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Background: The usefulness of prophylactic surgery and surveillance for hereditary breast cancer has been demonstrated, and germline testing for BRCA1 and BRCA2 had been covered by insurance since 2020 in Japan. In addition to BRCA1 and BRCA2, several other genes are also associated with an increased risk of developing breast cancer, such as PALB2, ATM, BARD1, CHEK2, PTEN, and TP53. Next-generation sequencing-enabled multigene panel tensing provides information about these gene variants at the same time, and at a low cost. Although germline testing of BRCA1 and BRCA2 has become widespread in Japan, multi-panel gene testing for germline variants has been conducted only in a limited number of facilities, partly due to the difficulty associated with dealing with the gene variant information obtained from the test. The aim of this study was to clarify the current status of multigene panel testing in our institute, and reveal the characteristics of the variants detected in patients with, or predisposed to, hereditary breast cancer. Methods: This retrospective study included 37 individuals who underwent next-generation sequencing-based multigene panel testing in order to investigate any inherited genetic variants due to a suspicion of hereditary breast cancer. Eighteen patients had a diagnosis of breast cancer with a family history of breast and/or ovarian cancer, nine patients had a diagnosis of breast cancer without family history of breast or ovarian cancer, and 10 patients had a family history of breast cancer but had not developed breast cancer themselves. Results: Utilizing mutigene panel testing, at least one alteration was found in 24 genes, and a total of 39 variants were found in the 37 patients. Of these 37 patients, nine (24.3%) had a pathogenic/likely pathogenic variant with or without other variants of uncertain significance (VUS), 15 (40.5%) had VUS, and 13 (35.1%) had negative genetic test results. Among the nine patients with pathogenic/likely pathogenic variants, seven had variants in either BRCA1 or BRCA2 (one BRCA1 pathogenic variant, five BRCA2 pathogenic variants, and one BRCA2 likely pathogenic variant), while the remaining positive results were attributed to other genes (one MLH1 pathogenic variant, and one SDHB pathogenic variant). VUS included BRCA1 and BRCA2, as well as other breast cancer-associated genes, such as ATM (n=2), CDH1 (n=2), NF1 (n=2), PALB2 (n=1), CHEK2 (n=1), NBN (n=1), and RAD51D (n=1). VUS also included other cancer syndrome-related genes, such as MLH1 (n=2), MUTYH (n=2), APC (n=1), and RET (n=1). Conclusion: Multigene panel tests in our institute revealed pathogenic/likely pathogenic variants in 24.3% of individuals who suspected hereditary breast cancer. As expected, multigene panel tests also revealed more VUS than pathogenic variants and 40.5% individuals were detected with VUS, which included many genes associated with hereditary breast cancer and other cancer syndromes, in addition to BRCA1 and BRCA2. Individuals with VUS will need to cope with new information if the interpretation of the variant changes in the future. We need to be aware of the characteristics and limitations of this type of panel testing, and to properly utilize the test results and information obtained for good quality patient care. Citation Format: Yusa Atake, Masayuki Nagahashi, Haruka Kanaoka, Akira Hattori, Ayako Bun, Reiko Fukui, Hiromi Ozawa, Yukie Fujimoto, Tomoko Higuchi, Michiko Imamura, Keiko Murase, Yuichi Takatsuka, Mina Kashima, Chiho Okada, Chinatsu Kinjo, Mikako Miyata, Ayako Miyazaki, Mako Ueda, Hiroshi Tsubamoto, Hideaki Sawai, Yasuo Miyoshi. Germline variants detected by next-generation sequencing-based multigene panel testing in patients with suspected hereditary breast cancer at a University Hospital in Japan [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-12-02.

The aim of this study was to elucidate the clinical significance of peripheral blood biomarkers, including absolute lymphocyte count (ALC), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and C-reactive protein (CRP) in patients with estrogen receptor-positive human epidermal growth factor receptor 2-negative advanced breast cancer treated with the CDK4/6 inhibitors, abemaciclib and palbociclib.A total of 83 patients treated with fulvestrant plus abemaciclib or palbociclib were included in this study. Progression-free survival (PFS) and overall survival (OS) were compared in relation to baseline levels of ALC, NLR, PLR and CRP.The cut-off values of ALC, NLR, PLR, and CRP for PFS were determined from the receiver operating characteristic curve using the Youden index for area under the curve and set at 1,212/μl, 1.964, 170 and 0.220 mg/dl, respectively. In the abemaciclib-treated group, ALC-high patients showed significantly better PFS than ALC-low patients (p=0.0151) and multivariate analysis revealed that ALC was an independent prognostic factor for PFS (p=0.0085). In the palbociclib-treated group, there was no significant relationship between any peripheral blood biomarkers and PFS. In both treatment groups, ALC-high patients showed significantly better OS than ALC-low patients (p=0.0169 and 0.0290, respectively). Multivariate analysis revealed ALC was an independent prognostic factor for OS in both abemaciclib- and palbociclib-treated groups (p=0.0112 and 0.0202, respectively).ALC is an independent prognostic factor for estrogen receptor-positive human epidermal growth factor receptor 2-negative advanced breast cancer patients treated with the CDK4/6 inhibitors abemaciclib and palbociclib.

Background/aim S-1, a 5-fluorouracil(5-FU) oral anti-cancer drug, has been traditionally used with a schedule of 4-week oral administration followed by 2-week rest for breast cancer treatment. We, herein, aimed to investigate the clinical efficacy and safety of a schedule of 2-week oral administration followed by 1-week rest for patients with metastatic breast cancer. Patients and methods We enrolled patients with HER2-negative metastatic breast cancer who had not received prior chemotherapy. S-1 was administered consecutively for 2-weeks followed by a 1-week rest. Results Between September 1, 2013 and August 31, 2016, 32 patients were enrolled. The median follow-up time was 32.1 months. The median progression-free survival (PFS) was 9.4 months. Overall survival (OS) was 41.0 months, time to treatment failure (TTF) was 7.8 months, response rate (RR) was 31.3%, and disease control rate (DCR) was 78.1%. The incidence of grade 3 side-effects was not high. Conclusion The 3-week schedule of S-1 can be considered useful as a treatment for patients with metastatic breast cancer, helping in maintaining a high quality of life.

Background: It has been well demonstrated that patients who achieved pathological complete response (pCR) after neoadjuvant chemotherapy (NAC) had a favorable prognosis compared with patients who did not (non-pCR). Even though pCR was not attained, reduction in tumor volume after chemotherapy may be associated with improved prognosis for a certain number of patients. However, the association between residual tumor volume and prognosis is not necessarily consistent. In order to identify substitute markers for breast cancer patients with non-pCR after NAC, we investigated the impact of serum levels of carcinoembryonic antigen (CEA) and carbohydrate antigen (CA15-3) at baseline as well as post-NAC. Patients and Methods: Ninety-six breast cancer patients treated with NAC and operated on at the Hyogo College of Medicine were recruited for this study. Serum CEA and CA15-3 were measured prior to chemotherapy as well as at completion of pre-operative treatment. The optimal cutoff points for CEA (1.55ng/m, normal range: Results: pCR and non-pCR was attained by 21 and 75 patients, respectively. For the non-pCR patients, serum CEA levels at baseline were classified into high (n=35) and low (n=38) and serum CA15-3 levels at baseline into high (n=31) and low (n=43). RFS of non-pCR patients with high serum CA15-3 levels was significantly worse than of those with low levels (3-year RFS: 0.47 vs 0.93; p=0.0009). RFS for patients with high and low serum levels of CA15-3 after NAC was also significantly different (p=0.037). As for CEA, no significant association with RFS was observed either at baseline or post-NAC. Univariate analysis demonstrated that tumor size and baseline CA15-3 were significant prognostic factors for RFS. Multivariate analysis showed that both tumor size (hazard ratio (HR): 3.88, 95% confidence interval (CI): 1.21-12.35, p=0.023) and baseline CA15-3 (HR: 13.51, 95% CI: 1.74-105.08, p=0.013) were significant and independent risk factors for relapse. As for lymph node metastasis, tumor grade, residual tumor size and pre- and post-NAC Ki67 expression levels of patients with non-pCR showed no significant association with RFS. Conclusion and discussion: High levels of serum CA15-3 at baseline constituted a significantly worse prognosis for breast cancer patients with non-pCR. Tumor size at baseline but not residual size and baseline CA15-3 seems to suitable as a substitute for prediction of outcome for patients with non-pCR. Our findings suggest that these markers may be useful for identifying patients with poor prognosis who may be candidates for additional adjuvant treatment. Citation Format: Fujimoto Y, Imamura M, Higuchi T, Nishimukai A, Yanai A, Miyagawa Y, Murase K, Takatsuka Y, Miyoshi Y. Baseline serum CA15-3 levels are associated with prognosis for breast cancer patients with non-complete pathological response to neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-05-27.

Adjuvant treatments for operable breast cancers are determined according to subtypes defined based on estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) status. The ER+/HER2− subtype can be divided into luminal A and luminal B usually by Ki67 expression levels. Although tumor size, lymph node metastasis and tumor grade have been widely accepted in daily clinical practice, the identification of further prognostic indicators especially in the ER+/HER2− subtype is warranted. A total of 387 operated breast cancers for which maximum standardized uptake value (SUVmax) on the 18 F-fluorodeoxyglucose positron-emission tomography/computed tomography (FDG PET/CT) were available at baseline were retrospectively analyzed. The optimal cutoff value of SUVmax for relapse-free survival (RFS) was determined to be 3.585 by means of the receiver operating characteristics curve with an area under the curve of 0.6795 (95% CI: 0.5972 to 0.7618, p = 0.0006, sensitivity: 78.8%, specificity: 57.1%). The RFS of patients with SUVmax-high (n = 178) was significantly (p = 0.0003) worse compared with those with SUVmax-low (n = 209). This significant association was prominently recognized in the ER+/HER2− subtype. By multivariable analysis, SUVmax (hazard ratio: 3.83, 95% confidence interval: 1.28–11.51, p = 0.017), tumor size (4.22, 1.39–12.82, p = 0.011) and lymph node metastasis (4.44, 1.81–10.87, p = 0.0012) were significant and independent prognostic factors for RFS. The ER+/HER2− subtype demonstrated consistently worse RFS in the SUVmax-high patients both in the luminal A (p = 0.037) and luminal B (p = 0.047) subtypes. Combination of Ki67 and SUVmax appears to be useful for selecting patients who have inferior prognosis and need further adjuvant treatment of the ER+/HER2− subtype.

Background When considering treatment for metastatic and recurrent breast cancer, it is necessary to select drugs with emphasis on side effects and QOL. In Japan, oral 5-FU drugs have often been used for initial treatment. In the conventional schedule of S-1 for 4-week administration period followed by 2-week rest, the inferiority of S-1 to T has already been proved. In this study, we examined the efficacy and safety of the schedule of S-1 for 2-week administration period followed by 1-week rest, which is considered to have less side effects and better compliance. Methods We enrolled individuals with HER2-negative breast cancer who had not received chemotherapy after diagnosis of metastatic breast cancer. S-1 (40-60 mg, twice daily) was administered consecutively for 14 days followed by 7 days of rest (1 course). The primary endpoint was progression-free survival (PFS); the secondary endpoints were overall survival (OS), time to treatment failure (TTF), response rate (RR), disease control rate (DCR), and adverse events. Results Between September 1, 2013 and August 31, 2016, 32 patients were enrolled. Median PFS was 7.8 months (1.4-35.4 months), and median OS was 25.2 months (4.8-47.8 months). TTF was 9.1 months. RR was 31.3%, and the DCR was 78%. The cumulative rates of the relative total administration dose of S-1 was 95.6%. Incidence of grade 3 side effects were neutropenia (9.4%), leukopenia (3.1%), anorexia (3.1%), ocular symptoms (3.1%), and an increase in total bilirubin levels (3.1%). Conclusions The schedule of 2-week administration period followed by 1-week rest seems to be safe and effective for primary treatment of metastatic and recurrent breast cancer. Legal entity responsible for the study Kobe Breast Cancer Oncology Group. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Background: Bone-modifying agents have demonstrated their efficacy for treatment by suppressing osteoclast function. The activity of bone-modifying agents can be monitored by means of bone resorption markers such as c-terminal crosslinking telopeptide of type I collagen (1CTP) and N-telopeptide of type I collagen (NTX) as well as bone forming marker bone-specific alkaline phosphatase (BAP). In contrast to these markers which indirectly indicate bone turnover, tartrate-resistant acid phosphatase-5b (Tracp-5b) has been established as a direct marker showing osteoclast number and activity. The aim of this study was to identify the relative significance of these bone turnover markers as indicators of treatment efficacy induced by bone-modifying agents for breast cancer patients with bone metastases. Patients and Methods: For this study, 52 breast cancer patients with bone metastases treated with bone-modifying agents were recruited. Zoledronic acid and denosumab were administered as bone-modifying agents to 36 and 22 patients, respectively (for 6 patients, denosumab was used after zoledronic acid). Serum Tracp-5b, 1CTP, NTX and BAP were measured with, respectively, the EIA (enzyme immunoassay), RIA (two-antibody radioimmunoassay), ELISA (enzyme-linked immunosorbent assay) and CLEIA (chemiluminescent enzyme immunoassay) method. Blood samples were obtained pretreatment and 1, 3 and 6 months after treatment. Changes in these bone turnover markers were statistically analyzed with Friedman's test, and correlation between serum markers and clinicopathological factors was calculated with Mann-Whitney's test. Results: Serum tracp-5b decreased significantly after treatment (p Conclusion and discussion: Although baseline values of the bone turnover markers Tracp-5b, NTX and BAP decreased significantly after treatment with bone-modifying agents, the pattern of reduction for these three markers varied. Tracp-5b appears to reflect efficacy of bone-modifying agents most quickly and sensitively, possibly due to its direct link to the number and activity of osteoclasts. These findings may prove usefulness of Tracp-5b when considering the efficacy of various bone-modifying agents in clinical practice. Citation Format: Higuchi T, Nishimukai A, Yanai A, Miyagawa Y, Murase K, Imamura M, Ozawa H, Takatsuka Y, Miyoshi Y. Differences in patterns of change of bone turnover markers during treatment with bone-modifying agents of breast cancer patients with bone metastases. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-16-01.

Background: It has been demonstrated that adjuvant treatment using bisphosphonate may reduce recurrence among breast cancer patients. However, these improved prognoses of patients are reportedly limited to breast cancers of estrogen receptor (ER)-positive and postmenopausal women. Although the mechanisms of the effects of bisphosphonate remain unknown, this finding seems to represent support for the hypothesis that suppression of bone resorption by bisphosphonate results in favorable prognoses at least for patients in this subset. In order to determine the prognostic significance of bone resorption in breast cancer patients, we investigated these markers c-terminal crosslinking telopeptide of type I collagen (1CTP) and N-telopeptide of type I collagen (NTX). Patients and Methods: 469 breast cancer patients were recruited who were operated on Hyogo College of Medicine and histologically confirmed to have invasive carcinoma. Serum 1CTP and NTX were measured preoperatively with the two-antibody radioimmunoassay and enzyme-linked immunosorbent assay methods, respectively, and blood samples were obtained before treatment from patients who were treated with neoadjuvant chemotherapy or endocrine therapy. The area under receiver operating characteristic curves were applied and optimal cutoff values were set at 3.6ng/ml for 1CTP, and 10.55nmolBCE/L premenopausal and 14.05nmolBCE/L postmenopausal for NTX. The relationships between these bone turnover markers and various clinicopathological characteristics were evaluated with the chi square or Fisher's exact test. The log-rank test was used to compare relapse-free survival (RFS) in Kaplan-Meier plots. Associations of RFS were assessed with a Cox proportional-hazards model based on the results of univariate and multivariate analyses. Differences were considered statistically significant if p Results: There were significantly more 1CTP-high patients among postmenopausal women and RFS of 1CTP-high patients was significantly worse than that of 1CTP-low patients (5-year RFS: 0.65 vs 0.86; p=0.0002). Similarly, NTX-high patients were significantly associated with postmenopausal status, but there was no significant association between NTX-high worse RFS (p=0.0976). Multivariate analysis of tumor size, lymph node metastasis and nuclear grade identified 1CTP (hazard ratio: 2.04, 95% confidence interval: 1.13-3.68; p=0.018) as a significant independent prognostic factor. Subset analyses of 1CTP showed that prognosis was consistently worse recognized for postmenopausal (p=0.0002), but not premenopausal (p=0.37) patients. Furthermore, prognosis for 1CTP-high patients was worse for the estrogen receptor (ER)-positive subset (p=0.0005) but not for the ER-negative subset (p=0.22). Conclusion and discussion: High levels of serum bone resorption markers at baseline were identified as significant unfavorable prognostic factors for breast cancer patients. The prognostic significance of 1CTP seems to be prominent for postmenopausal patients with ER-positive breast cancers. These findings suggest the use of bone-modifying agents as an adjuvant therapy may be beneficial for breast cancer patients, especially for patients with high serum levels of 1CTP. Citation Format: Imamura M, Nishimikai A, Yanai A, Miyagawa Y, Higuchi T, Ozawa H, Murase K, Takatsuka Y, Miyoshi Y. High levels of serum C-terminal crosslinking telopeptide of type 1 collagen at baseline are associated with poor prognosis for breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-08-24.

It is speculated that adjuvant use of bisphosphonate reduces recurrence in breast cancer patients through suppression of bone resorption. To determine the prognostic impact of bone resorption markers, we investigated serum levels of the pyridinoline crosslinked carboxyterminal telopeptide of type I collagen (1CTP) and N-terminal crosslinking telopeptides of type I collagen (NTX). 1CTP and NTX were measured at baseline (before operation or neoadjuvant therapies) and afterward in 469 patients operated on breast cancer. The optimal cutoff value of 1CTP for relapse-free survival (RFS) was set at 3.6 ng/ml with an area under the receiver operating characteristics curve of 0.641 [95% confidence interval (CI) = 0.560-0.721; p = 0.0011]. However, we were unable to determine a significant cutoff value for NTX. RFS was significantly worse for 1CTP-high patients with than for those with low levels of 1CTP (p = 0.0002). Multivariate analysis with tumor size, lymph node metastasis, and nuclear grade showed that 1CTP was a significant independent prognostic factor (hazard ratio = 2.04, 95% CI = 1.13-3.68; p = 0.018). Worse prognosis for the subset with high 1CTP levels applied only to postmenopausal patients (p = 0.0002). RFS of 130 patients whose 1CTP changed from low at baseline to high at 6 months postoperatively showed RFS almost as poor as that for patients with high 1CTP throughout. These findings suggest that 1CTP may be useful not only for identifying patients with unfavorable prognosis, but also for selecting patients who may benefit from administration of bone-modifying agents in an adjuvant setting.

Residual cancer burden or Ki67 expression levels in residual tumors reportedly provided significant prognostic information for a non-pathological complete response subset after neoadjuvant chemotherapy (NAC). However, the significance of Ki67 reduction for clinical response during chemotherapy in each subtype or menopausal status is yet to be determined. A total of 183 breast cancers surgically removed after chemotherapy were recruited for this study. Expression levels of estrogen receptor (ER), progesterone receptor (PgR), and Ki67 were determined immunohistochemically for semiquantitative measurement and these biomarkers were compared in pre- and post-NAC samples from pathological non-responders (n = 125). Responses to chemotherapy were evaluated both clinically and pathologically. Ki67 expression levels after NAC (median 5 %, range 0–70 %) were significantly reduced compared with before NAC (25, 1–80 %, P

Background Although the prognosis for operable breast cancers is reportedly worse if serum carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA15-3) levels are above normal, the usefulness of this prognosis is limited due to the low sensitivity and specificity; in addition, the optimal cutoff levels remain unknown. Methods A total of 1076 patients who were operated for breast cancers (test set = 608, validation set = 468) without evidence of metastasis were recruited, and their baseline and postoperative serum CEA and CA15-3 levels were analyzed. The optimal cutoff values of CEA and CA15-3 for disease-free survival (DFS) were 3.2 ng/mL and 13.3 U/mL, respectively, based on receiver operating characteristic curve and area under the curve analyses. Results The DFS of patients with high CEA levels (CEA-high: n = 191, 5-year DFS 70.6%) was significantly worse (p

Indication for chemotherapy in estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancers is determined on the basis of Ki67 expression level. However, since Ki67-high cancers are not necessarily sensitive to chemotherapy, identification of such patients who do not need chemotherapy is an important issue. We used immunohistochemical staining to examine the expression levels of ER, progesterone receptor (PgR), Ki67, and geminin, a marker of S to G2/M phases, in 80 ER-positive/HER2-negative breast cancers. The labeling indices of Ki67 and geminin were determined and cutoff values were set at 15 and 6 %, respectively. Ki67 and geminin expression levels were significantly associated with nuclear grade. In the Ki67-low subset, 26 out of 28 (92.9 %) cancers were geminin low and in the Ki67-high subset, 31 out of 52 (59.6 %) were geminin high. Distant disease-free survival (DDFS) of the geminin-high subset was significantly poorer than that of the geminin-low subset (P = 0.009). In the Ki67-low subset, only one patient showed recurrence. Metastasis was detected in eight out of 31 (25.8 %) patients in the geminin-high group of the Ki67-high subset, but no recurrence was observed in the geminin-low group of the Ki67-high subset. Geminin-high breast cancers are significantly associated with worse prognosis. Since poorer prognosis was recognized only in the geminin-high group in Ki67-high cancers, we speculate that geminin may be useful for identifying patients in the Ki67-high subset who can avoid unnecessary chemotherapy.

Adjuvant treatments for operable breast cancers are determined according to subtypes defined based on estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) status. The ER+/HER2- subtype can be divided into luminal A and luminal B usually by Ki67 expression levels. Although tumor size, lymph node metastasis and tumor grade have been widely accepted in daily clinical practice, the identification of further prognostic indicators especially in the ER+/HER2- subtype is warranted. A total of 387 operated breast cancers for which maximum standardized uptake value (SUVmax) on the

Bone-modifying agents are effective for treatment of breast cancer patients with bone metastases. Since their action is mediated through suppression of the osteoclast function, their efficacy can be determined by monitoring bone turnover markers. However, the clinical significance of these markers is yet to be compared. For this study, 52 breast cancer patients with bone metastases treated with zoledronic acid (n = 36) or denosumab (n = 22) were enrolled (6 patients were treated sequentially with both agents). Serum tartrate-resistant acid phosphatase-5b (TRACP-5b), pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (1CTP), N-terminal cross-linking telopeptides of type I collagen (NTX) and bone-specific alkaline phosphatase (BAP) were measured at pretreatment and 1, 3 and 6 months after treatment. Serum TRACP-5b (p

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency causes episodic ketoacidotic crises and no apparent symptoms between them. Here, we report a Japanese case of neonatal-onset SCOT deficiency. The male patient presented a severe ketoacidotic crisis, with blood pH of 7.072 and bicarbonate of 5.8 mmol/L at the age of 2 days and was successfully treated with intravenous infusion of glucose and sodium bicarbonate. He was diagnosed as SCOT deficient by enzymatic assay and mutation analysis. At the age of 7 months, he developed a second ketoacidotic crisis, with blood pH of 7.059, bicarbonate of 5.4 mmol/L, and total ketone bodies of 29.1 mmol/L. He experienced two milder ketoacidotic crises at the ages of 1 year and 7 months and 3 years and 7 months. His urinary ketone bodies usually range from negative to 1+ but sometimes show 3+ (ketostix) without any symptoms. Hence, this patient does not show permanent ketonuria, which is characteristic of typical SCOT-deficient patients. He is a compound heterozygote of c.1304C > A (T435N) and c.658-666dupAACGTGATT p.N220_I222dup. mutations in the OXCT1 gene. The T435N mutation was previously reported as one which retained significant residual activity. The latter novel mutation was revealed to retain no residual activity by transient expression analysis. Both T435N and N220_I222 lie close to the SCOT dimerization interface and are not directly connected to the active site in the tertiary structure of a human SCOT dimer. In transient expression analysis, no apparent interallelic complementation or dominant negative effects were observed. Significant residual activity from the T435N mutant allele may prevent the patient from developing permanent ketonuria.

We determined the activation of the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways in 108 cases of estrogen receptor-positive and human epidermal growth factor receptor 2-negative breast cancer with high and low Ki-67 expression. The expression levels of Ki-67, p53, phosphorylated MAPK (pMAPK), and protein S6 (pS6; downstream molecule of PI3K/Akt/mammalian target of rapamycin/S6 kinase pathway) were determined immunohistochemically. pS6 positivity, but not pMAPK positivity, was significantly associated with the high Ki-67 expression subset.Evaluation of luminal A and luminal B characteristics of estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancer is considered important. Although the phosphoinositide 3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways are thought to be involved in the luminal B subtype, the details of their contribution to breast cancer remain unclear.We determined the activation of these pathways (phosphorylated MAPK [pMAPK] and protein S6 [pS6; a downstream molecule of PI3K/Akt/mammalian target of rapamycin (mTOR)/S6 kinase (S6K)]) in 108 ER(+), HER2(-) breast cancer cases with high and low Ki-67 expression. The ER, progesterone receptor (PgR), Ki-67, p53 expression levels were also determined immunohistochemically. The cutoff value for Ki-67 was set at 15%.A significantly greater percentage of cancer cases with high Ki-67 expression showed pS6 positivity than did those with low Ki-67 expression (53.2% vs. 19.7%; P = .0003). No significant differences were found between the cases with high and low expression levels were detected for p53 (23.4% vs. 11.5%; P = .12) or pMAPK (36.2% vs. 34.4%; P = .85) positivity. Multivariate analysis showed that pS6 positivity (odds ratio 5.16, 95% confidence interval 1.95-13.63; P = .0009), nuclear grade 2 and 3, and low PgR expression (≤ 20%) were independently associated with the high Ki-67 subset.From our findings, we have concluded that the pS6 expression level is associated with the characteristics of breast cancer with high Ki-67 expression. Because these associations were observed, irrespective of menopausal status, the biologic difference seems to be less affected by estrogen signaling than by activation of S6 protein, especially in terms of proliferation. Our findings have also indicated that targeting the mTOR/S6K pathway might be a useful strategy for the treatment of ER(+)/HER2(-) breast cancer with high Ki-67 expression.

We examined the prognostic significance of progesterone receptor (PgR) expression in immunohistochemical-based luminal subtypes defined by Ki-67 expression, taking menopausal status into consideration. The study included 327 surgically removed estrogen receptor-positive and human epidermal growth factor receptor 2-negative breast cancers. High Ki-67 expression (15%) and low PgR expression (£ 20%) were significant independent factors resulting in worse distant relapse-free survival. This association was observed in postmenopausalwomen but not in premenopausal women.Accurate classification of luminal A and luminal B characteristics of estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancer is considered clinically important for determining effective adjuvant treatment. Although Ki-67 expression has been identified as an essential constituent for this classification, the role of progesterone receptor (PgR) expression has yet to be fully elucidated. Because PgR expression is influenced by the estrogen milieu, we examined the prognostic significance of PgR expression in immunohistochemical (IHC)-based luminal subtypes defined by Ki-67 expression, taking menopausal status into consideration.We examined 327 surgically removed ER(+) and HER2(-) breast cancer specimens. ER, PgR, and Ki67 expression was determined IHC for semiquantitative measurement. We used 1%, 20%, and 15% as the cutoff value for ER, PgR, and Ki-67, respectively.Breast cancer with low PgR (≤ 20%) expression was significantly associated with postmenopausal status, a large tumor size, and low ER expression. The low PgR expression subset had significantly worse distant relapse-free survival (DRFS) than the high PgR expression subset (P = .0067). This association was observed consistently in postmenopausal women but not in the premenopausal women. Multivariate analysis demonstrated that high Ki-67 expression (hazard ratio [HR], 3.80; 95% confidence interval [CI], 1.57-10.58; P = .003) and low PgR expression (HR, 2.54; 95% CI, 1.08-6.40; P = .038) were significant independent factors affecting DRFS.Low PgR expression was independently associated with a poorer prognosis for ER(+) and HER2(-) breast cancer. Determination of PgR expression combined with that of Ki-67 could thus improve the accuracy of IHC-based classification of luminal A and luminal B breast cancer, especially for postmenopausal women.

Mitochondrial acetoacetyl-CoA thiolase deficiency is an autosomal recessive disorder, characterized by intermittent ketoacidosis. We developed a multiplex ligation-dependent probe amplification method for mutation detection in the ACAT1 gene, which encodes this enzyme, and validated it using DNAs from two previously reported patients having partial deletion and duplication in this gene. Using this method, we identified a heterozygous deletion including exons 3–4 in a third patient, likely due to Alu-mediated non-equal homologous recombination between Alu sequences.

The impact of body mass index (BMI) on the risk of postmenopausal estrogen receptor (ER)-positive breast cancers has been well documented. However, the mechanism for the impact of BMI on the etiology of luminal A and luminal B subtypes has not yet been identified.We analyzed associations between BMI and breast cancers stratified by immunohistochemically defined intrinsic subtypes, and 1,297 Japanese women (615 breast cancer patients and 682 healthy women from a breast cancer screening program) were enrolled in a case-control study. ER-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancers were classified into luminal A and B subtypes according to Ki67 expression levels.Higher BMI was significantly positively associated with postmenopausal breast cancer risk for one-unit increase in BMI (adjusted odds ratio (aOR) 1.09, 95 % confidence interval (CI) 1.04-1.15; P = 0.0008). Analyses of postmenopausal women revealed that BMI was consistently and exclusively associated with luminal A incidence (aOR 1.18, 95 % CI 1.10-1.26; P0.0001). When BMI was divided into three categories corresponding to those of controls, among postmenopausal women, the observed positive association was confined to luminal A (high vs low, aOR 2.98, 95 % CI 1.53-5.80; P0.005), but not luminal B (aOR 0.95, 95 % CI 0.47-1.91) subtypes.We observed that BMI was significantly positively associated with increased risk of postmenopausal breast cancer for Japanese women with luminal A, but not with luminal B tumor subtype.

The aim of our study was to investigate the association of C4.4A expression in breast tumors with both patients’ clinicopathological characteristics and outcomes in order to clarify the significance of C4.4A in breast cancer. Primary breast cancer patients (n = 125, stage I–III) who had undergone breast mastectomy or breast-conserving surgery at our hospital between 2005 and 2011 were recruited for this study. Tumor samples were obtained from surgical specimens and expression status of C4.4A, estrogen receptors, progesterone receptors, human epidermal growth factor receptor 2 (HER2) and Ki67 was analyzed immunohistochemically, while HER2 amplification was examined using fluorescence in situ hybridization. Multivariate analysis showed that HER2 positivity was the only independent predictive factor for C4.4A expression (odds ratio 5.31, 95 % confidence interval 2.04–15.72; P

High body mass index (BMI) is associated not only with a higher incidence of breast cancers but also with poorer prognosis. It is speculated that both enhanced production of estrogens and other factors associated with obesity are involved in these associations, but the biological characteristics associated with high BMI have yet to be thoroughly identified. We studied 525 breast cancers, focusing on biological differences between tumors associated with high and low BMI and by immunohistochemically defined intrinsic subtype. Ki67 expression levels were used to differentiate luminal A from luminal B estrogen receptor (ER)+/human epidermal growth factor receptor 2 (HER2)-breast cancers. Premenopausal patients with high BMI showed a significantly higher frequency of lymph node metastasis (46.4 % vs. 22.9 %, P = 0.005) and tended to have a larger tumor size (P = 0.05) and higher nuclear grade (P = 0.07) than those with low BMI. These differences were not observed among postmenopausal patients. BMI was not associated with distribution of breast cancer subtypes, and ER, progesterone receptor (PR), and Ki67 expression levels of each subtype showed no differences between high and low BMI among premenopausal patients. Higher BMI might influence aggressive tumor characteristics among premenopausal patients, but its influence on ER, PR, and Ki67 expression levels seems to be limited.

The molecular basis of simultaneous two-exon skipping induced by a splice-site mutation has yet to be completely explained. The splice donor site mutation c.1248+5g>a (IVS13) of the OXCT1 gene resulted predominantly in skipping of exons 12 and 13 in fibroblasts from a patient (GS23) with succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency. We compared heteronuclear RNA (hnRNA) intermediates between controls' and GS23's fibroblasts. Our strategy was to use RT-PCR of hnRNA to detect the presence or absence of spliced exon clusters in RNA intermediates (SECRIs) comprising sequential exons. Our initial hypothesis was that a SECRI comprising exons 12 and 13 was formed first followed by skipping of this SECRI in GS23 cells. However, such a pathway was revealed to be not a major one. Hence, we compared the intron removal of SCOT transcript between controls and GS23. In controls, intron 11 was the last intron to be spliced and the removal of intron 12 was also rather slow and occurred after the removal of intron 13 in a major pathway. However, the mutation in GS23 cells resulted in retention of intron 13, thus causing the retention of introns 12 and 11. This "splicing paralysis" may be solved by skipping the whole intron 11-exon 12-intron 12-exon 13-mutated intron 13, resulting in skipping of exons 12 and 13.

Estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancers can be divided into luminal A and luminal B subtypes based on Ki67 expression levels. However, the biological differences in ER and progesterone receptor (PR) expression levels between these luminal subtypes are not clear.We examined immunohistochemical expression levels of ER, PR, and Ki67 in 180 ER-positive/HER2-negative breast cancers while taking menopausal status into account. Breast cancers were divided according to ER and PR levels (H:50%, L: ≤ 50%), and luminal A and B were classified by the Ki67 labeling index (A: Ki6714%, B: Ki67 ≥ 14%).When breast cancers were classified based on ER and PR levels, the distribution of pre- and postmenopausals was significantly different for luminal A (P0.0001), but not for luminal B cancers. As for luminal A, ER-H/PR-L cancers were rare among premenopausals (8%), but frequent among postmenopausals (54%). Correlation between ER and PR levels among luminal A cancers was strong in premenopausals but weak in postmenopausals. Since crosstalk with growth factor signaling is unlikely in luminal A, we speculate that intratumoral estrogen insufficiency contributed to the characteristics of postmenopausal ER-H/PR-L cancers.We speculate that the biological characteristics of luminal A cancers are influenced by the estrogen environment, but its influence on luminal B cancers may be limited. We believe these considerations constitute useful information for a better understanding of the biology of ER-positive-HER2-negetive breast cancers.

A flow injection (FI) spectrophotometric method is proposed for the determination of low concentration of formaldehyde (HCHO) in liquid media. It is based on the condensation of HCHO with hydroxylamine sulfate, followed by the reduction reaction of iron(III)-ferrozine complex with the residual hydroxylamine to form a purple iron(II)-ferrozine complex (λ(max)=562 nm). In the first reaction, hydroxylamine decreases proportionally to the concentration of HCHO, and therefore the produced purple iron(II)-ferrozine complex decreases with increasing HCHO (a negative FI peak is obtained). The detection limit (S/N=3) was 1.6 μg L(-1). The method can be applied to the determination of HCHO in industrial wastewater.

The most critical step for initiation and progression of estrogen receptor-α (ERα)-positive breast cancers is thought to be upregulation of ERα expression. There are several factors involved in this mechanism, i.e., increased promoter activity of the ERα gene (ESR1) at the transcriptional level, ESR1 gene amplification, and diminished degradation of ERα protein through ubiquitination and proteasomal pathways. Mediating these factors, ERα protein levels seem to be controlled, although the details of the mechanism remain to be clarified. In addition, for upregulation of estrogen signaling, functional changes in its action in cancer cells originating from normal epithelial cells, i.e., estrogen stimulation, which then leads to proliferation of ERα-positive cancer cells, has been recognized, but this action has not been observed in normal epithelial cells. These alterations are therefore likely to contribute to the pathogenesis of ERα-positive breast cancers.

The classic action that leads to transcriptional activation of estrogen response genes mediated through estrogen receptors (ER) and the estrogen complex plays a pivotal role in the development of ER-positive breast cancers. In addition to this pathway, non-classic action and non-genomic action, both estrogen-dependent and estrogen-independent genomic actions have also been found to contribute to ER-positive tumor growth. Although the details of these mechanisms are not well known, participation of the growth factor signaling pathway is likely to be the most significant factor for acquisition of resistance to hormonal therapy. This resistance is mediated not only directly through cell growth promotion by growth factor signaling, but also through enhancement of alternative ER signaling pathways in addition to classic action. The reason why tamoxifen-insensitive ER-positive breast cancers respond to aromatase inhibitors may be explained, at least in part, by the different estrogen-related signaling pathways in which aromatase inhibitors may block estrogen signaling. In this paper we discuss the molecular mechanisms for resistance to hormonal therapy based on an understanding of estrogen signaling pathways.

We report a case of neuroendocrine carcinoma in a 40-year-old woman who presented with two lumps in her left breast. Mammography failed to reveal any lesions because she had so-called dense breasts, but ultrasonography showed 4 irregular hypoechoic masses. Magnetic resonance imaging also showed 4 homogeneous lobulated tumors with early contrast enhancement, suggesting malignancy. Core needle biopsy and subsequent immunohistochemical examination of the specimens was performed. Neuroendocrine carcinoma was diagnosed. The tumor cells were diffusely positive for chromogranin A and synap-tophysin, and some were positive for CD56. We performed total mastectomy with sentinel lymph node biopsy, which showed no metastasis. Recurrence has not been detected at 36 months after surgery.