Using the DNA Integrity Number (DIN) to analyze DNA quality in specimens collected from liquid-based cytology after fine needle aspiration of breast tumors and lesions.

BACKGROUND: Cancer genome analysis using next-generation sequencing requires adequate and high-quality DNA samples. Genomic analyses were conventionally performed using formalin-fixed paraffin-embedded (FFPE) sections rather than cytology samples such as cell block or smear specimens. Specimens collected from liquid-based cytology (LBC) have the potential to be sources of high-quality DNA suitable for genetic analysis even after long-term storage. METHODS: We collected breast tumor/lesion fractions from 92 residual LBC specimens using fine needle aspiration (FNA) biopsy, including breast carcinoma (1 invasive carcinoma and 4 ductal carcinomas in situ), papillomatous lesion (5 intraductal papillomas), and fibroepithelial lesion (19 phyllodes tumors and 53 fibroadenomas) samples, and others (1 ductal adenoma, 1 hamartoma, 1 fibrocystic disease, and 7 unknown). DNA was extracted from all samples and subjected to DNA Integrity Number (DIN) score analysis. RESULTS: Average DIN score collected from 92 LBC specimens was significantly higher score. In addition, high-quality DNA with high DIN values (7.39 ± 0.80) was successfully extracted more than 12 months after storage of residual LBC specimens. CONCLUSION: Residual LBC specimens collected from FNA of the breast were verified to carry high-quality DNA and could serve as an alternate source for genetic analysis.

The Clinicopathological and Prognostic Significance of HER2-Low Breast Cancer: A Comparative Analysis Between HER2-Low and HER2-Zero Subtypes.

BACKGROUND: HER2-low breast cancer (BC) is a newly defined subset of HER2-negative BC. However, it is still uncertain whether HER2-low BC can be categorized as a distinct biological/clinical subgroup with any prognostic significance. METHODS: Invasive BC cases (n = 10,215) with Stage I-III were retrospectively analyzed to determine the HER2 status. The HER2 status was then divided into 3 groups: HER2-0, HER2-low, and HER2-positive. RESULTS: The HER2 status was classified as HER2-0 in 1,227 cases (12.0%), HER2-low in 7,209 cases (70.6%), and HER2-positive in 1779 cases (17.4%). HER2-low cases had more positive nodes and were significantly associated with positive ER/PgR, lower nuclear grade, and lower Ki-67 index. HER2-0 had the lowest OS rate in the primary cases and after recurrence. HER2-0 in the node positive group had the lowest OS and was significantly different from HER2-low in the same group. The pathological complete response (pCR) rate for NAC was lowest in the HER2-low group. The DFS after NAC was significantly better in all the pCR cases, regardless of the HER2 status. However, the DFS was significantly lower in the HER2-low non-pCR cases. CONCLUSION: HER2-low accounted for 70% of the cases and correlated with favorable biological markers. The HER2-low group had a significantly better OS than the HER2-0 group. However, the response to NAC was low in the HER2-low group, and this group had the poorest prognosis among all the non-pCR cases. These findings indicate that HER2-low may have a different biology and prognosis and therefore should be classified as a new entity.

Potential role of Fbxo22 in resistance to endocrine therapy in breast cancer with invasive lobular carcinoma.

BACKGROUND: Invasive lobular carcinoma (ILC) is distinct from invasive ductal carcinoma (IDC) in terms of their hormonal microenvironments that may require different therapeutic strategies. We previously reported that selective estrogen receptor modulator (SERM) function requires F-box protein 22 (Fbxo22). Here, we investigated the role of Fbxo22 as a potential biomarker contributing to the resistance to endocrine therapy in ILC. METHODS: A total of 302 breast cancer (BC) patients including 150 ILC were recruited in the study. Fbxo22 expression and clinical information were analyzed to elucidate whether Fbxo22 negativity could be a prognostic factor or there were any correlations among clinical variables and SERM efficacy. RESULTS: Fbxo22 negativity was significantly higher in ILC compared with IDC (58.0% vs. 27.0%, P < 0.001) and higher in postmenopausal patients than premenopausal patients (64.1% vs. 48.2%, P = 0.041). In the ILC cohort, Fbxo22-negative patients had poorer overall survival (OS) than Fbxo22-positive patients, with 10-year OS rates of 77.4% vs. 93.6% (P = 0.055). All patients treated with SERMs, Fbxo22 negativity resulted in a poorer outcome, with 10-year OS rates of 81.3% vs. 92.3% (P = 0.032). In multivariate analysis regarding recurrence-free survival (RFS) in ILC patients, Fbxo22 status was independently predictive of survival as well as lymph node metastasis. CONCLUSION: Fbxo22 negativity significantly impacts on survival in BC patients with IDC and ILC, and the disadvantage was enhanced among ILC postmenopausal women or patients treated with SERMs. The findings suggest that different therapeutic strategies might be needed according to the different histopathological types when considering adjuvant endocrine therapy.

Long-term prognostic value of the GenesWell BCT score in Asian women with hormone receptor-positive/HER2-negative early breast cancer.

BACKGROUND: Accurate prediction of the risk of recurrence is crucial for optimal treatment decisions in hormone receptor (HR)-positive/human epidermal growth factor receptor 2 (HER2)-negative early breast cancer. The GenesWell BCT is a molecular assay to predict the 10-year risk of distant metastasis. In this study, we evaluated the long-term prognostic value of the GenesWell BCT assay. METHODS: The BCT score was assessed in patients with HR-positive/HER2-negative early breast cancer who did not receive chemotherapy. We compared the 15-year distant metastasis-free survival (DMFS) between risk groups classified based on the BCT score. The risk of early (0-5 years) and late (5-15 years) recurrence was evaluated based on the BCT score classification. RESULTS: According to the BCT score, 366 patients from Japan and Korea were categorized as BCT low risk (83.6%) and high risk (16.4%) for distant metastasis. Median follow-up time was 17.4 years. The 15-year DMFS rate was significantly lower in the BCT high-risk group (63.3%) than in the BCT low-risk group (93.6%) (P < 0.001). The BCT risk group was an independent prognostic factor for 15-year DMFS (hazard ratio, 4.59; 95% confidence interval 2.13-9.88; P < 0.001). Furthermore, the BCT score was a significant predictor of late recurrence (5-15 years) in patients aged ≤ 50 years and those aged > 50 years, and added prognostic information to traditional clinical prognostic factors. CONCLUSION: The BCT score can identify patients at low risk for recurrence who may not require adjuvant chemotherapy or extended endocrine therapy, regardless of age.

Evaluation of RASSF1A methylation in the lysate of sentinel lymph nodes for detecting breast cancer metastasis: A diagnostic accuracy study.

The restriction enzyme-based digital methylation-specific polymerase chain reaction (RE-dMSP) assay is useful for diagnosing sentinel lymph node (SN) metastasis in patients with breast cancer, by detecting tumor-derived methylated Ras association domain-containing protein 1 (RASSF1A). In addition, this assay has high concordance (95.0%) with one-step nucleic acid amplification (OSNA). The present study aimed to perform RE-dMSP using OSNA lysate from more patients and to re-evaluate its clinical usage. Overall, 418 SNs from 347 patients were evaluated using both OSNA and RE-dMSP. The concordance rate was 83.3% (348/418). RASSF1A methylation of the primary tumors was negative in 36 patients. When these patients were excluded, the concordance rate improved to 88.2% (330/374). Of the 79 OSNA-negative cases, 19 were RE-dMSP-positive, although all were positive for cytokeratin 19 expression in the primary tumor, suggesting that RE-dMSP can detect tumor-derived DNA with a higher sensitivity. The percent of methylated reference of the breast tumors showed a wide variety in the 16 OSNA-positive/RE-dMSP-negative cases, and such variability of methylation could have affected the results in these patients. In conclusion, although RE-dMSP can diagnose SN metastasis with high sensitivity and accuracy, and can be a supplementary tool to OSNA in breast cancer, RE-dMSP showed certain discordance with OSNA and critically depended on the absence or heterogeneity of DNA methylation in breast tumors. Further research is expected to develop an assay targeting other DNA alterations, such as mutations.

Induction of SGK1 via glucocorticoid-influenced clinical outcome of triple-negative breast cancer patients.

PURPOSE: Triple-negative breast cancer (TNBC) is a highly heterogeneous and aggressive breast malignancy. Glucocorticoid (GC)-glucocorticoid receptor (GR) pathway plays a pivotal role in the cellular responses to various stresses including chemotherapy. Serum- and glucocorticoid-induced kinase-1 (SGK1) is known as an important downstream effector molecule in the GR signaling pathway, we attempted to explore its clinicopathological and functional significance in TNBC in which GR is expressed. METHODS: We first immunolocalized GR and SGK1 and correlated the results with clinicopathological variables and clinical outcome in 131 TNBC patients. We also evaluated the effects of SGK1 on the cell proliferation and migration in TNBC cell lines with administration of dexamethasone (DEX) to further clarify the significance of SGK1. RESULTS: The status of SGK1 in carcinoma cells was significantly associated with adverse clinical outcome in TNBC patients examined and was significantly associated with lymph node metastasis, pathological stage, and lymphatic invasion of the patients. In particular, SGK1 immunoreactivity was significantly associated with an increased risk of recurrence in GR-positive TNBC patients. Subsequent in vitro studies also demonstrated that DEX promoted TNBC cell migration and the silencing of gene expression did inhibit the cell proliferation and migration of TNBC cells under DEX treatment. CONCLUSIONS: To the best of our knowledge, this is the first study to explore an association between SGK1 and clinicopathological variables and clinical outcome of TNBC patients. SGK1 status was significantly positively correlated with adverse clinical outcome of TNBC patients and promoted carcinoma cell proliferation and migration of carcinoma cells.

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.

A prediction model for early systemic recurrence in breast cancer using a molecular diagnostic analysis of sentinel lymph nodes: A large-scale, multicenter cohort study.

BACKGROUND: The one-step nucleic acid amplification (OSNA) assay can quantify the cytokeratin 19 messenger RNA copy number as a proxy for sentinel lymph node (SN) metastasis in breast cancer. A large-scale, multicenter cohort study was performed to determine the prognostic value of the SN tumor burden based on a molecular readout and to establish a model for the prediction of early systemic recurrence in patients using the OSNA assay. METHODS: SN biopsies from 4757 patients with breast cancer were analyzed with the OSNA assay. The patients were randomly assigned to the training or validation cohort at a ratio of 2:1. On the basis of the training cohort, the threshold SN tumor burden value for stratifying distant recurrence was determined with Youden's index; predictors of distant recurrence were investigated via multivariable analyses. Based on the selected predictors, a model for estimating 5-year distant recurrence-free survival was constructed, and predictive performance was measured with the validation cohort. RESULTS: The prognostic cutoff value for the SN tumor burden was 1100 copies/µL. The following variables were significantly associated with distant recurrence and were used to construct the prediction model: SN tumor burden, age, pT classification, grade, progesterone receptor, adjuvant cytotoxic chemotherapy, and adjuvant anti-human epidermal growth factor receptor 2 therapy. The values for the area under the curve, sensitivity, specificity, and accuracy of the prediction model were 0.83, 63.4%, 81.7%, and 81.1%, respectively. CONCLUSIONS: Using the OSNA assay, the molecular readout-based SN tumor burden is an independent prognostic factor for early breast cancer. This model accurately predicts early systemic recurrence and may facilitate decision-making related to treatment.

Nuclear grade and comedo necrosis of ductal carcinoma in situ as histopathological eligible criteria for the Japan Clinical Oncology Group 1505 trial: an interobserver agreement study.

OBJECTIVE: The Japan Clinical Oncology Group 1505 trial is a single-arm multicentre prospective study that examined the possibility of non-surgical follow-up with endocrine therapy for patients with low-grade ductal carcinoma in situ. In that study, the eligible criteria included histopathological findings comprising low to intermediate nuclear grade and absence of comedo necrosis, and cases were entered according to the local histopathological diagnosis. Nuclear grade is largely based on the Consensus Conference criteria (1997), whereas comedo necrosis is judged according to the Rosen's criteria (2017). The purpose of this study was to standardize and examine the interobserver agreement levels of these histopathological criteria amongst the participating pathologists. METHODS: We held slide conferences, where photomicrographs of haematoxylin-eosin-stained slides from 68 patients with ductal carcinoma in situ were presented using PowerPoint. The nuclear grade and comedo necrosis statuses individually judged by the pathologists were analysed using κ statistics. RESULTS: In the first and second sessions, where 22 cases each were presented, the interobserver agreement levels of nuclear grade whether low/intermediate grade or high grade were moderate amongst 29 and 24 participating pathologists, respectively (κ = 0.595 and 0.519, respectively). In the third session where 24 cases were presented, interobserver agreement levels of comedo necrosis or non-comedo necrosis were substantial amongst 25 participating pathologists (κ = 0.753). CONCLUSION: Although the concordance rates in nuclear grade or comedo necrosis were not high in a few of the cases, we believe that these results could provide a rationale for employing the present criteria of nuclear grade and comedo necrosis in the clinical study of ductal carcinoma in situ.

Significance of glucocorticoid signaling in triple-negative breast cancer patients: a newly revealed interaction with androgen signaling.

PURPOSE: Chemotherapy is the only current effective systemic treatment for triple-negative breast cancer (TNBC) patients. Therefore, the identification of active biological pathways that could become therapeutic targets is crucial. In this study, considering the well-reported biological roles of glucocorticoid and androgen receptors (GR, AR) in TNBC, we attempted to explore the effects of glucocorticoids (GCs) on cell kinetics as well as the potential interaction between GR and AR in TNBC. METHODS: We first explored the association between the status of GR, AR, and/or GCs-metabolizing enzymes such as 11ß-hydroxysteroid dehydrogenase (11ßHSD) 1 and 2 and the clinicopathological variables of the TNBC patients. Thereafter, we also studied the effects of dexamethasone (DEX) with/without dihydrotestosterone (DHT) on TNBC cell lines by assessing the cell proliferation, migration and GC response genes at the transcriptional level. RESULTS: GR positivity in carcinoma cells was significantly associated with adverse clinical outcome of the patients and AR positivity was significantly associated with lower histological grade and Ki-67 labeling index of the cases examined. In particular, AR positivity was significantly associated with decreased risks of developing recurrence in GR-positive TNBC patients. The subsequent in vitro studies revealed that DEX-promoted cell migration was inhibited by the co-treatment with DHT in GR/AR double-positive HCC38 cells. In addition, DHT inhibited the DEX-increased serum and glucocorticoid-regulated kinase-1 (SGK1) mRNA expression. CONCLUSION: This is the first study to reveal that the interaction of GR and AR did influence the clinical outcome of TNBC patients and GCs induced cell migration in TNBC cells.

Correlation between 18F-FDG uptake on PET/MRI and the level of tumor-infiltrating lymphocytes (TILs) in triple-negative and HER2-positive breast cancer.

PURPOSE: The tumor microenvironment is related to the metabolism of cancer cells and local immune reactions. Previous studies have established TILs could be a significant prognostic factor, especially for triple-negative breast cancers (TNBC) and human epithelial growth factor receptor 2 (HER2)-positive breast cancers. We explore the association between metabolic information on PET/MRI with TILs in TNBC and HER2-positive breast cancer. METHOD: We retrospectively reviewed the cases of 55 women with triple-negative or HER2-positive invasive ductal carcinomas who had undergone 18F-FDG PET/MRI without neo-adjuvant treatment for pre-operative evaluation. FDG uptake was quantified as standardized uptake value (SUV) max. The existence of peritumoral edema on PET/MRI was also recorded. The TIL score of the breast cancers was defined histologically on the basis of the proportion of the area infiltrated by lymphocytes, and classified as low (<10 %), intermediate (10-50 %), and high (>50 %). The association between PET/MRI findings and TILs was assessed using Kruskal-Wallis test and Wilcoxon signed-rank test. RESULTS: There were 14 high TIL, 20 intermediate TIL and 21 low TIL lesions. Higher values of SUVmax were found in the high and intermediate TIL group as compared to the low TIL group (P = 0.013). On the other hand, the lesions with peritumoral edema in the low TIL group tended to show high SUVmax (P = 0.014). CONCLUSIONS: 18F-FDG uptake on PET/MRI correlated with TIL levels in patients with TNBC and HER2-positive breast cancer. This finding suggests that preoperative PET/MRI may be useful as a non-invasive tool for guiding the treatment plan.

Diffuse distribution of tumor-infiltrating lymphocytes is a marker for better prognosis and chemotherapeutic effect in triple-negative breast cancer.

PURPOSE: High-density tumor-infiltrating lymphocytes (TILs) are a prognostic marker for triple-negative breast cancer (TNBC). However, lymphocytic infiltration is heterogeneous in its pattern. We aimed to explore the utility of TIL distribution patterns against TIL density for predicting TNBC prognosis and chemotherapeutic effects. METHODS: Primary invasive TNBC cases were retrieved from a single institutional cohort, and archived samples were reviewed by two board-certificated pathologists. We used 154 consecutive surgical specimens from patients with standard adjuvant therapy, and 80 biopsies taken before primary systemic chemotherapy. The average density of stromal TILs was scored at 10% intervals, while the distribution pattern of TILs was evaluated as diffuse or non-diffuse. The association between TILs and prognosis or pathological complete response (pCR) was statistically analyzed. RESULTS: A diffuse pattern of TILs at primary surgery correlated with better prognosis (relapse-free survival [RFS], hazard ratio [HR] 3.71, 95% confidence interval [CI] 1.60-8.57; overall survival [OS], HR 3.87, 95% CI 1.46-10.27), as well as high TIL density (≥ 50%; RFS, HR 4.51, 95% CI 2.06-9.90; OS, HR 3.28, 95% CI 1.32-8.14). Diffuse TIL pattern and nodal status were independent prognostic factors in multivariate analysis. Diffuse TIL pattern upon biopsy was associated with higher pCR rate (diffuse, 46%; non-diffuse, 21%; P = 0.032). All high TIL cases had diffuse patterns and the best outcome. Interobserver concordance was moderate (k = 0.53-0.55; distribution pattern) to good (weighted k = 0.67-0.69; density), and it was faster to assess the distribution pattern than to assess the density of TIL. CONCLUSIONS: Showing similar clinical impacts to the TIL density, diffuse TILs could be a predictive marker for better prognosis and higher pCR. The assessment of TIL distribution pattern is simple, faster, and practical. Heterogeneous tumor immunity may contribute to further stratification of TNBC treatment.

Apocrine papillary lesion: comparison of pathological findings from 22 years previously and the present.

Apocrine papillary lesion (APL) is difficult to diagnose as benign or malignant. We experienced an APL remaining in the body for 22 years. We present a case of a 71-year-old woman who had undergone excisional biopsy 22 years previously at the first hospital that she visited. 1 year previously, she had undergone fine-needle aspiration cytology at a second hospital, and the lesion was diagnosed as potentially malignant. She underwent core-needle biopsy at a third hospital, but whether the lesion was benign or malignant could not be definitively diagnosed. We performed right mastectomy and sentinel lymph-node biopsy, because her tumor was suspected to be malignant based on imaging means, and malignancy could not be ruled out on either biopsy or cytology. The histopathological diagnosis was tiny foci of apocrine proliferative lesion with massive hemorrhagic necrosis and no tumor metastasis in two sentinel lymph nodes. Retrospectively, we compared all of the patient's previous specimens with the present ones, and applied the recent pathological diagnostic criteria. Although the biopsy specimen excised 22 years ago suggested an encapsulated apocrine papillary carcinoma or a papilloma with apocrine ductal carcinoma in situ, neither infiltration nor metastasis has occurred. Furthermore, neither the pathological findings nor the clinical behavior has changed over time.

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.

Maspin mRNA expression in sentinel lymph nodes predicts non-SLN metastasis in breast cancer patients with SLN metastasis.

AIMS: Maspin is known to be a tumour suppressor protein, but its prognostic significance in breast cancer patients is controversial. There is no report focusing on maspin expression in metastatic carcinoma of sentinel lymph nodes (SLNs); we thus investigated maspin mRNA expression in SLNs using the remaining specimens after the one-step nucleic acid amplification (OSNA) assay. METHODS AND RESULTS: Ninety-three breast cancer patients with SLNs metastasis detected by the OSNA assay were enrolled. All patients experienced additional axillary lymph nodes (LNs) dissection and all dissected LNs were examined histopathologically. Maspin mRNA expression in SLNs was detected in 49.5% (46 of 93) and was correlated significantly with the presence of non-SLN metastasis (P < 0.0001) and ≥4 LN metastases (P = 0.029). In a multivariate logistic analysis, maspin mRNA expression in SLNs (P = 0.0015) had the most significant effect on predicting non-SLN metastasis, followed by pathological tumour size (P = 0.0039) and lymphovascular invasion (P = 0.009). The status of maspin mRNA expression in SLNs was correlated significantly with that of maspin protein expression in the primary carcinoma (P < 0.0001). CONCLUSIONS: This is the first study, to our knowledge, demonstrating that maspin mRNA expression in SLNs is an independent predictor of non-SLN metastasis and the presence of ≥4 LN metastases in breast cancer patients with SLN metastasis. The investigation of maspin mRNA expression in SLNs using the remaining specimens after the OSNA assay may be useful for predicting the further progression of metastatic carcinoma in breast cancer patients with SLNs metastasis.

PCP4/PEP19 upregulates aromatase gene expression via CYP19A1 promoter I.1 in human breast cancer SK-BR-3 cells.

The Purkinje cell protein 4/peptide 19 (PCP4/PEP19) is a novel breast cancer cell expressing peptide, originally found in the neural cells as an anti-apoptotic factor, could inhibit cell apoptosis and enhance cell migration and invasion in human breast cancer cell lines. The expression of PCP4/PEP19 is induced by estrogens in estrogen receptor-positive (ER+) MCF-7 cells but also highly expressed in ER- SK-BR-3 cells. In this study, we investigated the effects of PCP4/PEP19 on aromatase gene expression in MCF-7 and SK-BR-3 human breast cancer cells. In SK-BR-3 cells but not in MCF-7 cells, PCP4/PEP19 knockdown by siRNA silencing decreased the aromatase expression in gene transcriptional level. When PCP4/PEP19 was overexpressed by CMV promoter-driven PCP4/PEP19 expressing plasmid transfection, aromatase gene transcription increased in SK-BR-3 cells. This aromatase gene transcription is mainly mediated through promoter region PI.1, which is usually active in the placental tissue but not in the breast cancer tissue. These results indicate a new function of PCP4/PEP19 that would enhance aromatase gene upregulation to supply estrogens in heterogeneous cancer microenvironment.

Intraoperative Nomograms, Based on One-Step Nucleic Acid Amplification, for Prediction of Non-sentinel Node Metastasis and Four or More Axillary Node Metastases in Breast Cancer Patients with Sentinel Node Metastasis.

BACKGROUND: One-step nucleic acid amplification (OSNA) for cytokeratin 19 messenger RNA is an intraoperative diagnostic procedure for the detection of lymph node metastasis. OBJECTIVE: This study aimed to construct intraoperative nomograms using OSNA for the prediction of non-sentinel lymph node (NSLN) metastasis and four or more axillary lymph node (ALN) metastases. METHODS: Of the 4736 breast cancer patients (T1-3, N0) who underwent sentinel lymph node (SLN) biopsy and had SLNs examined intraoperatively with OSNA, 623 with SLN metastasis treated with completion ALN dissection (cALND) were retrospectively analyzed, and were randomly divided into training (n = 312) and validation (n = 311) sets. RESULTS: Of the clinicopathological parameters available preoperatively and intraoperatively, the multivariate analysis of the training set revealed that clinical tumor size and total tumor load (TTL) determined by OSNA were significantly associated with NSLN metastasis, and that clinical tumor size, number of macrometastatic SLNs, and TTL were significantly associated with four or more ALN metastases. Nomograms for NSLN metastasis and four or more ALN metastases were constructed using these parameters, and their area under the receiver operating characteristic curve (AUC) of the validation set were both 0.70, with a diagnostic accuracy similar to that of previously reported postoperative nomograms. CONCLUSIONS: We constructed intraoperative nomograms using OSNA for the prediction of NSLN metastasis and four or more ALN metastases. These nomograms are as accurate as the conventional postoperative nomograms and might be helpful for decision making regarding the indication for cALND or the choice of adjuvant chemotherapeutic regimens and radiation field.

Effectiveness of neo-adjuvant systemic therapy with trastuzumab for basal HER2 type breast cancer: results from retrospective cohort study of Japan Breast Cancer Research Group (JBCRG)-C03.

PURPOSE: While human epidermal growth factor receptor 2 (HER2) target therapies have significantly improved the prognosis of patients with HER2-enriched breast cancer, differing clinical benefits and gene expression analyses suggest a divergent HER2 subgroup. We aimed to investigate whether the basal HER2 subtype of breast cancer has distinguished characteristics. METHODS: We performed a substudy by using data from a retrospective multi-institutional cohort of JBCRG-C03. Between 2001 and 2011, we identified 184 eligible patients who received concurrent neo-adjuvant chemotherapy (NAC) with trastuzumab for hormone receptor-negative and HER2-positive breast cancer. We defined basal HER2 subtype breast cancer as HER2-positive, ER/PgR-negative, and basal markers (EGFR, CK14 or CK5/6) positive by immunohistochemistrical evaluation. The pathologic complete response (pCR) and disease-free survival (DFS) rates were compared between the two subtypes. RESULTS: A total of 127 (69.0%) patients achieved pCR after NAC and 29 (15.8%) patients experienced events of DFS within a 42 month median follow-up period (interquartile range 26-58 months). Although the basal HER2 subtype was related with poor DFS (3 year DFS: non-basal HER2, 95.0%; basal HER2, 86.9%; adjusted HR 3.4; 95% CI 1.2-14.5), neither the subtype (pCR: non-basal HER2, 75%; basal HER2, 66.7%; adjusted OR 0.60; 95% CI 0.27-1.28) nor the degree of expression of basal markers was significantly related with the pCR rate. CONCLUSION: Basal HER2 phenotype showed poor DFS, but equivalent pCR rate after concurrent neo-adjuvant chemotherapy with trastuzumab. A different treatment approach to basal-HER2 type is needed even for cases that achieved adequate clinical response after NAC.

Simultaneous whole-body and breast 18F-FDG PET/MRI examinations in patients with breast cancer: a comparison of apparent diffusion coefficients and maximum standardized uptake values.

PURPOSE: To compare standardized uptake value (SUV) and apparent diffusion coefficient (ADC) values acquired using a PET/MRI scanner in breast cancer patients. MATERIALS AND METHODS: Whole-body PET/MRI and breast PET/MRI were performed in 108 consecutive patients. Ninety-four patients who had a total of 100 breast cancers were analyzed. SUVmax and ADCmean acquired using breast PET/MRI were compared with pathologic prognostic factors. RESULTS: All the lesions were visually detectable using PET and diffusion-weighted imaging (DWI) on breast PET/MRI; however, lesions were visually undetectable on whole-body DWI in 13 patients (13%) or on whole-body PET in 7 patients (7%). An analysis of ADCmean and SUVmax demonstrated a statistically significant correlation between whole-body imaging and breast imaging (rho = 0.613, p < 0.001 and rho = 0.928, p < 0.001, respectively). In a univariate analysis, SUVmax was significantly correlated with HER2 status (p < 0.001), Ki-67 (p = 0.014), tumor size (p = 0.0177), and nuclear grade (p = 0.0448). In multiple regression analysis, only tumor size (p = 0.00701) was shown to independently influence SUVmax. CONCLUSION: Prone breast imaging was more sensitive than whole-body PET/MRI for detection of breast cancers. Both SUVmax and ADCmean showed limited correlation with pathologic prognostic factors.

Effects of cytokines derived from cancer-associated fibroblasts on androgen synthetic enzymes in estrogen receptor-negative breast carcinoma.

PURPOSE: The tumor microenvironment plays pivotal roles in promotion of many malignancies. Cancer-associated fibroblasts (CAFs) have been well-known to promote proliferation, angiogenesis, and metastasis but mechanistic understanding of tumor-stroma interactions is not yet complete. Recently, estrogen synthetic enzymes were reported to be upregulated by co-culture with stromal cells in ER positive breast carcinoma (BC) but effects of co-culture on androgen metabolism have not been extensively examined. Therefore, we evaluated roles of CAFs on androgen metabolism in ER-negative AR-positive BC through co-culture with CAFs. METHODS: Concentrations of steroid hormone in supernatant of co-culture of MDA-MB-453 and primary CAFs were measured using GC-MS. Cytokines derived from CAFs were determined using Cytokine Array. Expressions of androgen synthetic enzymes were confirmed using RT-PCR and Western blotting. Correlations between CAFs and androgen synthetic enzymes were analyzed using triple-negative BC (TNBC) patient tissues by immunohistochemistry. RESULTS: CAFs were demonstrated to increase expressions and activities of 17ßHSD2, 17ßHSD5, and 5α-Reductase1. IL-6 and HGF that were selected as potential paracrine mediators using cytokine array induced 17ßHSD2, 17ßHSD5, and 5α-Reductase1 expression. Underlying mechanisms of IL-6 paracrine regulation of 17ßHSD2 and 17ßHSD5 could be partially dependent on phosphorylated STAT3, while phosphorylated ERK could be involved in HGF-mediated 5α-Reductase1 induction. α-SMA status was also demonstrated to be significantly correlated with 17ßHSD2 and 17ßHSD5 status in TNBC tissues, especially AR-positive cases. CONCLUSIONS: Results of our present study suggest that both IL-6 and HGF derived from CAFs could contribute to the intratumoral androgen metabolism in ER-negative BC patients.