Interleukin (IL)-17A in triple-negative breast cancer: a potent prognostic factor associated with intratumoral neutrophil infiltration.

BACKGROUND: Triple-negative breast cancer (TNBC) is characterized as highly immunogenic and lacks specific targeted therapies. Interleukin 17A (IL-17A) is a controversial cytokine and is known to have anti-tumor and pro-tumor roles depending on the tumor microenvironment. In addition, IL-17A has been recently implicated in the recruitments of neutrophil into tumor tissues. Although IL-17A is considered tumor-promoting in breast cancer, its significance in the possible regulation of neutrophil infiltration in TNBC is not clearly defined. MATERIALS AND METHODS: We immunolocalized IL-17A, CD66b (neutrophil marker), and chemokine (C-X-C motif) ligand 1 (CXCL1, neutrophil chemoattractant) in 108 TNBC specimens and assessed their correlation among each other. The correlation between these markers and clinicopathological parameters was also assessed. We subsequently performed in vitro study to address the possible regulation of CXCL1 by IL-17A using TNBC cell lines, MDA-MB-231 and HCC-38. RESULTS: It was revealed that IL-17A correlated significantly with CXCL1 and CD66b, also CD66b with CXCL1. Furthermore, IL-17A was significantly associated with shorter disease-free and overall survival, especially in a high density CD66b group of patients. In vitro results revealed that IL-17A upregulated CXCL1 mRNA expression in a dose and time dependent manner, and this induction was significantly suppressed by an Akt inhibitor. CONCLUSION: IL-17A was considered to contribute to neutrophil infiltration by inducing CXCL1 in TNBC tissues and educating neutrophils to promote tumor progression. IL-17A might therefore serve as a potent prognostic factor in TNBC.

Heterogeneous Nuclear Ribonucleoprotein K Is Involved in the Estrogen-Signaling Pathway in Breast Cancer.

Heterogeneous nuclear ribonucleoprotein K (hnRNPK) transcripts are abundant in estrogen receptor (ER)- or progesterone receptor (PR)-positive breast cancer. However, the biological functions of hnRNPK in the ER-mediated signaling pathway have remained largely unknown. Therefore, this study analyzes the functions of hnRNPK expression in the ER-mediated signaling pathway in breast cancer. We initially evaluated hnRNPK expression upon treatment with estradiol (E2) and ICI 182,780 in the ERα-positive breast carcinoma cell line MCF-7. The results revealed that E2 increased hnRNPK; however, hnRNPK expression was decreased with ICI 182,780 treatment, indicating estrogen dependency. We further evaluated the effects of hnRNPK knockdown in the ER-mediated signaling pathway in MCF-7 cells using small interfering RNAs. The results revealed that hnRNPK knockdown decreased ERα expression and ERα target gene pS2 by E2 treatment. As hnRNPK interacts with several other proteins, we explored the interaction between hnRNPK and ERα, which was demonstrated using immunoprecipitation and proximity ligation assay. Subsequently, we immunolocalized hnRNPK in patients with breast cancer, which revealed that hnRNPK immunoreactivity was significantly higher in ERα-positive carcinoma cells and significantly lower in Ki67-positive or proliferative carcinoma cells. These results indicated that hnRNPK directly interacted with ERα and was involved in the ER-mediated signaling pathway in breast carcinoma. Furthermore, hnRNPK expression could be an additional target of endocrine therapy in patients with ERα-positive breast cancer.

The significance of lipid accumulation in breast carcinoma cells through perilipin 2 and its clinicopathological significance.

Both systemic and intratumoral lipid metabolism have been recently reported to play pivotal roles in both tumor development and progression in various human malignancies including breast cancer. However, its details have remained largely unknown in breast cancer patients. Therefore, in this study, we focused on perilipin 2, which is involved in constituting the intracellular lipid composition. Perilipin 2 was first immunolocalized in 105 cases of breast cancer. The status of perilipin 2 immunoreactivity was significantly positively associated with histological grade, Ki-67 labeling index and HER2 status and negatively with estrogen receptor status of these patients. Subsequent in vitro study also revealed that its mRNA expression in triple negative breast carcinoma cells was higher than cells of other subtypes. We then examined the correlation between perilipin 2 immunoreactivity and intracellular lipid droplet evaluated by Oil-red O stating in 13 cases of breast carcinoma tissues. A significantly positive correlation was detected between the status of perilipin 2 and Oil-red O staining. These findings above did indicate that perilipin 2 could represent the status of intracellular lipid droplets in surgical pathology specimens of breast cancer and perilipin 2 was also associated with its more aggressive biological phenotypes.

Diagnostic Accuracy of Positron Emission Mammography with 18F-fluorodeoxyglucose in Breast Cancer Tumor of Less than 20 mm in Size.

OBJECTIVES: To investigate the diagnostic accuracy of positron emission mammography (PEM) and positron emission tomography/computed tomography (PET/CT) for small breast tumors of less than 20 mm in size. METHODS: The study was conducted on a total of 100 subjects (i.e., 50 patients with pathologically proven breast cancer and 50 normal cases of medical screening). The total number of tumors was 54 (mean size: 11±5.1 mm, range: 4-20 mm). The diagnostic accuracy of PEM alone, PET/CT alone, and combined PET/CT and PEM was evaluated by two nuclear medicine physicians based on visual inspection. The two groups (i.e., tumors of ≤ 10 mm and > 10-20 mm) were compared in terms of the diagnostic capability of the three modalities (PEM alone, PET/CT alone, and PET/CT+PEM). RESULTS: The sensitivities of PEM alone, PET/CT alone, and combined PET/CT and PEM were 72%, 60%, and 76%, respectively. The specificities of these tests were 98%, 100%, and 98%, respectively. Furthermore, the accuracies of these diagnostic modalities were 85%, 79%, and 87%, respectively. The combined PET/CT and PEM showed significantly higher sensitivity and accuracy than PET/CT alone (P=0.005 and P=0.02, respectively). In addition, PEM demonstrated a significantly higher sensitivity than PET/CT in the ≤ 10 mm group (P=0.03); however, no difference was observed between the two modalities in the > 10 mm group in terms of sensitivity. CONCLUSION: 18F-fluorodeoxyglucose PET had limited capability for the detection of small breast cancers of < 10 mm. Combined PET/CT and PEM showed higher sensitivity and accuracy, compared to PET/CT alone.

Cancer stem-like properties of hormonal therapy-resistant breast cancer cells.

BACKGROUND: Presently, hormonal therapy targeting estrogen receptors is the most effective treatment available for luminal breast cancer. However, many patients relapse after the therapy. It has been suggested that cancer stem-like cells are involved with hormonal therapy resistance; in the present study, we evaluated this hypothesis. METHODS: In the present study, we used our previously established hormonal therapy-resistant cell lines, including aromatase inhibitor (AI)-resistant cells (Type 1 and Type 2) and fulvestrant-resistant cells (MFR). RESULTS: AI-resistant cell lines expressing ER (Type 1 V1 and V2) showed high cancer stemness in terms of their CD44/CD24 expression and side populations, which were stimulated by the addition of estrogen and inhibited by fulvestrant. However, ALDH activity was lower than in the ER-negative resistant cells, suggesting that the stemness of luminal cells is distinct from that of basal-like breast cancer cells. The migration and invasion activity of the ER-positive Type 1 V1 and V2 cells were higher than in the ER-negative cell lines, Type 2 and MFR. CONCLUSIONS: Fractionation of parental cells based on CD44/CD24 expression and colony formation assay indicated that CD44+/CD24+ cells might be the origin of hormonal therapy-resistant cells. This population reconstituted various other subpopulations under estrogen deprivation. These results indicate that hormonal therapy resistance is closely related to the cancer stem cell-like properties of luminal breast cancer.

Impact of Topoisomerase IIα, PTEN, ABCC1/MRP1, and KI67 on triple-negative breast cancer patients treated with neoadjuvant chemotherapy.

PURPOSE: Triple-negative breast cancer (TNBC) patients with residual disease following neoadjuvant chemotherapy (NAC) harbor higher risk of relapse, and eventual demise compared to those who achieve pathologic complete response. Therefore, in this study, we assessed a panel of molecules involved in key pathways of drug resistance and tumor progression before and after NAC in TNBC patients, in order to clarify the underlying mechanisms. METHODS: We studied 148 TNBC Japanese patients treated with anthracycline/taxane-based NAC. KI67, Topoisomerase IIα (TopoIIα), PTEN, p53, Bcl2, vimentin, ABCG2/BCRP1, ABCB1/MDR1, and ABCC1/MRP1 were immunolocalized in surgical pathology materials before and after NAC. RESULTS: The status of vimentin and increasing labeling index (LI) of TopoIIα and KI67 in biopsy specimens were significantly associated with those who responded to NAC treatment. The abundance of p53 (p = 0.003), ABCC1/MRP1 (p = 0.033), ABCB1/MDR1 (p = 0.022), and a loss of PTEN (p < 0.0001) in surgery specimens following treatment were associated with pathologic parameters. TopoIIα, PTEN, and ABCC1/MRP1 status predicted pathologic response. In addition, the status of PTEN, ABCC1/MRP1, ABCB1/MDR1, Bcl2, and vimentin in surgical specimens was also significantly associated with adverse clinicopathological factors in surgery specimens, suggesting that these alterations could be responsible for tumor relapse in TNBC patients. CONCLUSION: KI67, TopoIIα, PTEN, and ABCC1/MRP1 status could predict treatment response and/or eventual clinical outcomes. These results could also provide an insight into the mechanisms of drug resistance and relapse of TNBC patients receiving NAC.

The interaction between carcinoembryonic antigen-related cell adhesion molecule 6 and human epidermal growth factor receptor 2 is associated with therapeutic efficacy of trastuzumab in breast cancer.

Human epidermal growth factor receptor 2 (HER2) is a target of the HER2 inhibitor trastuzumab, which has been administered to HER2-positive breast cancer patients. However, the therapeutic effects of HER2 inhibitor monotherapy are not always clinically effective as compared with cotreatment with chemotherapy. Therefore, it has become pivotal to predict the therapeutic efficacy of trastuzumab monotherapy prior to administration. Recently, carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) has been reported to be a HER2-related factor. The aim of the present study was to explore the therapeutic mechanism of trastuzumab, including the relevance of CEACAM6 expression. CEACAM6/HER2-double-positive human breast carcinoma cell lines BT-474, HCC-1419 and MDA-MB-361 were used in this study. CEACAM6 knockdown decreased the inhibitory effects of trastuzumab in the trastuzumab-sensitive BT-474 and HCC-1419 cells, but not in the trastuzumab-resistant MDA-MB-361 cells. We examined the interaction between CEACAM6 and HER2 by using a proximity ligation assay (PLA). The interaction was detected in BT-474 and HCC-1419 cells, but not in MDA-MB-361 cells, and was significantly associated with in vitro trastuzumab therapeutic sensitivity. We further analysed the status of CEACAM6 and HER2 and their interaction in archival pathology specimens, also using PLA. The interaction was detected only in CEACAM6/HER2-double-positive breast cancer cases, and their PLA score was significantly associated with the efficacy of trastuzumab treatment. Therefore, evaluation of the CEACAM6-HER2 interaction could serve as a marker to predict the efficacy of trastuzumab monotherapy in breast cancer patients. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Newly-Developed Positron Emission Mammography (PEM) Device for the Detection of Small Breast Cancer.

Positron emission mammography (PEM) has higher detection sensitivity for breast cancer compared with whole-body positron emission tomography (PET) due to higher spatial resolution. We have developed a new PEM device with high resolution over a wide field of view. This PEM device comprises novel scintillation crystals, praseodymium-doped lutetium aluminum garnet (Pr:LuAG). In the present study, the clinical use of the newly developed PEM for the detection of small breast cancer was compared with that of the conventional PET-computed tomography (PET/CT). Eighty-two patients with breast cancer less than 20 mm (UICC T1) participated in this study, including 23 patients with T1a or T1b breast cancer (less than 10 mm). Histologically-proved lesions were examined by PET/CT and PEM on the same day after injection of [18F]fluoro-2-deoxy-2-fluoro-D-glucose ([18F]FDG), a marker of glycolytic activity. The newly developed PEM showed better sensitivity of cancer detection compared with PET/CT especially in case of the small T1a or T1b lesions. Moreover, when the conventional PET/CT and new PEM were combined, the detection sensitivity with [18F]FDG molecular imaging for T1 (N = 82) and T1a plus T1b breast cancer (N = 23) were 90% and 70%, respectively. The uptake of [18F]FDG was proportional to the histological malignancy of breast cancer. Using the newly-developed PEM with [18F]FDG, we are able to identify and characterize exactly the small breast tumors less than 10 mm in combination with the conventional PET/CT. These data indicate that PEM and PET/CT are synergic and complementary for the detection of small breast cancer.

Effect of the normal mammary differentiation regulator ELF5 upon clinical outcomes of triple negative breast cancers patients.

BACKGROUND: Elf5 is a transcription factor previously shown to be involved in regulating cell differentiation in both normal and pathological breast tissues. Pertinently, Elf5 was reported to interact with the FOXA1 transcription factor, a pivotal regulatory factor in a subset of AR overexpressing triple negative cancer (TNBC) cases. METHODS: We examined the correlation among AR, FOXA1, and Elf5 expression in a series of TNBC cases. The cases were retrieved from surgical pathological files of Tohoku University Hospital Japan and consisted of 60 cases operated between the year 1999 and 2007. An additional cohort cases of 51 TNBC ductal carcinoma in situ was used to compare invasive and non-invasive TNBC. RESULTS: In our cohort, 47% of all carcinomas were positive for Elf5, with a significantly higher proportion of Elf5 positive cases occurring in the younger age groups (p = 0.0061). Elf5 immunoreactivity was not associated with any other clinicopathological factors examined in this study. However, Elf5 expression was associated with decreased overall and disease-free survival of the patients (Peto-Peto modification of Gehan-Wilcoxon test, OS p = 0.132, DFS p = 0.1 (LI cutoff 10%); OS p = 0.038, DFS p = 0.021 (LI cutoff 50%)). Of particular interest, its effects on survival were more pronounced in the EGFR-/CK5/6- (non-basal surrogate) than the EGFR+ and/or CK5/6+ (basal-surrogate) subtype of TNBC. CONCLUSIONS: Elf5 is present in TNBC and its status was significantly correlated with overall survival of the patients. Further studies examining possible interactions between Elf5 and other factors in TNBC could contribute to disentangling TNBC biology.

Metastasis in the mandibular condyle: a case report.

BACKGROUND: Most bone metastases are observed in the trunk of the body. Metastasis in the mandibular condyle is rare. In many case reports, temporary common temporomandibular joint disorder-like symptoms can be a sign of relapse and metastasis. CASE PRESENTATION: We report a rare case of breast carcinoma metastatic to the left mandibular condyle in a 55-year-old Japanese woman, who visited our department for a dental check-up prior to chemotherapy. She had almost no symptoms, but radiographs suggested the existence of metastasis. CONCLUSIONS: In many case reports, patients had some symptoms. In this case report, our patient had slight symptoms, but we were able to confirm the metastasis from the symptoms and panoramic dental radiograph. When patients complain about discomfort of the temporomandibular joint, we need to consider the possibility of metastasis and notice changes on the panoramic dental radiograph.

Quantitative diagnostic imaging of cancer tissues by using phosphor-integrated dots with ultra-high brightness.

The quantitative sensitivity and dynamic range of conventional immunohistochemistry (IHC) with 3,3'-diaminobenzidine (IHC-DAB) used in pathological diagnosis in hospitals are poor, because enzyme activity can affect the IHC-DAB chromogenic reaction. Although fluorescent IHC can effectively increase the quantitative sensitivity of conventional IHC, tissue autofluorescence interferes with the sensitivity. Here, we created new fluorescent nanoparticles called phosphor-integrated dots (PIDs). PIDs have 100-fold greater brightness and a more than 300-fold greater dynamic range than those of commercially available fluorescent nanoparticles, quantum dots, whose fluorescence intensity is comparable to tissue autofluorescence. Additionally, a newly developed image-processing method enabled the calculation of the PID particle number in the obtained image. To quantify the sensitivity of IHC using PIDs (IHC-PIDs), the IHC-PIDs method was compared with fluorescence-activated cell sorting (FACS), a method well suited for evaluating total protein amount, and the two values exhibited strong correlation (R = 0.94). We next applied IHC-PIDs to categorize the response to molecular target-based drug therapy in breast cancer patients. The results suggested that the PID particle number estimated by IHC-PIDs of breast cancer tissues obtained from biopsy before chemotherapy can provide a score for predicting the therapeutic effect of the human epidermal growth factor receptor 2-targeted drug trastuzumab.

Minimal impact of postmastectomy radiation therapy on locoregional recurrence for breast cancer patients with 1 to 3 positive lymph nodes in the modern treatment era.

INTRODUCTION: Given modern treatment strategies, controversy remains regarding whether postmastectomy radiation therapy (PMRT) is necessary for breast cancer patients with 1-3 positive axillary lymph nodes (ALN). Our aim was to assess the significance of PMRT in the modern treatment era for these patients. MATERIAL AND METHODS: We have conducted the retrospective multicenter study and identified 658 patients with 1-3 positive ALN who were treated with mastectomy and ALN dissection between 1999 and 2012. Propensity score weighting was used to minimize the influence of confounding factors between the PMRT and no-PMRT groups. The variables including tumor size, lymph nodes status, skin and/or muscle invasion, histological grade, lymphovascular invasion and ER positivity which were statistically unbalanced between the groups were used to define the propensity scores. RESULTS: The median follow-up time was 7.3 years. In the modern era (2006-2012), no significant difference in locoregional recurrence (LRR)-free survival was noted between the PMRT and no-PMRT groups (P = 0.3625). The 8-year LRR-free survival rates of the PMRT and no-PMRT groups were 98.2% and 95.3%, respectively. After matching patients by propensity scores, the PMRT group, compared to the no-PMRT group, exhibited significantly better locoregional control (P = 0.0366) in the entire cohort. The 10-year LRR-free survival rates were 97.8% and 88.4% in the PMRT and no-PMRT groups, respectively. In contrast, no significant difference in LRR-free survival was noted between the PMRT and no-PMRT groups in the modern era (P = 0.5298). The 8-year LRR-free survival rates of patients treated in the modern era were approximately the same between the groups (98.0% and 95.7% in the PMRT and no-PMRT groups, respectively). Particularly, LRR-free survival of HER2 positive breast cancer significantly improved in the modern treatment era, compared with that of the old treatment era (P = 0.0349). CONCLUSION: PMRT had minimal impact on LRR for breast cancer patients with 1-3 positive ALN in the modern treatment era.

In situ detection of estrogen receptor dimers in breast carcinoma cells in archival materials using proximity ligation assay (PLA).

Estrogen receptor (ER) is required for carcinoma cell proliferation in the great majority of breast cancer and also functions as a dimer. ER dimeric proteins have been largely identified by BRET/FRET analyses but their in situ visualization have not yet been reported. Recently, in situ Proximity Ligation Assay (PLA) has been developed as the methods detecting protein interactions in situ. Therefore, in this study we firstly demonstrated the dimerization of ERα in breast carcinoma cell lines and tissues using PLA. The human breast carcinoma cell lines MCF-7, T-47D and MDA-MB-231 were used in this study. Cells were treated with ER agonist or antagonist and fixed in 4% PFA, and ER dimers were subsequently detected using PLA. The evaluation of ER dimers in breast carcinoma cell lines were quantified by measuring the area of dots localized in the nuclei using image analysis. We also firstly demonstrated the visualization of ER dimer patterns in 10% formalin-fixed paraffin-embedded tissues of breast cancer using PLA technique. Estradiol (E2) administration induced ERα homodimers in the nuclei of MCF-7 and T-47D but not in ER-negative MDA-MB-231. 4-OH tamoxifen also induced ERα homodimers but the subcellular localization of these ERα homodimers was predominant in cytoplasm instead of the nuclei induced by E2 treatment. ICI182,780 treatment did decrease the number of formation of ERα homodimers in MCF-7. In breast cancer patients, ERα PLA score was significantly correlated positively with ERα- or PgR (progesterone receptor) immunohistochemical scores and inversely with Ki-67-labeling index, respectively. We also demonstrated the ERα/ß heterodimer as well as ERα homodimers in both breast carcinoma cell lines and surgical pathology specimens. In summary, we did firstly succeed in the visualization of ER dimeric proteins using PLA method. The evaluation of ER dimer patterns could provide pivotal information as to the prediction of response to endocrine therapy of breast cancer patients.

The presence and impact of estrogen metabolism on the biology of triple-negative breast cancer.

PURPOSE: While triple-negative breast cancer (TNBC) is negative for estrogen receptor alpha, a substantial proportion of carcinomas express estrogen receptor beta (ERß); consequently, estrogen actions and metabolism may be relevant in this cancer subtype. METHODS: A cohort of 81 TNBC patients from Tohoku University Hospital, Japan were characterised with regard to the expression of estrogen receptor beta and enzymes known to modulate levels of estrogens in breast and other tissues (Aromatase, 17-beta- Hydroxysteroid dehydrogenases 1, 2 and 6). This was done at the protein level by means of immunohistochemistry. As this cohort has been previously characterised for androgens, this also allows for comparison between the expressions of estrogen-related proteins and of androgen-related proteins. Preliminary mechanistic studies in cell culture were also undertaken. RESULTS: 17ßHSD2 was detected in the highest number of cases followed by 17ßHSD1, 17ßHSD6 and aromatase. When comparing the expression of ERß with that of the enzymes, it was positively correlated with the expression of 17ßHSD6 (p < 0.05) and trended towards correlation with dual expression of 17ßHSD1 and 2 (p < 0.07). 17ßHSD1 was associated with significantly reduced tumour volume (p = 0.0025), while ERß was associated with a trend towards reduced lymphovascular invasion, (p < 0.061). Interestingly, in survival analysis, 17ßHSD6 expression was the only one of these five factors that influenced survival, with positive samples being associated with longer disease-free survival compared to those that were negative for 17ßHSD6 (p < 0.05). In assessing associations with expression of proteins in the androgenic pathway, expression of aromatase appeared to be associated with androgenic pathways in TNBC patients (p < 0.05). Due to this association and the potential relevance to androgen-directed therapies in TNBC, we evaluated this interaction in vitro. We observed androgen-dependent upregulation of aromatase and ERß in a subset of AR expressing TNBC cell lines (MDA-MB-453, SUM-185-PE and MFM-223). CONCLUSION: Overall this study suggests the presence of, and a potential protective effect of estrogens in TNBC.

Intratumoral estrogen production and actions in luminal A type invasive lobular and ductal carcinomas.

The great majority of invasive lobular carcinoma (ILC) is estrogen-dependent luminal A type carcinoma but the details of estrogen actions and its intratumoral metabolism have not been well studied compared to invasive ductal carcinoma (IDC). We first immunolocalized estrogen-related enzymes including estrogen sulfotransferase (EST), estrogen sulfatase (STS), 17ß-hydroxysteroid dehydrogenase (HSD) 1/2, and aromatase. We then evaluated the tissue concentrations of estrogens in ILC and IDC and subsequently estrogen-responsive gene profiles in these tumors in order to explore the possible differences and/or similarity of intratumoral estrogen environment of these two breast cancer subtypes. The status of STS and 17ßHSD1 was significantly lower in ILCs than IDCs (p = 0.022 and p < 0.0001), but that of EST and 17ßHSD2 vice versa (p < 0.0001 and p = 0.0106). In ILCs, tissue concentrations of estrone and estradiol were lower than those in IDCs (p = 0.0709 and 0.069). In addition, the great majority of estrogen response genes tended to be lower in ILCs. Among those genes above, FOXP1 was significantly higher in ILCs than in IDCs (p = 0.002). FOXP1 expression was reported to be significantly higher in relapse-free IDC patients treated with tamoxifen. Therefore, tamoxifen may be considered an option of endocrine therapy for luminal A type ILC patients. This is the first study to demonstrate the detailed and comprehensive status of intratumoral production and metabolism of estrogens and the status of estrogen response genes in luminal A-like ILC with comparison to those in luminal A-like IDCs.

Tumor microenvironment in invasive lobular carcinoma: possible therapeutic targets.

Invasive ductal and lobular carcinomas (IDC and ILC) are the two most common histological types of breast cancer, and have been considered to develop from terminal duct lobular unit but their molecular, pathological, and clinical features are markedly different between them. These differences could be due to different mechanisms of carcinogenesis and tumor microenvironment, especially cancer-associated fibroblasts (CAFs) but little has been explored in this aspect. Therefore, in this study, we evaluated the status of angiogenesis, maturation of intratumoral microvessels, and proliferation of CAFs using immunohistochemistry and PCR array analysis to explore the differences of tumor microenvironment between ILC and IDC. We studied grade- and age-matched, luminal-like ILC and IDC. We immunolocalized CD34 and αSMA for an evaluation of CAFs and CD31, Vasohibin-1, a specific marker of proliferative endothelial cells and nestin, a marker of pericytes for studying the status of proliferation and maturation of intratumoral microvessel. We also performed PCR array analysis to evaluate angiogenic factors in tumor stromal components. The number of CAFs, microvessel density, and vasohibin-1/CD31 positive ratio were all significantly higher in ILC than IDC but nestin immunoreactivity in intratumoral microvessel was significantly lower in ILC. These results did indicate that proliferation of CAFs and endothelial cells was more pronounced in ILC than IDC but newly formed microvessels were less mature than those in IDC. PCR array analysis also revealed that IGF-1 expression was higher in ILC than IDC. This is the first study to demonstrate the differences of tumor microenvironment including CAFs and proliferation and maturation of intratumoral vessels between ILC and IDC.

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.

INTRODUCTION: The 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). METHODS: One 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. RESULTS: TNBC 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). CONCLUSIONS: This 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.

11ß-Prostaglandin F2α, a bioactive metabolite catalyzed by AKR1C3, stimulates prostaglandin F receptor and induces slug expression in breast cancer.

Prostaglandins are a group of lipid compounds involved in inflammation and cancer. We focused on PGF2α and its stereoisomer 11ß-PGF2α and examined the expression and functions of their cognate receptor (FP receptor) and metabolizing enzymes (AKR1B1 and AKR1C3 respectively) in breast cancer. In immunohistochemical analysis FP receptor status associated with adverse clinical outcome only in the AKR1C3 positive cases. Therefore, we studied FP receptor-mediated functions of 11ß-PGF2α using FP receptor expressed MCF-7 cell line (MCF-FP). 11ß-PGF2α treatment phosphorylated ERK and CREB and induced Slug expression through FP receptor in MCF-FP, and MCF-FP cells demonstrated decreased chemosensitivity compared to parental controls. Finally, the correlation between FP receptor and Slug was also confirmed immunohistochemically in breast cancer cases. Overall these results indicated that the actions of AKR1C3 can produce FP receptor ligands whose activation results in carcinoma cell survival in breast cancer.

Correlation of miRNA expression profiling in surgical pathology materials, with Ki-67, HER2, ER and PR in breast cancer patients.

BACKGROUND: New molecular markers related to prognosis and/or clinical outcome have been extensively studied in breast cancer. In particular, microRNA (miRNA) has attracted the interest of both basic and clinical investigators as one of the promising molecular markers of breast cancer patients. MiRNAs are a class of short noncoding RNAs that regulate mRNAs at posttranscriptional level and are deregulated in various human malignancies. Previous studies have reported that miRNAs were stably conserved in 10% formalin-fixed paraffin-embedded tissues without significant degradation, in contrast to more fragile RNA. METHODS: Therefore, in this study, we examined 21 surgical breast cancer specimens using the Human Cancer microRNA PCR Array system (QIAGEN) to explore potential molecular targets of miRNAs. RESULTS: Profiling of miRNA expression in archival materials demonstrated that a group of deregulated miRNAs was associated with clinicopathological parameters of the patients, such as Ki-67, HER2, ER and PR. For instance, an abundant expression of multiple let-7 miRNA family, also known as tumor suppressor, was detected in low Ki-67 and HER2 groups. Elevated expression of 8 miRNAs overlapped between Ki-67+/HER2+/ER+/PR+ groups, including several known oncogenic miRNAs such as miR-148b, miR-15b, miR-200c, miR-150, miR-191, miR-96, miR-25 and miR-21. CONCLUSIONS: These results all indicated that when analyzing miRNAs in surgical pathology specimens of breast cancer as a biomarker, they should be examined as a cluster through miRNA profiling, rather than relying on the analysis of a single miRNA.

Immunolocalization of thymidylate synthase as a favorable prognostic marker in estrogen receptor-positive breast carcinoma.

BACKGROUND: Thymidylate synthase (TS) is an enzyme involved in DNA synthesis, and it is a target for 5-fluorouracil. Previous studies have demonstrated that TS is a potent estrogen-induced gene in breast carcinoma cells, suggesting the importance of TS in estrogen-receptor (ER)-positive breast carcinoma. TS immunolocalization has been reported previously, but the clinicopathological significance of TS in ER-positive breast carcinoma still remains unclear. PATIENTS AND METHODS: We immunolocalized TS in 178 breast carcinoma tissues in total, and examined its significance according to the ER-status. RESULTS: TS status was positive in 58% of ER-positive ductal carcinoma in situ (DCIS) cases, and it was significantly associated with the Ki-67 and progesterone receptor (PR). Moreover, in ER-positive DCIS patients who received aromatase inhibitor (AI) before surgery, TS immunoreactivity was significantly decreased after AI treatment. In ER-positive invasive ductal carcinoma (IDC) cases, TS status was significantly associated with PR, and it turned out an independent favorable prognostic factor for recurrence of the patients by multivariate analysis. On the other hand, TS status was positively correlated with pathological T factor in ER-negative IDC cases, and tended to have a worse prognosis for disease-free survival of the patients. CONCLUSION: These results suggest that TS expression is mainly regulated by estrogen in ER-positive breast carcinoma and is associated with estrogen-mediated proliferation. TS status is a favorable prognostic factor in ER-positive IDC patients, which is different from the ER-negative cases.