurrence for whom serial sections of resected primary breast cancer specimens were available and made comparisons of the time to in-breast recurrence.ResultsCancer cells at the surgical margin were present at a rate of 4.1 % on the surface and 15.2% within 5 mm on the areolar side of the surgical margin. Histopathologically, the reasons for local recurrence after breast conserving therapy included a positive surgical margin (21/30), lymphatic permeation (4/30), and others (5/30). The last category included cases with an inadequate margin diagnosis because of a biopsy scar. Disease-free intervals for the patients without postoperative radiotherapy decreased as the volume of cancer cell nests in the surgical margin increased (P=0.06). On the other hand, this trend was not observed in the group with postoperative radiotherapy.ConclusionAdequate materials are essential for accurate evaluation of surgical margin status. Quantitative evaluation of surgical margin status, apart from whether or not radiotherapy was performed, is important for estimating the risk and disease-free period to in-breast recurrence.