Sasagu Kurozumi

Recent trends in microRNA research into breast cancer with particular focus on the associations between microRNAs and intrinsic subtypes.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate the function of target genes at the post-transcriptional phase. miRNAs are considered to have roles in the development, progression and metastasis of cancer. Recent studies have indicated that particular miRNA signatures are correlated with tumor aggressiveness, response to drug therapy and patient outcome in breast cancer. On the other hand, in routine clinical practice, the treatment regimens for breast cancer are determined based on the intrinsic subtype of the primary tumor. Previous studies have shown that miRNA expression profiles of each intrinsic subtypes of breast cancer differ. In hormone receptor-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer, miRNA expressions are found to be correlated with endocrine therapy resistance, progesterone receptor expression and heat shock protein activity. Some miRNAs are associated with resistance to HER2-targeted therapy and HER3 expression in HER2-positive breast cancer. In triple-negative breast cancer, miRNA expressions are found to be associated with BRCA mutations, immune system, epithelial–mesenchymal transition, cancer stem cell properties and androgen receptor expression. As it has been clarified that the expression levels and functions of miRNA differ among the various subtypes of breast cancer, and it is necessary to take account of the characteristics of each breast cancer subtype during research into the roles of miRNA in breast cancer. In addition, the discovery of the roles played by miRNAs in breast cancer might provide new opportunities for the development of novel strategies for diagnosing and treating breast cancer.

HER2 intratumoral heterogeneity analyses by concurrent HER2 gene and protein assessment for the prognosis of HER2 negative invasive breast cancer patients

HER2 gene-protein assay (GPA) is a new method for the simultaneous evaluation of HER2 immunohistochemistry (IHC) and HER2 dual in situ hybridization (DISH) on single tissue sections of breast cancer. We investigated the presence of HER2 gene and protein discrepancy and HER2-heterogeneity using HER2-GPA. HER2 status was analyzed for the correlation between the presence of HER2-heterogeneity and patient prognosis. Consecutive 280 invasive breast cancer were examined. Statuses of HER2 protein and gene were evaluated in whole tumor sections of HER2 GPA slides. HER2 protein and gene combination patterns were classified to six phenotypic and genotypic types for each case, as well as at individual cell levels: (A) IHC and DISH positive; (B) IHC positive and DISH negative; (C) IHC equivocal and DISH positive; (D) IHC equivocal and DISH negative; (E) IHC negative and DISH positive; and (F) IHC and DISH negative. The presence of HER2-heterogeneity was determined by the existence of at least two of six types within one tumor. HER2-IHC positive patients had significantly worse survival than IHC negative patients and HER2-DISH positive patients had significantly worse survival than DISH negative patients. HER2 IHC negative and DISH positive patients had significantly worse recurrence-free survival than IHC and DISH negative patients. In the HER2 IHC and DISH negative group, the HER2 heterogeneous group had significantly worse survival than the nonheterogeneous group. Notably, among triple negative breast cancer (TNBC), the HER2 heterogeneous group had significantly worse survival than the nonheterogeneous group. Our study suggests that the presence of HER2-heterogeneity might be a prognostic factor in HER2 negative breast cancer patients, especially in TNBC.

Prognostic value of the ubiquitin ligase carboxyl terminus of the Hsc70-interacting protein in postmenopausal breast cancer

The carboxyl terminus of the Hsc70‐interacting protein (CHIP) is considered to induce the ubiquitination and degradation of several oncogenic proteins, and play a role in the inhibition of tumor progression and invasion under experimental conditions. However, the impact of CHIP expression on the prognosis of breast cancer patients has not yet been established. In this study, using an immunohistochemical method, 272 patients with invasive breast cancer were assessed for the expression of CHIP (graded scores 0‐3) and the statuses of biomarkers, such as estrogen receptor (ER), progesterone receptor (PgR), and HER2. The relationships between the statuses of CHIP and biomarkers as well as clinical features were also evaluated, and that between the expression of CHIP and patient prognosis was analyzed. We revealed that the strong expression of CHIP correlated with positive ER (P < 0.001), positive PgR (P < 0.001), and negative HER2 (P = 0.02). In postmenopausal patients, relapse‐free survival (RFS) was significantly better in the high CHIP group than in the low CHIP group (P = 0.042). In addition, RFS and cancer‐specific survival (CSS) were significantly better in patients with ER‐positive/CHIP score 3 tumors than in those with ER‐negative/CHIP score 0 tumors (RFS: P = 0.038, CSS: P = 0.0098). The methylation status of CHIP gene promoter did not always account for the down‐regulation of its expression. In conclusion, the overexpression of CHIP is a potent prognostic factor of a good prognosis in ER‐positive breast cancer patients in the postmenopausal phase.

Significance of evaluating tumor-infiltrating lymphocytes (TILs) and programmed cell death-ligand 1 (PD-L1) expression in breast cancer

The immune system affects all phases of tumor growth from initiation to progression and dissemination. Tumor-infiltrating lymphocytes (TILs) are mononuclear immune cells that infiltrate tumor tissue. Several retrospective studies have suggested the potential of TILs as a prognostic as well as predictive factor of chemotherapy in some breast cancers. On the other hand, programmed cell death protein-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) eliminate T cell activation in various types of cancers. Prospective trials to evaluate the efficacy of antibody agents to PD-1 and PD-L1 are ongoing in patients with breast cancer. The findings of these studies appear to support the potential of immune checkpoint inhibitors targeting the PD-1/PD-L1 axis in triple negative breast cancer. Further studies are needed in order to confirm previous findings on TILs and promote the development of new immune therapy approaches for breast cancer patients. Furthermore, the search for TILs will soon be introduced into actual clinical practice, for which the standardization of evaluation methods and establishment of a simple evaluation method are expected.

Impact of combining the progesterone receptor and preoperative endocrine prognostic index (PEPI) as a prognostic factor after neoadjuvant endocrine therapy using aromatase inhibitors in postmenopausal ER positive and HER2 negative breast cancer

The preoperative endocrine prognostic index (PEPI) predicts survival after neoadjuvant endocrine therapy (NAE) using aromatase inhibitors (AIs) for women with postmenopausal estrogen receptor (ER)-positive breast cancer irrespective of the human epidermal growth factor receptor 2 (HER2) status. Although the progesterone receptor (PgR) is also a prognostic factor for ER-positive breast cancer, the PgR status was not considered a prognostic factor in the original PEPI scoring system. In this study, we investigated the utility of a modified PEPI including the PgR status (PEPI-P) as a prognostic factor after NAE for postmenopausal patients with ER-positive and HER2-negative breast cancer. We enrolled 107 patients with invasive ER-positive and HER2-negative breast cancer treated with exemestane for ≥4 months as NAE. We initially assessed PEPI and compared survival between the groups. Additionally, we obtained an effective cutoff for PgR through survival analysis. Then, we assessed the survival significance of PEPI-P. A PgR staining rate of 50% was the most significant cutoff for predicting recurrence-free survival (RFS) and cancer-specific survival (CSS). PEPI was a significant prognostic factor; moreover, PEPI-P was the most significant prognostic indicator for RFS and CSS. PEPI-P is a potent prognostic indicator of survival after NAE using AIs for postmenopausal patients with ER-positive and HER2-negative breast cancer. This modified PEPI may be useful for therapeutic decision-making regarding postmenopausal ER-positive and HER2-negative breast cancer after NAE.

Comparing protein and mRNA expressions of the human epidermal growth factor receptor family in estrogen receptor-positive breast cancer

The human epidermal growth factor receptor (HER) family plays a vital role in the development of resistance to treatments in estrogen receptor (ER)-positive breast cancer. This study investigated the correlation between protein and mRNA expressions of the HER family in ER-positive breast cancer. We dissected regions of invasive cancer from the frozen tissues of 34 patients with ER-positive breast cancer using laser-capture microdissection, followed by evaluation of the mRNA levels of the ER and HER family (EGFR, HER2, HER3, and HER4) using the quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. In addition, we assessed the protein expressions of the ER and HER family using an immunohistochemical (IHC) assay. A significant correlation was observed between the ER protein and mRNA expressions. For HER2, HER3, and HER4, protein expressions significantly correlated with mRNA levels. We established significant correlations of the mRNA level between EGFR versus HER2, as well as EGFR versus HER3. Furthermore, a significant correlation of the mRNA level between HER2 and HER3 was illustrated. In conclusion, IHC evaluation may be reliable and representable for mRNA. Hence, this study established a marked correlation between the mRNA expressions of HER family members in patients with ER-positive breast cancer.

Molecular pathogenesis of triple-negative breast cancer based on microRNA expression signatures: antitumor miR-204-5p targets AP1S3

Triple-negative breast cancer (TNBC) is an aggressive type of cancer associated with a poor prognosis. Identification of novel therapeutic targets in TNBC is urgently needed. Here, we investigated the microRNA (miRNA) expression signature of TNBC using clinical specimens. In total, 104 miRNAs (56 upregulated and 48 downregulated) were significantly dysregulated in TNBC tissues; miR-204-5p showed the most dramatic downregulation. We then examined the antitumor roles of miR-204-5p in breast cancer (BC) cells. Notably, cancer cell migration and invasion were significantly reduced by ectopic expression of miR-204-5p in BC cells. Genome-wide gene expression analysis and in silico database search revealed that 32 genes were putative miR-204-5p targets. High expression of AP1S3, RACGAP1, ELOVL6, and LRRC59 was significantly associated with poor prognosis in patients with BC, and adaptor-related protein complex 1 sigma 3 subunit (AP1S3) was directly regulated by miR-204-5p, as demonstrated by luciferase reporter assays. AP1S3 overexpression was detected in TNBC clinical specimens and enhanced cancer cell aggressiveness. We further analyzed downstream RNA networks regulated by AP1S3 in BC cells. Overall, this miRNA signature is expected to be an effective tool for identification of miRNA-mediated molecular mechanisms of TNBC pathogenesis.

Relationship Between FDG Uptake and Neutrophil/Lymphocyte Ratio in Patients with Invasive Ductal Breast Cancer

Background: 18F-Fluorodeoxyglucose-positron-emission tomography (FDG-PET) is used to evaluate the glucose metabolic rates of tumors. Several studies have reported that high FDG uptake is predictive of poor prognosis and aggressive features in patients with breast cancer. FDG uptake is influenced by many factors, including inflammation. In this study, the relationship between FDG uptake and neutrophil/lymphocyte ratio (NLR), which is an indicator of systemic inflammation, was investigated. Patients and Methods: A retrospective investigation of the cases of 143 consecutive patients with invasive ductal carcinoma who had undergone surgery and FDG-PET preoperatively. PET was evaluated using standardized uptake value max (SUVmax). The median SUVmax was 2.5 (range=0-10.5). The cases were divided into two groups based on the value of SUVmax: low (<2.5) and high (≥2.5). The relationships between SUVmax and clinicopathological features, including NLR, were investigated. Results: Among the 143 patients, 73 (51.0%) had high SUVmax in the primary tumor. The analysis revealed that large tumor size (p<0.001), high nuclear grade (p<0.001), the presence of lymphovascular invasion (p<0.001), high C-reactive protein (p=0.046) and high NLR (p<0.001) were significantly associated with high SUVmax in the primary tumor. SUVmax and NLR were significantly positively correlated (r=0.323, p<0.001). Among the 70 cases with low SUVmax, there was no recurrent disease, while out of the 73 cases with high SUVmax had disease recurrence. It is interesting to note that the group with high SUVmax and low NLR had no recurrent disease. Conclusion: The present study demonstrated that the finding of high preoperative FDG uptake in breast cancer may be reflective of poor prognosis and that a high NLR may be predictive of aggressive features among patients with breast cancer. On the other hand, among patients with breast cancer with high SUVmax in the primary tumor, it will be useful to identify those with a low NLR in order to improve prognostic accuracy.

Abstract P4-14-17: Prognostic value of tumor-infiltrating lymphocytes in residual tumors after neoadjuvant chemotherapy concomitant with trastuzumab for HER2-positive breast cancer

Background: Neoadjuvant chemotherapy (NAC) with taxanes, followed by fluorouracil, epirubicin, and cyclophosphamide (FEC), with concurrent trastuzumab is known to achieve a high pCR rate of more than 60% for HER2-positive breast cancer (BC) as well as good prognoses in those obtaining pCR. On the other hand, the prognostic significance of tumor-infiltrating lymphocytes (TILs) has recently been described in triple-negative BC. However, the prognostic and predictive values of TILs in HER2-positive BC remain unclear. In the present study, we examined the grades of TILs in pre-treatment cancer tissues and residual tumors after NAC with trastuzumab, and also investigated its predictive utility for pCR and prognostic power for HER2-positive BC. Patients and Methods: A total of 128 Japanese women with HER2-positive BC received either paclitaxel or docetaxel followed by FEC, with concomitant trastuzumab. The proportional grades of stromal (Str)-TILs in pre-treatment biopsy specimens and residual tumors after NAC with trastuzumab were determined as follows: low grade (0-10%), intermediate grade (10-40%), and high grade (40-90%), using the criteria of the International Working Group for TILs in BC. Analysis 1: The relationship between the grades of Str-TILs in pre-treatment tumors and pCR rates was investigated. Relapse-free survival (RFS) and cancer-specific survival (CSS) were analyzed for a correlation with pre-treatment Str-TILs. Analysis 2: Alterations in the grade of Str-TILs were examined in the residual tumors of non-pCR patients, and RFS and CSS were analyzed for a correlation with residual Str-TILs. Results: pCR was achieved in 83 out of the 128 patients (pCR rate, 64.8%) who received NAC with trastuzumab, and RFS was significantly better in the pCR group than in the non-pCR group (p = 0.0071). Analysis 1: The patient distribution of the Str-TILs grade in pre-treatment tumors was as follows: high: 24 (18.8%); intermediate: 38 (29.7%); and low: 66 (51.6%). pCR rates correlated with the Str-TILs grade in pre-treatment tumors: 83.3% in the high group, 71.1% in the intermediate group, and 54.5% in the low group (p = 0.026); however, the Str-TILs grade in pre-treatment tumors did not correlate with survival. Analysis 2: In 45 non-pCR patients, the distribution of the Str-TILs grade in residual tumors was as follows: high: 9 (20.0%); intermediate: 8 (17.8%); and low: 28 (62.2%), respectively. In non-pCR patients, the rate of a high Str-TILs grade was greater in residual tumors than in pre-treatment tumors (residual, 20.0%, pre-treatment, 8.9%). RFS was significantly better with a high grade than with a low grade of residual Str-TILs (p = 0.033). Conclusions: The status of TILs in pre-treatment tumors predicted responses to NAC concomitant with trastuzumab in HER2-positive BC. The grade of TILs was higher in residual tumors than in pre-treatment tumors, and, among non-pCR patients, the prognosis of patients with a high residual-TILs grade was better prognosis than that of patients with a low residual-TILs grade. We speculate that an examination of TILs in residual tumors after NAC with trastuzumab may be necessary for selecting patients with a good prognosis from non-pCR patients. Citation Format: Kurozumi S, Inoue K, Matsumoto H, Hayashi Y, Tozuka K, Kubo K, Komatsu K, Takai K, Nagai SE, Oba H, Horiguchi J, Takeyoshi I, Kurosumi M. Prognostic value of tumor-infiltrating lymphocytes in residual tumors after neoadjuvant chemotherapy concomitant with trastuzumab for HER2-positive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-14-17.

Abstract P2-02-20: Enumeration of heterogeneous circulating tumor cells (CTCs) in metastatic breast cancer patients based on size and deformability

Background : The detection of circulating tumor cells (CTCs) in peripheral blood is an independent predictor of the efficacy of systemic therapy and a prognostic marker for patients with metastatic breast cancer. One of the leading techniques to detect CTCs uses immune-magnetic separation followed by immunocytochemistry. A microdevice can capture and enumerate CTCs using distinctive physiological difference (size and deformability) between cancer cells and blood cells. This microdevice thus obtains a larger CTC yield than that of affinity based separation which enriches the samples from a particular subgroup of cells based on biomarker (EpCAM) used. In this study, we investigated CTCs in peripheral blood from metastatic breast cancer patients using this microdevice. Patients and methods: We examined blood samples of 9 patients with heavily treated locally recurrent or metastatic breast cancer. Informed consent from these patients was obtained before blood extraction. Blood samples were taken into sodium EDTA tubes after discarding the first 1ml of blood samples. Two ml whole blood were subjected to the microdevice (Clear cell system), and CTCs were trapped in the microwells: Trapped cells were analyzed by immunocytochemistry with monoclonal antibodies specific for leukocytes (CD45) and epithelial cells (CK8/18), along with 4,2-diamidino-2-phenylndole dihydrochloride (DAPI) for nuclei. CK8/18- positive, DAPI-positive and CD45-negative cells were defined as CTCs. Three patients were examined using both this microdevice and affinity-based separation with EpCAM, to compare the yield of CTCs. Results: Of the 9 patients: 7 had ER-positive primary tumors, and 6 had PgR-positive ones, HER2 overexpression was detected in 2 primary tumors. CTCs were detected in 8 patients. The single patient in whom CTCs were not detected suffered from local recurrence (axillary lymph node metastasis) only, with no distant metastases. We were also unable to detect CTCs using EpCAM affinity method for this patient. The number of detected CTCs in the other patients ranged from 19/2ml to 156/2ml (mean 90/2ml), and the sizes of CTCs varied from 5 to 16μm. CK8/18-negative and DAPI positive were detected in most patients, and these cells tended to be larger than CK8/18-positive cells, suggesting that epithelial–mesenchymal transition (EMT) might occur in CTCs. The total number of CTCs detected by the microdevice from 2 patients was larger than that of CTCs detected by EpCAM affinity method (107/2ml vs 1/7.5ml, and 19/2ml vs 39/7.5ml). Conclusion: CTCs detected by this microdevice varied in regard to the size of trapped cells and characteristics examined by immunochemistry, suggesting the heterogeneity of CTCs. Further research on this heterogeneity is vital in order to develop personalized treatment for patients with metastatic breast cancer. Citation Format: Tozuka K, Nagai SE, Inoue K, Komatsu K, Matsumoto H, Hayashi Y, Kurozumi S, Suganuma M. Enumeration of heterogeneous circulating tumor cells (CTCs) in metastatic breast cancer patients based on size and deformability. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-20.