Kentaro Tamaki

Vasohibin-1 in human breast carcinoma: a potential negative feedback regulator of angiogenesis.

Vasohibin‐1 is a recently identified negative feedback inhibitor or suppressor of angiogenesis induced by vascular endothelial growth factor (VEGF)‐A. The status of vasohibin‐1 in human breast carcinoma has not been examined. We examined 151 breast specimens including 98 cases of invasive ductal carcinoma (IDC), 12 of ductal carcinoma in situ (DCIS), 16 of fibroadenoma (FA), six of inflammatory lesion, nine of fibrocystic change and seven of non‐pathological breast tissue. We immunolocalized vasohibin‐1 and compared its immunoreactivity to that of VEGF‐A, basic fibroblastic growth factor (bFGF), VEGF receptor 2 (Flk‐1), CD31, CD34 and Ki‐67/MIB‐1. The correlation of vasohibin‐1 immunoreactivity with overall survival (OS), and disease‐free survival (DFS) of the patients with breast carcinoma was also evaluated. In addition, we evaluated Ki‐67 and CD31, and Ki‐67 and vasohibin‐1 double‐immunostaining for further characterization of neovascularization. Vasohibin‐1 was detected in endothelial cells of human breast and its immunodensity was significantly higher in IDC and inflammatory lesions than the other types (P < 0.001). In addition, a significant positive correlation was detected between vasohibin‐1 and VEGF‐A, bFGF or Flk‐1 (P < 0.001). There was also positive associations between vasohibin‐1 and OS (P = 0.004) and between vasohibin‐1 and DFS (P ≤ 0.001) in carcinoma cases. Results of double‐immunostaining demonstrated the ratio of Ki‐67‐positive cells among vasohibin‐1‐positive endothelial cells (46.5%) was significantly higher than those among CD31‐positive cells (23.5%). This is the first study demonstrating the status of vasohibin‐1 in human breast lesions, which indicates that vasohibin‐1 is associated with neovascularization and may especially play important roles in the regulation of intratumoral angiogenesis in human breast cancer. (Cancer Sci 2009; 100: 88–94)

Comparison of core needle biopsy (CNB) and surgical specimens for accurate preoperative evaluation of ER, PgR and HER2 status of breast cancer patients

The roles of core needle biopsy (CNB) have become well established as an important preoperative diagnostic method for breast lesions. We examined the concordance of histological types, nuclear grades, hormone receptors, and human epidermal growth factor receptor 2 (HER2) status between CNB and surgical specimens in 353 cases. In addition, we analyzed the correlation between the number of CNB specimens obtained and accuracy of histological factors in order to explore the optimal number of CNB specimens. Between CNB and surgical specimens, concordance rates of histological type, nuclear grade, estrogen receptor (ER), and progesterone receptor (PgR) status (cut‐off 0–<1%, 1–10%, and 10%<), and HER2 were 84.4%, 81.3%, 92.9%, and 89.3%, respectively. In 52 of 353 patients who were histopathologically diagnosed as ductal carcinoma in situ (DCIS) by CNB, final diagnosis was changed in to invasive ductal carcinoma (IDC) in surgical specimens. Statistically significant differences were detected in the discrepancy of the following factors between CNB and subsequent surgical specimens: histological types, nuclear grade, and PgR, between patients who received four or more cores and those who had received three or less cores. In addition, a similar tendency was also detected in estrogen receptor (ER) and HER2 as in the above, and the cases that received four cores reached to 100% concordance in diagnosis between CNB and surgical specimens. Therefore, the optimal numbers of CNB were considered four at least in assessing the histological type, invasion, nuclear grade, hormone receptor status, and HER2 status of individual patients in the preoperative setting. (Cancer Sci 2010)

Prognostic significance of tumor-infiltrating CD8 + and FOXP3 + lymphocytes in residual tumors and alterations in these parameters after neoadjuvant chemotherapy in triple-negative breast cancer: a retrospective multicenter study

IntroductionThe status of tumor-infiltrating lymphocytes (TILs) has been recently proposed to predict clinical outcome of patients with breast cancer. We therefore studied the prognostic significance of CD8+ TILs and FOXP3+ TILs in residual tumors after neoadjuvant chemotherapy (NAC) and the alterations in these parameters before and after NAC in patients with triple-negative breast cancer (TNBC).MethodsOne hundred thirty-one TNBC patients who received NAC at three institutions were examined. CD8+ TIL and FOXP3+ TIL in residual tumors and biopsy specimens were evaluated by double-staining immunohistochemistry. The CD8+ TIL and FOXP3+ TIL status of the residual tumors was assessed, and the rates of their changes before and after NAC were calculated.ResultsTNBC patients with high CD8+ TIL levels or a high CD8/FOXP3 ratio in residual tumors had significantly better recurrence-free survival (RFS) and breast cancer-specific survival (BCSS) than patients with low values of these parameters. In multivariate analyses, CD8+ TIL exhibited strong prognostic significance for RFS, with a hazard ratio (HR) of 3.09 (95 % confidence interval (CI) 1.537–6.614, P=0.0013). The CD8/FOXP3 ratio was also significantly correlated with RFS (HR=2.07, 95 % CI 1.029–4.436, P=0.0412). TNBC with larger residual tumor size and positive lymph node status, which are known prognostic factors, was independently associated with worse RFS (P=0.0064 and P=0.0015, respectively). High CD8+ TIL levels were a markedly powerful indicator of improved BCSS, with an HR of 3.59 (95 % CI 1.499–9.581, P=0.0036). Nodal status was also associated with BCSS (P=0.0024). TNBC with a high rate of CD8+ TIL changes was associated with significantly better RFS compared with the low group (P=0.011). Higher rates of changes in the CD8/FOXP3 ratio were significantly correlated with both better RFS and BCSS compared with lower rates (P=0.011 and P=0.023, respectively).ConclusionsThis is the first study to demonstrate that high CD8+ TIL and a high CD8/FOXP3 ratio in residual tumors and increment of these parameters following NAC and accurately predict improved prognosis in TNBC patients with non-pathological complete response following NAC. These parameters could serve as a surrogate one for adjuvant treatment in patients with residual disease in the neoadjuvant setting.

Vasohibin-1 as a potential predictor of aggressive behavior of ductal carcinoma in situ of the breast.

Vasohibin‐1 is a recently identified negative feedback regulator of angiogenesis induced by VEGF‐A and bFGF. In this study, we first evaluated mRNA expression of vasohibin‐1 and CD31 in 39 Japanese female breast carcinoma specimens including 22 invasive ductal carcinoma (IDC) and 17 ductal carcinoma in situ (DCIS) using a real‐time quantitative RT‐PCR (QRT‐PCR) with LightCycler system. In addition, we also immunolocalized vasohibin‐1 and CD31 and compared their immunoreactivity to nuclear grades and histological grades of 100 carcinoma cases (50 IDC and 50 DCIS). There were no statistically significant differences of CD31 mRNA expression and the number of CD31 positive vessels between DCIS and IDC (P = 0.250 and P = 0.191, respectively), whereas there was a statistically significant difference in vasohibin‐1 mRNA expression and the number of vasohibin‐1 positive vessels in DCIS and IDC (P = 0.022 and P ≤ 0.001, respectively). There was a significant positive correlation between vasohibin‐1 mRNA level and Ki‐67 labeling index in DCIS (r2 = 0.293, P ≤ 0.001). In addition, vasohibin‐1 mRNA expression was correlated with high nuclear and histological grades in DCIS cases and a significant positive correlation was detected between the number of vasohibin‐1 positive vessels and Ki‐67 labeling index or nuclear grade or Van Nuys classification of carcinoma cells (P ≤ 0.001, respectively). These results all indicate the possible correlation between aggressive biological features in DCIS including increased tumor cell proliferation and the status of neovascularization determined by vasohibin‐1 immunoreactivity.

Androgenic pathway in triple negative invasive ductal tumors: Its correlation with tumor cell proliferation

Triple negative breast cancer (TNBC) is defined by estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 negativity. Patients with TNBC frequently undergo an aggressive clinical course due to the unavailability of specific targeted therapies. Androgen receptor (AR) was reported to be expressed in up to 60% of TNBC cases but there have been controversies as to the roles of androgen signaling through AR in TNBC. Therefore, in this study, we analyzed the status of AR in combination with androgen synthesizing enzymes (5α‐reductase type 1 (5αR1) and 17β‐hydroxysteroid dehydrogenase type 5 (17βHSD5)] in order to further understand androgenic actions in TNBC. Androgen receptor, 5αR1, and 17βHSD5 were immunolocalized in a cohort of 203 TNBC patients from Thailand and Japan. We then correlated the findings with clinicopathological characteristics (age, stage, tumor diameter, lymph node invasion, metastatic spread, Ki‐67 labeling index, disease‐free survival, and overall survival) of the patients. Univariate analysis revealed that AR+/enzyme+ cases were associated with a significantly lower Ki‐67 labeling index than AR−/enzyme− samples. Multivariate analysis indicated the presence of significant positive correlations between AR and enzyme status in tumor cells, and between tumor diameter, lymph node invasion, and distant metastasis. Significant negative correlations were also detected between Ki‐67 labeling index and AR status (P = 0.04) or 5αR1 (P < 0.001). Cox proportional hazards analysis showed that Ki‐67 labeling index and stage were the only factors predicting disease‐free and overall survival of the patients, although univariate Kaplan–Meier analysis revealed AR/5αR1 negativity suggested a more adverse clinical course up to 80 months after surgery. These results suggest that the presence of androgen synthesizing pathways in addition to AR expression in tumor cells could confer a better clinical outcome through suppression of cell proliferation.

Correlation between mammographic findings and corresponding histopathology: Potential predictors for biological characteristics of breast diseases

The present study retrospectively evaluated the mammographic findings of 606 Japanese women with breast cancer (median age 50 years; range 27–89 years) and correlated them with histopathological characteristics. Mammographic findings were evaluated with an emphasis on mass shape, margin, density, calcification, and the presence of architectural distortion; these findings were correlated with histopathological characteristics such as intrinsic subtype, histological grade, lymphovascular invasion, and the Ki‐67 labeling index. An irregular mass shape and masses with a spiculated margin were significantly higher in the group of patients with luminal A breast cancer than in patients with masses that were lobular or round, or in tumors with an indistinct or microlobulated periphery (P = 0.017, P = 0.024, P < 0.001, and P = 0.001, respectively). Irregular mass shape and spiculated periphery were significantly lower in patients with Grade 3 cancer (P < 0.001 for both). In terms of lymphovascular invasion, there were significant differences between oval and irregular or round mass shape (P = 0.008 and P = 0.034), between tumors with a microlobulated and indistinct periphery (P = 0.014), between tumors with a punctate and amorphous or pleomorphic calcification shape (P = 0.030 and 0.038), and between the presence and absence of architectural distortion (P = 0.027). Equivalent or low‐density masses were also higher in Grade 1 breast cancers (P = 0.007). There were significant differences in the Ki‐67 labeling index between irregular and lobular or round tumors (P < 0.001 and P = 0.014), as well as between spiculated and indistinct or microlobulated tumors (P < 0.001 for both). Significant differences were noted in the mammographic features of different primary breast cancer subtypes. These proposed mammographic diagnostic criteria based on biological characteristics may contribute to a more accurate prediction of biological behavior of breast malignancies. (Cancer Sci 2011; 102: 2179–2185)

Hexokinase II in breast carcinoma: a potent prognostic factor associated with hypoxia-inducible factor-1α and Ki-67.

Hypoxia‐inducible factor‐1α (HIF‐1α) mediates adaptive responses to changes under tissue hypoxia in carcinoma cells by controlling the expression of various target genes. Previous studies have demonstrated that HIF‐1α is associated with adverse clinical outcome in breast carcinoma patients, but details of HIF‐1αs role have remained largely unknown. Therefore, in this study, we examined the expression profiles of HIF‐1α‐induced genes in 10 breast carcinoma cases using microarray data. As a result, we demonstrated that the status of hexokinase II (HKII) was associated with carcinoma recurrence in patients with these genes. The enzyme HKII is involved in the first, and rate‐limiting, step of glycolysis, but its clinical significance has not yet been examined in breast carcinoma. Therefore, we immunolocalized HKII in 118 breast carcinomas, and HKII immunoreactivity was detected in 44% of the cases. It is significantly associated with histological grade, Ki‐67 labeling index and HIF‐1α immunoreactivity. Also, HKII status is significantly associated with increased risk of recurrence and adverse clinical outcome in breast cancer patients. Subsequent multivariate analysis demonstrated that HKII status was an independent prognostic factor for disease‐free survival of patients. These results all suggest that HKII is induced by HIF‐1α and plays important roles in the proliferation and/or progression of breast carcinoma possibly through increased glycolytic activity. The status of HKII is therefore considered a potent prognostic factor in human breast cancer patients.

Proliferation and maturation of intratumoral blood vessels in non-small cell lung cancer

Non-small cell lung carcinoma is one of the most common leading causes of cancer mortality, and studying the features of intratumoral vessels, especially their generation and maturation, has become more important because of the recent application of antiangiogenic therapy. Vasohibin-1 has been recently considered one of the immunohistochemical markers for identifying neovascularization in archival materials. In addition, the functional maturation of blood vessels is considered to be related to pericyte formation around endothelial cells. Therefore, in this study, we evaluated the status of angiogenesis and maturation of intratumoral blood vessels in 93 patients with non-small cell lung carcinoma (50 with adenocarcinoma and 43 with squamous cell carcinoma) using immunohistochemistry of vasohibin-1, endoglin, CD31, and nestin. The vasohibin-1/CD31-positive ratio was significantly (P = .03) correlated with the Ki-67/CD31 ratio, confirming that the vasohibin-1/CD31-positive ratio represented the status of neovascularization in lung cancer. There were no statistically significant differences in vasohibin-1/CD31 ratios between adenocarcinoma and squamous cell carcinoma in both inner (P = .39) and outer areas (P = .36) of the tumor. The vasohibin-1/nestin-positive ratio, which represents the degrees of vascular maturation in proliferative vessels, was significantly lower in inner areas of adenocarcinoma (0.4 ± 0.1) than those in squamous cell carcinoma (0.8 ± 0.1) (P = .02). These results demonstrated that the degrees of maturation in newly formed blood vessels were less developed in inner areas of squamous cell carcinoma than adenocarcinoma, which may account partly for the complications of antivascular endothelial growth factor therapy more frequently detected in patients with squamous cell carcinoma.

Androgen receptor and enzymes in lymph node metastasis and cancer reoccurrence in triple-negative breast cancer.

Background Triple-negative breast cancer (TNBC) is characterized by the absence of estrogen receptor, progesterone receptor and HER2. TNBCs are a diverse subgroup, but one promising marker and therapeutic target of this breast cancer is the androgen receptor (AR). Previously we demonstrated that AR and cognate intracrine pathways are associated with decreased proliferation in invasive ductal carcinoma with their decrease also detected between organ-confined and invasive diseases. Therefore, in this study, we examined the status of AR and androgen-producing enzymes during the process of metastasis to lymph nodes and cancer recurrence. Materials and Methods We studied 2 series of patients with TNBC, one from Kumamoto University Hospital composed of 16 matched cases of primary and locally or distal recurrences and the other from Tohoku University Hospital examining 46 lymph node metastasis from 23 patients. In addition to studying concordance in AR expression, we also examined the interactions between AR and Ki-67 labeling index and AR and site of distal metastasis. Results In both series, AR status was concordant between primary and recurrent/metastatic disease, but coordinated expression of AR and androgenic enzymes was lost during the process. The inverse association between AR and Ki-67, previously reported in invasive ductal carcinoma (IDC), was markedly potentiated in both lymph node and recurrent cancers. In addition, AR expression appeared to have little effect on visceral metastasis but was associated directly with bone metastasis and inversely with brain metastasis. Conclusions The results of our present study demonstrated that AR remained in the majority of metastatic samples from AR-positive primary TNBCs and that AR manipulation could be exploited in the metastatic settings of TNBC.

A Non-invasive Modality: The US Virtual Touch Tissue Quantification (VTTQ) for Evaluation of Breast Cancer

OBJECTIVE We evaluated the biologic features of breast tissues using a newly developed non-invasive diagnostic system, named virtual touch tissue quantification. METHODS A total of 180 patients including 115 invasive ductal carcinoma, 30 ductal carcinoma in situ, 4 mucinous carcinoma, 7 invasive lobular carcinoma, 8 fibroadenoma, 12 fibrocystic change and 4 intraductal papilloma were studied at Nahanishi Clinic, Okinawa. We first compared the results of virtual touch tissue quantification according to each histologic subtype and determined the optimal cutoff values for virtual touch tissue quantification to distinguish benign from malignant tissues, using the receiver operating characteristic method. In addition, we also examined the correlation between virtual touch tissue quantification velocities and Ki-67, estrogen receptor, progesterone receptor or human epidermal growth factor receptor 2 in cases of invasive ductal carcinoma using linear regression analyses and Students t-test. RESULTS Virtual touch tissue quantification velocities were statistically higher in malignant cases than in benign cases (P < 0.05, respectively) and the best cutoff value for the virtual touch tissue quantification velocity which could differentiate benign from malignant cases was 2.89 m/s. There were statistically significant correlations between the virtual touch tissue quantification velocity and the Ki-67 labeling index (r = 0.338, r(2) = 0.114 and P < 0.001) and significant inverse correlations between virtual touch tissue quantification and the estrogen receptor (r = -0.311, r(2) = 0.097 and P < 0.001) or progesterone receptor (r = -0.361, r(2) = 0.131 and P < 0.001) status of invasive ductal carcinoma. There were also significant differences of the average velocities between human epidermal growth factor receptor 2-positive (6.39 ± 1.44 m/s) and -negative (4.43 ± 1.41 m/s) cases (P < 0.001). CONCLUSION Virtual touch tissue quantification could be a valuable clinical tool for estimating breast cancer pathology in a non-invasive fashion.