Takanori Ishida

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.

Luminal-type breast cancer: correlation of apparent diffusion coefficients with the Ki-67 labeling index.

PURPOSEnTo evaluate the correlation between apparent diffusion coefficient ( ADC apparent diffusion coefficient ) values and the Ki-67 labeling index for luminal-type (estrogen receptor-positive) breast cancer not otherwise specified ( NOS not otherwise specified ) diagnosed by means of biopsy.nnnMATERIALS AND METHODSnThe institutional review board approved this retrospective study, and the requirement for informed consent was waived. Between December 2009 and December 2012, 86 patients with 86 lesions with luminal-type invasive breast cancer NOS not otherwise specified underwent magnetic resonance imaging, including dynamic contrast material-enhanced imaging and diffusion-weighted imaging with b values of 0 and 1000 sec/mm(2). Conventional measurement of the minimum and mean ADC apparent diffusion coefficient s by placing regions of interest and histogram analysis of pixel-based ADC apparent diffusion coefficient data of the entire tumor were performed by two observers independently and correlated with the Ki-67 labeling index of surgical specimens.nnnRESULTSnFor the interobserver reliability, interclass correlation coefficients for all parameters with the exception of the minimum ADC apparent diffusion coefficient exceeded 0.8, indicating almost perfect agreement. The minimum ADC apparent diffusion coefficient and mean ADC apparent diffusion coefficient and the 25th, 50th, and 75th percentiles of the histograms showed negative correlations with the Ki-67 labeling index (r = -0.49, -0.55, -0.54, -0.53, and -0.48, respectively). Receiver operating characteristic curve analysis for the differential diagnosis between the high-proliferation (Ki-67 ≥ 14; n = 44) and low-proliferation (Ki-67 < 14; n = 42) groups revealed that the most effective threshold for the mean ADC apparent diffusion coefficient was lower than 1097 × 10(-6) mm(2)/sec, with sensitivity and specificity of 82% and 71%, respectively. The area under the receiver operating characteristic curve (AUC) was 0.81 for the mean ADC apparent diffusion coefficient . There were no significant differences in the AUC among the parameters.nnnCONCLUSIONnConsidering convenience for routine practice, the authors suggest that the mean ADC apparent diffusion coefficient of the conventional method would be practical to use for estimating the Ki-67 labeling index.

Tumor-infiltrating CD8+ and FOXP3+ lymphocytes in triple-negative breast cancer: its correlation with pathological complete response to neoadjuvant chemotherapy

The anti-tumor immune response was recently reported to play a critical role in the chemotherapeutic sensitivity of breast cancer. Therefore, we investigated the correlation between CD8+ and FOXP3+ tumor-infiltrating lymphocytes and the pathological complete response (pCR) following neoadjuvant chemotherapy (NAC) in triple-negative breast cancer (TNBC), in conjunction with neoangiogenesis, basal and proliferation markers. CD8+ and FOXP3+ lymphocytes were assessed in biopsy specimens by double-staining immunohistochemistry, in combination with immunostaining of vasohibin-1, CD31, EGFR, CK5/6, and Ki-67. Earlier age, pre-menopausal status, smaller tumor size, and high Ki-67 were significantly associated with pCR, as in high CD8+, high CD8+/FOXP3+ ratio, and low vasohibin-1 positive ratio. Multivariate analysis did reveal that a high CD8+/FOXP3+ ratio was a strong predictor of pCR with an odds ratio of 5.32 (Pxa0=xa00.005). High Ki-67 was also significantly associated with pCR (Pxa0=xa00.002). TNBCs with a high CD8+/FOXP3+ ratio and high Ki-67 had the highest pCR rate (70xa0%) following NAC. However, the pCR rate of the patients with low CD8+/FOXP3+ ratio and low Ki-67 was only 5xa0%. The pCR rates of a high CD8+/FOXP3+ ratio and low Ki-67 patients and those with a low CD8+/FOXP3+ ratio and high Ki-67 were 24 and 21xa0%, respectively. TNBCs with a high CD8+/FOXP3+ ratio were more sensitive to anthracycline and taxane-based chemotherapeutic regimens, and the CD8+/FOXP3+ ratio in conjunction with Ki-67 could predict pCR following NAC in TNBC. This predictor may represent a new surrogate for testing the efficacy of investigational agents in the neoadjuvant setting.

Increased androgen receptor activity and cell proliferation in aromatase inhibitor-resistant breast carcinoma

Aromatase inhibitors (AI) are commonly used to treat postmenopausal estrogen-receptor (ER)-positive breast carcinoma. However, resistance to AI is sometimes acquired, and the molecular mechanisms underlying such resistance are largely unclear. Recent studies suggest that AI treatment increases androgen activity during estrogen deprivation in breast carcinoma, but the role of the androgen receptor (AR) in breast carcinoma is still a matter of controversy. The purpose of this study is to examine the potential correlation between the AR- and AI-resistant breast carcinoma. To this end, we performed immunohistochemical analysis of 21 pairs of primary breast carcinoma and corresponding AI-resistant recurrent tissue samples and established two stable variant cell lines from ER-positive T-47D breast carcinoma cell line as AI-resistance models and used them in in vitro experiments. Immunohistochemical analysis demonstrated that the expression of prostate-specific antigen (PSA) and Ki-67 were significantly higher and ER and progesterone receptor (PR) were lower in recurrent lesions compared to the corresponding primary lesions. Variant cell lines overexpressed AR and PSA and exhibited neither growth response to estrogen nor expression of ER. Androgen markedly induced the proliferation of these cell lines. In addition, the expression profile of androgen-induced genes was markedly different between variant and parental cell lines as determined by microarray analysis. These results suggest that in some cases of ER-positive breast carcinoma, tumor cells possibly change from ER-dependent to AR-dependent, rendering them resistant to AI. AR inhibitors may thus be effective in a selected group of patients.

Androgenic pathways in the progression of triple-negative breast carcinoma: a comparison between aggressive and non-aggressive subtypes

One of the active intracellular pathways/networks in triple-negative breast carcinoma (TNBC) is that of the androgen receptor (AR). In this study, we examined AR and androgen-metabolising enzyme immunoreactivity in subcategories of TNBC to further elucidate the roles of androgenic pathways in TNBC. We utilised formalin-fixed paraffin-embedded breast cancer samples from ductal carcinoma in situ (DCIS) and invasive ductal carcinoma patient cohorts. We then used immunohistochemistry to classify these samples into basal-like and non-basal samples and to assess interactions between AR, androgen-metabolising enzymes and proliferation. To further substantiate our hypothesis and provide mechanistic insights, we also looked at the expression and regulation of these factors in publically available microarray data and in a panel of TNBC AR-positive cell lines. DCIS was associated with higher levels of AR and enzymes (pxa0<xa00.02), although a similar difference was not noticed in basal and non-basal samples. AR and enzymes were correlated in all states. In TNBC cell lines (MDA-MD-453, MFM-223 and SUM185-PE), we found that DHT treatment up-regulated 5αR1 and 17βHSD5 suggesting a mechanistic explanation for the correlations observed in the histological samples. Publicly available microarray data in TNBC cases suggested similar patterns to those observed in histological samples. In the majority of settings, including publically available microarray data, an inverse association between AR and proliferation was detected. These findings suggest that decreases in AR and androgen-metabolising enzymes may be involved in the increased biological aggressiveness in TNBC development.

Aryl Hydrocarbon Receptor in Breast Cancer—A Newly Defined Prognostic Marker

Aryl hydrocarbon receptor (AhR) has been reported to exert various anticancer effects upon breast carcinoma cells in vitro but its details have remained largely unknown. Therefore, we first examined the AhR status in 90 invasive ductal carcinoma patients using immunohistochemistry. We then performed in vitro studies including wound healing assay, invasion assay, and matrix metalloproteinase (MMP) protein array in order to further elucidate the roles of AhR signaling in breast carcinoma. The status of AhR immunoreactivity was inversely correlated with histological grade (Pu2009=u20090.0135) and Ki-67 labeling index (LI; Pu2009=u20090.0087) of the patients. In addition, results of both uni- and multivariate analyses revealed that AhR in carcinoma cells turned out an independent prognostic factor with a protective relative risk (Pu2009=u20090.0179). An administration of 10xa0nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a ligand of AhR, significantly decreased Ki-67 LI in an AhR-dependent fashion in MCF-7, T47D, ZR75-1, and MDA-MB-231. Wound healing and invasion assays performed in T47D and ZR75-1 further demonstrated that 10xa0nM TCDD inhibited estrogen-induced migration and invasion of cells. MMP proteins associated with AhR in breast carcinoma cells were also firstly identified. These results demonstrated that AhR in breast carcinoma cells is considered a newly defined histological prognostic parameter of the breast cancer patients and effects of AhR activation on proliferation and MMPs expression may be related to the relatively good clinical outcome of AhR-positive breast cancer patients.

A Randomized Controlled Trial to Verify the Efficacy of the Use of Ultrasonography in Breast Cancer Screening Aged 40–49 (J-START): 76 196 Women Registered

OBJECTIVEnThe objective of the Japan Strategic Anti-cancer Randomized Trial was to verify the efficacy of the use of ultrasonography in breast cancer screening among women aged 40-49 years. The purpose of this paper was to report the design and recruitment result of this study.nnnMETHODSnIn this study of women in their 40s, the participants were divided into two groups, one of which (the intervention group) was subjected to mammography and ultrasonography (using a standardized ultrasonography examination), while the other (the control group) was examined with mammography, in a randomized controlled trial, with the objective of verifying the accuracy and efficacy of examinations by comparing the two groups.nnnRESULTSnThe cumulative total number of participants registered in the study was 76 196 (38 313 in the intervention group and 37 883 in the control group). 71.0% of participants registered to the study were under individual randomized controlled trial, 25.0% were under cluster randomized controlled trial and 3.9% were under non-randomized controlled group. The study was designed so that participants registered at their first examination underwent examinations by the same method for the subsequent two years. 74.1% of participants scheduled for a second examination had undertaken it, while information regarding the presence of interval cancer had been obtained from a further 20.6% using a questionnaire. At July 2013, the status of 5.3% of all participants was unclear.nnnCONCLUSIONSnIt was the first large-scale randomized controlled trial carried out in Japan. The scheduled second examinations were completed at the end of fiscal 2012. Once the proportion of participants whose status is unclear has fallen to ≤5%, the authors plan to collate the data relating to the primary end points, and publish the results.

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.

Controversies in Breast Cancer Screening for Women Aged 40–49 Years

Mammography is the only method of breast cancer screening that has established evidence of a mortality reduction. However, mammography does not achieve sufficient accuracy in the high-density breasts of patients<50 years of age. In 2009, the US Preventive Services Task Force revised its recommendation for breast cancer screening in women aged 40-49 years from Grade B to C because the net benefit was relatively small for this age bracket. The net benefit of screening is the sum of benefits and harm and should always be monitored especially in population screening. A high recall rate, an inefficient number needed to invite for screening to prevent one breast cancer death, a high false-positive rate and unnecessary additional imaging for women aged 40-49 years are great concerns of mammography screening. Overdiagnosis is also a detriment of mammography screening; however, it may have a limited effect on women aged 40-49 years. Establishment of new evidence for breast cancer screening, such as ultrasonography screening, is needed in order to create a more effective screening system.

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

OBJECTIVEnWe evaluated the biologic features of breast tissues using a newly developed non-invasive diagnostic system, named virtual touch tissue quantification.nnnMETHODSnA 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.nnnRESULTSnVirtual 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).nnnCONCLUSIONnVirtual touch tissue quantification could be a valuable clinical tool for estimating breast cancer pathology in a non-invasive fashion.