Modular Design Platform for Activatable Fluorescence Probes Targeting Carboxypeptidases Based on ProTide Chemistry.

Carboxypeptidases (CPs) are a family of hydrolases that cleave one or more amino acids from the C-terminal of peptides or proteins and play indispensable roles in various physiological and pathological processes. However, only a few highly activatable fluorescence probes for CPs have been reported, and there is a need for a flexibly tunable molecular design platform to afford a range of fluorescence probes for CPs for biological and medical research. Here, we focused on the unique activation mechanism of ProTide-based prodrugs and established a modular design platform for CP-targeting florescence probes based on ProTide chemistry. In this design, probe properties such as fluorescence emission wavelength, reactivity/stability, and target CP can be readily tuned and optimized by changing the four probe modules: the fluorophore, the substituent on the phosphorus atom, the linker amino acid at the P1 position, and the substrate amino acid at the P1' position. In particular, switching the linker amino acid at position P1 enabled us to precisely optimize the reactivity for target CPs. As a proof-of-concept, we constructed probes for carboxypeptidase M (CPM) and prostate-specific membrane antigen (also known as glutamate carboxypeptidase II). The developed probes were applicable for the imaging of CP activities in live cells and in clinical specimens from patients. This design strategy should be useful in studying CP-related biological and pathological phenomena.

Dynamic Changes in Peripheral Systemic Immunity Markers During Chemotherapy in HER2-negative Advanced Breast Cancer.

BACKGROUND/AIM: The immune system has a pivotal role in modulating the response to chemotherapy in breast cancer (BC). However, the immune status during chemotherapy remains unclear. We evaluated the sequential changes in peripheral systemic immunity markers in BC patients treated with various chemotherapeutic agents. MATERIALS AND METHODS: We examined the correlation between the peripheral systemic immunity markers, neutrophil-to-lymphocyte ratio (NLR), absolute lymphocyte count (ALC) and the local cytolytic activity (CYT) score obtained by quantitative reverse-transcription polymerase chain reaction of 84 preoperative BC patients. Next, we observed the sequential changes in the peripheral systemic immunity markers during treatment with four anticancer drugs: oral 5-fluorouracil derivative; S-1, epirubicin plus cyclophosphamide; paclitaxel plus the anti-vascular endothelial growth factor antibody bevacizumab, and eribulin in 172 HER2-negative advanced BC patients. Finally, we examined the correlation between the changes in the peripheral systemic immunity markers, time to treatment failure (TTF) and progression-free survival (PFS). RESULTS: A negative correlation was found between ALC and NLR. ALC-low and NLR-high cases were positively associated with CYT score-low cases. The ratio of ALC-increase and NLR-decrease varies depending on the anticancer drugs used. The responder group (TTF ≥3 months) had a higher NLR-decrease ratio than the nonresponder group (TTF <3 months). Patients with a high NLR-decrease ratio showed higher PFS. CONCLUSION: The change in ALC or NLR varies according to the anticancer drugs, suggesting differential immunomodulatory effects of the drugs. Furthermore, the change in NLR reflects the therapeutic efficacy of chemotherapy in advanced BC.

[Efficacy and Safety of Scalp Cooling Device RV21-01 for Suppression of Chemotherapy-Induced Alopecia in Breast Cancer Patients].

We evaluated the efficacy and safety of the RV21-01 scalp cooling device in controlling hair loss during chemotherapy in this study. Thirty-nine breast cancer patients who underwent anthracycline- and/or taxane-based chemotherapy were assigned to the scalp cooling group(27 patients)and the hair loss observation group(12 patients). The alopecia rate using the NCI alopecia toxicity criteria and the quantitative alopecia toxicity grade was 51.9%(14/27 patients)and 100%(12/12 patients)in the scalp cooling and hair loss observation groups, respectively. Regarding safety, all subjects in both the scalp cooling and hair loss observation groups experienced adverse events; only 1 subject in each group experienced a severe adverse event due to chemotherapy and majority of the subjects in both groups experienced minor adverse events. RV21-01 scalp cooling therapy was demonstrated to be effective in reducing hair loss in patients undergoing standard chemotherapy for breast cancer. In addition, the adverse events associated with the scalp cooling therapy were minor and mild, and hence, deemed acceptable.

Development of an intraoperative breast cancer margin assessment method using quantitative fluorescence measurements.

Breast-conserving surgery has become the preferred treatment method for breast cancer. Surgical margin assessment is performed during surgery, as it can reduce local recurrence in the preserved breast. Development of reliable and lower-cost ex vivo cancer detection methods would offer several benefits for patient care. Here, a practical and quantitative evaluation method for the ex vivo fluorescent diagnosis of breast lesions was developed and confirmed through a three-step clinical study. Gamma-glutamyl-hydroxymethyl rhodamine green (gGlu-HMRG) has been reported to generate fluorescence in breast lesions. Using this probe, we constructed a reliable and reproducible procedure for the quantitative evaluation of fluorescence levels. We evaluated the reliability of the method by considering reproducibility, temperature sensitivity, and the effects of other clinicopathological factors. The results suggest that the fluorescence increase of gGlu-HMRG is a good indicator of the malignancy of breast lesions. However, the distributions overlapped. A 5 min reaction with this probe could be used to distinguish at least part of the normal breast tissue. This method did not affect the final pathological examination. In summary, our results indicate that the methods developed in this study may serve as a feasible intraoperative negative-margin assessment tool during breast-conserving surgery.

Rapid and Accurate Visualization of Breast Tumors with a Fluorescent Probe Targeting α-Mannosidase 2C1.

Accurate detection of breast tumors and discrimination of tumor from normal tissues during breast-conserving surgery are essential to reduce the risk of misdiagnosis or recurrence. However, existing probes show substantial background signals in normal breast tissues. In this study, we focus on glycosidase activities in breast tumors. We synthesized a series of 12 fluorescent probes and performed imaging-based evaluation on surgically resected human breast specimens. Among them, the α-mannosidase-reactive fluorescent probe HMRef-αMan detected breast cancer with 90% sensitivity and 100% specificity. We identified α-mannosidase 2C1 as the target enzyme and confirmed its overexpression in various breast tumors. We found that fibroadenoma, the most common benign breast lesion in young woman, tends to have higher α-mannosidase 2C1 activity than malignant cancer. Combined application of green-emitting HMRef-αMan and a red-emitting γ-glutamyltranspeptidase probe enabled efficient dual-color, dual-target optical discrimination of malignant and benign tumors.

N-Cadherin mRNA Levels in Peripheral Blood Could Be a Potential Indicator of New Metastases in Breast Cancer: A Pilot Study.

BACKGROUND: There is growing evidence that patients with metastatic breast cancer whose disease progresses from a new metastasis (NM) have a worse prognosis than that of patients whose disease progresses from a pre-existing metastasis. The aim of this pilot study is to identify a blood biomarker predicting NM in breast cancer. METHODS: The expression of epithelial (cytokeratin 18/19) or mesenchymal (plastin-3, vimentin, and N-cadherin) markers in the peripheral blood (PB) of recurrent breast cancer patients undergoing chemotherapy with eribulin or S-1 was measured over the course of treatment by RT-qPCR. The clinical significance of preoperative N-cadherin expression in the PB or tumor tissues of breast cancer patients undergoing curative surgery was assessed by RT-qPCR or using public datasets. Finally, N-cadherin expression in specific PB cell types was assessed by RT-qPCR. RESULTS: The expression levels of the mesenchymal markers N-cadherin and vimentin were high in the NM cases, whereas that of the epithelial marker cytokeratin 18 was high in the pre-existing metastasis cases. High preoperative N-cadherin expression in PB or tumor tissues was significantly associated with poor recurrence-free survival. N-cadherin was expressed mainly in polymorphonuclear leukocytes in PB. CONCLUSION: N-cadherin mRNA levels in blood may serve as a novel prognostic biomarker predicting NM, including recurrence, in breast cancer patients.

Long-term outcome of adipose-derived regenerative cell-enriched autologous fat transplantation for reconstruction after breast-conserving surgery for Japanese women with breast cancer.

PURPOSE: More effective methods are needed for breast reconstruction after breast-conserving surgery for breast cancer. The aim of this clinical study was to assess the perioperative and long-term outcomes of adipose-derived regenerative cell (ADRC)-enriched autologous fat grafting. METHODS: Ten female patients who had undergone breast-conserving surgery and adjuvant radiotherapy for breast cancer were enrolled. An ADRC-enriched fat graft prepared from the patient's adipose tissue was implanted at the time of adipose tissue harvest. The perioperative and long-term outcomes of the grafts, which included safety, efficacy, and questionnaire-based patient satisfaction, were investigated. RESULTS: The mean operation time was 188 ± 30 min, and the mean duration of postoperative hospitalization was 1.2 ± 0.4 days. No serious postoperative complications were associated with the procedure. Neither recurrence nor metastatic disease was observed during the follow-up period (7.8 ± 1.5 years) after transplantation. Of 9 available patients, "more than or equal to average" satisfaction with breast appearance and overall satisfaction were reported by 6 (66.7%) and 5 (55.6%) patients, respectively. CONCLUSIONS: ADRC-enriched autologous fat transplantation is thus considered to be safe perioperatively, with no long-term recurrence, for patients with breast cancer treated by breast-conserving surgery, and it may be an option for breast reconstruction, even after adjuvant radiotherapy.

Erratum to immediate breast volume replacement using a free dermal fat graft after breast cancer surgery: multi-institutional joint research of short-term outcomes in 262 Japanese patients.

[This corrects the article on p. 179 in vol. 4, PMID: 26005649.].

Rapid diagnosis of lymph node metastasis in breast cancer using a new fluorescent method with γ-glutamyl hydroxymethyl rhodamine green.

Sentinel lymph node biopsy is performed as a standard procedure in breast cancer surgery, and the development of quick and simple methods to detect metastatic lesions is in high demand. Here, we validated a new fluorescent method using γ-glutamyl hydroxymethyl rhodamine green to diagnose metastatic lymph nodes in breast cancer. One hundred and forty-nine lymph nodes from 38 breast cancer patients were evaluated in this study. Comparison of fluorescent and pathological images showed that this fluorescent method was successful for visualizing breast cancer cells in lymph nodes. This method had a sufficiently high sensitivity (97%), specificity (79%) and negative predictive value (99%) to render it useful for an intraoperative diagnosis of cancer. These preliminary findings suggest that this novel method is useful for distinguishing non-cancerous specimens from those in need of careful examination and could help save time and cost for surgeons and pathologists.

Rapid intraoperative visualization of breast lesions with γ-glutamyl hydroxymethyl rhodamine green.

We previously developed γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) as a tool to detect viable cancer cells, based on the fact that the enzyme γ-glutamyltranspeptidase (GGT) is overexpressed on membranes of various cancer cells, but is not expressed in normal tissue. Cleavage of the probe by GGT generates green fluorescence. Here, we examined the feasibility of clinical application of gGlu-HMRG during breast-conserving surgery. We found that fluorescence derived from cleavage of gGlu-HMRG allowed easy discrimination of breast tumors, even those smaller than 1 mm in size, from normal mammary gland tissues, with 92% sensitivity and 94% specificity, within only 5 min after application. We believe this rapid, low-cost method represents a breakthrough in intraoperative margin assessment during breast-conserving surgery.

Immediate breast volume replacement using a free dermal fat graft after breast cancer surgery: multi-institutional joint research of short-term outcomes in 262 Japanese patients.

BACKGROUND: Immediate volume replacement using a free dermal fat graft (FDFG) has been proven safe with early postoperative benefits. The aims of the present study were to clarify adequate indications and risk factors associated with operative morbidity. PATIENTS AND METHODS: A multi-institutional analysis of partial mastectomy with immediate volume replacement with FDFG was undertaken in 14 hospitals specializing in breast cancer treatment. Clinical and oncological variables were analyzed to identify factors associated with postoperative complications. RESULTS: A total of 262 cases were analyzed. Considering the observation period and overlap of patients, 13 (5.4%) out of 242 patients had complications within 1 month of surgery while 7 (4.6%) out of 151 patients developed complications 1-12 months after surgery. Two hundred and eleven out of 242 patients were statistically examined using a multivariate analysis, which revealed that the weight of resected breast tissue, size of implanted FDFG (cranio-caudal length), and weight of implanted FDFG were associated with a higher likelihood of postoperative complications. CONCLUSIONS: Immediate breast volume replacement using a FDFG after breast cancer surgery should be done for selected patients with breast cancer to avoid postoperative complications. The prospective and larger investigations are warranted for the establishment of appropriate guidelines.

Diminished expression of MiR-15a is an independent prognostic marker for breast cancer cases.

BACKGROUND/AIM: MiR-15a targets Cyclin E1 (CCNE1), which regulates the cell cycle and promotes cell proliferation and progression. Herein, we investigated the clinicopathological significance of miR-15a as a prognostic marker in breast cancer (BC) cases. MATERIALS AND METHODS: We collected primary tumor samples of 230 BC cases, including 68 triple-negative cases. The expression levels of miR-15a in primary tumors were measured by qRT-PCR assay. RESULTS: Low expression of miR-15a in primary tumors was significantly correlated with shorter disease-free survival (p=0.0012) and overall survival (p=0.005) compared to the high miR-15a expression in triple-negative BC cases. Multivariate analysis indicated that low miR-15a expression was an independent prognostic factor for overall survival [RR=2.56(1.03-7.18), p=0.04]. CONCLUSION: MiR-15a expression levels could be a promising biological and prognostic marker for overall survival especially in triple-negative BC cases.

F-box protein FBXW7 inhibits cancer metastasis in a non-cell-autonomous manner.

The gene encoding F-box protein FBXW7 is frequently mutated in many human cancers. Although most previous studies have focused on the tumor-suppressive capacity of FBXW7 in tumor cells themselves, we determined that FBXW7 in the host microenvironment also suppresses cancer metastasis. Deletion of Fbxw7 in murine BM-derived stromal cells induced accumulation of NOTCH and consequent transcriptional activation of Ccl2. FBXW7-deficient mice exhibited increased serum levels of the chemokine CCL2, which resulted in the recruitment of both monocytic myeloid-derived suppressor cells and macrophages, thereby promoting metastatic tumor growth. Administration of a CCL2 receptor antagonist blocked the enhancement of metastasis in FBXW7-deficient mice. Furthermore, in human breast cancer patients, FBXW7 expression in peripheral blood was associated with serum CCL2 concentration and disease prognosis. Together, these results suggest that FBXW7 antagonizes cancer development in not only a cell-autonomous manner, but also a non-cell-autonomous manner, and that modulation of the FBXW7/NOTCH/CCL2 axis may provide a potential approach to suppression of cancer metastasis.

Effects of dietary genistein on hormone-dependent rat mammary carcinogenesis induced by ethyl methanesulphonate.

Genistein, a major soy isoflavone having weak estrogenic activities, has been suggested to reduce the risk of breast cancer incidence. However, many studies have yielded inconsistent results. We investigated the effects of dietary genistein on the development of breast cancer using ethyl methanesulphonate (EMS) chemically induced rat model of hormone-dependent mammary carcinoma. Female Wistar King A rats were orally given EMS for 12 wk and fed isoflavone-free NIH-07PLD diets with or without genistein, beginning immediately after weaning period. All EMS-treated rats fed either diet developed estrogen and/or progesterone receptor-positive mammary carcinoma by 24 wk. The addition of either low or high genistein, which produced the plasma concentrations comparable with those observed in humans consuming high soy diets, did not show any preventive activity. Soy-containing pellet food, exhibiting substantial plasma concentrations of isoflavones such as genistein, daidzein, equol, and glycitein, significantly increased the latency periods, compared to either NIH-07PLD diet with low (P = 0.027) or high (P = 0.034) genistein. Body weights, total EMS uptakes, and urinary estradiol concentrations were not significantly different among groups. These data indicate that genistein does not exert clear preventive effects and that isoflavone components other than genistein might be preventive against hormone-dependent mammary carcinogenesis.

Vascular endothelial growth factor receptor-1 mRNA overexpression in peripheral blood as a useful prognostic marker in breast cancer.

INTRODUCTION: Identification of useful markers associated with poor prognosis in breast cancer patients is critically needed. We previously showed that expression of vascular endothelial growth factor receptor-1 mRNA in peripheral blood may be useful to predict distant metastasis in gastric cancer patients. However, expression of vascular endothelial growth factor receptor-1 mRNA in peripheral blood of breast cancer patients has not yet been studied. METHODS: Real-time reverse transcriptase-PCR was used to analyze vascular endothelial growth factor receptor-1 mRNA expression status with respect to various clinical parameters in 515 patients with breast cancer and 25 controls. RESULTS: Expression of vascular endothelial growth factor receptor-1 mRNA in peripheral blood was higher in breast cancer patients than in controls. Increased vascular endothelial growth factor receptor-1 mRNA expression was associated with large tumor size, lymph node metastasis and clinical stage. Patients with high vascular endothelial growth factor receptor-1 mRNA expression also experienced a poorer survival rate than those with low expression levels, including those patients with triple-negative type and luminal-HER2(-) type disease. CONCLUSIONS: Expression of vascular endothelial growth factor receptor-1 mRNA in peripheral blood may be useful for prediction of poor prognosis in breast cancer, especially in patients with triple-negative type and luminal-HER2(-) type disease.

The relevance of intrinsic subtype to clinicopathological features and prognosis in 4,266 Japanese women with breast cancer.

BACKGROUND: Estrogen receptor (ER), progesterone receptor (PgR), and HER2 expression status in breast cancer function as prognostic and predictive factors that enable individualized treatment. Intrinsic subtype classification has also been performed based on these and other biological and prognostic characteristics. However, clinical analysis of such subtypes in a large number of Japanese breast cancer patients has not yet been reported. METHODS: Between January 2003 and December 2007, 4,266 patients with primary breast cancer were registered. Four subtypes based on immunohistochemically evaluated ER/PgR/HER2 status, clinicopathological features, and prognosis were analyzed retrospectively. RESULTS: The following subtype distribution was observed: luminal A type (ER+ and/or PgR+, HER2-), 3,046 cases (71%); luminal B type (ER+ and/or PgR+, HER2+), 321 cases (8%); HER2 type (ER-, PgR-, HER2+), 398 cases (9%); and triple negative (TN) type (ER-, PgR-, HER2-), 501 cases (12%). The HER2+ subtypes (luminal B and HER2 types) had a significantly higher incidence of lymph node metastasis and lymphatic permeation, while the hormone receptor negative subtypes (HER2 and TN types) showed a significantly higher nuclear grade. Overall, patients with HER2-type and TN-type disease had a significantly poorer prognosis than other subtypes. CONCLUSION: Intrinsic breast cancer subtypes are associated with clinicopathological features and prognosis in Japanese women. Long-term clinical observation of the relationship between each subtype and therapies used should provide useful information for selecting appropriately tailored treatments.

Psychosocial factors affecting the therapeutic decision-making and postoperative mood states in Japanese breast cancer patients who underwent various types of surgery: body image and sexuality.

OBJECTIVE: We conducted an empirical study to clarify how psychosocial factors (e.g. body image and sexuality) influence therapeutic decision-making and to identify the factors that affect post-operative mood states in Japanese women who underwent various types of surgery. METHODS: One hundred and two patients who had undergone surgical treatment for breast cancer participated in this study. Twenty-five patients had undergone mastectomy, 67 breast conserving treatment and 11 skin-sparing mastectomy and immediate breast reconstruction. The participants were evaluated based on a battery of questionnaires including value estimates of decision-making factors, a shortened version of the Profile of Mood States and self-efficacy. RESULTS: The patients regarded the possibility of cure and recurrence of cancer as well as the physician's support as important, regardless of the treatments they had chosen. The patients that had immediate breast reconstruction placed significantly more importance on their body image and sexuality (i.e. physical appearance, attractiveness to partner and self-evaluation of femininity and sexuality) than the mastectomy patients. After surgery, the former group tended to have a more negative mood on the shortened version of the Profile of Mood States than the latter group. Degree of self-efficacy had a marked influence on the patients' mood after surgery. CONCLUSIONS: This study suggests the importance of discussing body image and sexuality that has tended to be disregarded in therapeutic decision-making situations in breast cancer patients in Japan. Self-efficacy is a crucial variable for improving mood after treatment.

Risk factors of abdominal surgery in patients with collagen diseases.

Patients with collagen diseases have been reported to demonstrate a greater risk when undergoing surgical operations. To determine the risk factors in abdominal surgery for patients with collagen diseases, 32 patients with collagen diseases who underwent abdominal surgery were analyzed for their clinical features and surgical results by comparing 26 cases from the favorable prognosis group (Group A) and 6 cases resulting in hospital death (Group B). The analysis revealed that emergent operations tended to result in worse outcomes (P = 0.011) than elective operations and that cases undergoing operations for collagen disease-related problems, including intestinal perforation and acute pancreatitis, also showed a worse postoperative course than those who underwent operations for problems unrelated to collagen diseases, such as carcinomas and cholelithiasis (P = 0.0006). The dose of steroids administered at the time of operation was also significantly higher in Group B than in Group A (P = 0.03). These results suggested that the patients with collagen diseases should be followed periodically not only for the primary disease but also for any potential surgical diseases to identify such diseases at an early stage and to avoid an emergent operation, and that patients treated with high doses of steroids also need intensive care after abdominal surgery.

Prediction of survival from first recurrence of breast carcinoma in Japanese women.

The clinical courses of patients with recurrent breast carcinoma vary greatly. We retrospectively examined data on 1863 Japanese patients treated for a breast carcinoma from 1981 to 2000. Among them, 345 (18.5%) who had clearly died of recurrence were reviewed. Patients died most frequently (63.2%) up to 30 months after the first recurrence. Based on multivariate analysis, the four factors that were most predictive of survival after the first recurrence were disease-free interval, site of recurrence, progesterone receptor (PgR) status, and vascular involvement. These findings showed that the intrinsic tumor biology of the initial primary tumor plays a critical role in determining survival after the first recurrence in patients with a breast carcinoma. The combined analysis of disease-free interval, site of recurrence, PgR status, and vascular involvement may assist in estimating the median survival after first recurrence, and may assist with the designing of new therapeutic strategies for patients with recurrence for whom there is an unfavorable prognosis.

Prediction of early and late recurrence in patients with breast carcinoma.

BACKGROUND: The clinical course of patients with recurrent breast carcinoma varies greatly. Better characterization of an individual's clinical course for recurrent patients may aid in their clinical management. However, less attention has been paid to evaluating factors associated with the timing of recurrence in those patients. We investigated the clinicopathological indicators that determined the timing of recurrence by univariate and multivariate analysis. METHODS: We retrospectively examined data on 1428 curatively treated Japanese patients who had been surgically treated for breast cancer between 1983 and 2002. From these, 244(17.1%)who had clearly died of recurrence were entered into this study. RESULTS: By univariate analysis, tumor size, estrogen receptor(ER), and progesterone receptor(PgR)were significantly correlated with time to recurrence. Multivariate analysis indicated that the time between operation and recurrence was independently influenced by ER and PR. CONCLUSIONS: Our research shows that ER and PgR are independent factors influencing the timing of recurrence of breast carcinoma after curative resection. The combined analysis of these independent factors facilitates prediction of the time to recurrence for each patient.