Utility of human epidermal growth factor 2 heterogeneity as a prognostic factor in triple-negative breast cancer.

In some cases of human epidermal growth factor 2 (HER2)-negative breast cancer, including triple-negative breast cancer, HER2 expression is sporadically and strongly upregulated, a condition known as HER2 heterogeneity. We investigated the clinicopathological features of patients with HER2 heterogeneity in triple-negative breast cancers treated with neoadjuvant chemotherapy. Thirty-nine patients with triple-negative breast cancer who had undergone preoperative chemotherapy participated in this study. To assess for HER2 heterogeneity, we used dual in situ hybridization slides. We evaluated the association between HER2 heterogeneity and clinicopathological factors such as rates of pathologic complete response (pCR) and of recurrence-free survival. Of the 39 patients, 15 (38.5%) had cancers with HER2 heterogeneity. The pCR rates were 13.3% among patients with HER2 heterogeneity and 20.8% among those with HER2 nonheterogeneity, but the difference was not significant. The recurrence-free survival rate was significantly lower in patients with HER2 heterogeneity than in those without (P = 0.025). HER2 heterogeneity is a significant predictor of poor prognosis in patients with triple-negative breast cancer treated with neoadjuvant chemotherapy.

Identification of MicroRNAs Associated with Histological Grade in Early-Stage Invasive Breast Cancer.

This study aimed to identify microRNAs associated with histological grade using comprehensive microRNA analysis data obtained by next-generation sequencing from early-stage invasive breast cancer. RNA-seq data from normal breast and breast cancer samples were compared to identify candidate microRNAs with differential expression using bioinformatics. A total of 108 microRNAs were significantly differentially expressed in normal breast and breast cancer tissues. Using clinicopathological information and microRNA sequencing data of 430 patients with breast cancer from The Cancer Genome Atlas (TCGA), the differences in candidate microRNAs between low- and high-grade tumors were identified. Comparing the expression of the 108 microRNAs between low- and high-grade cases, 25 and 18 microRNAs were significantly upregulated and downregulated, respectively, in high-grade cases. Clustering analysis of the TCGA cohort using these 43 microRNAs identified two groups strongly predictive of histological grade. miR-3677 is a microRNA upregulated in high-grade breast cancer. The outcome analysis revealed that patients with high miR-3677 expression had significantly worse prognosis than those with low miR-3677 expression. This study shows that microRNAs are associated with histological grade in early-stage invasive breast cancer. These findings contribute to the elucidation of a new mechanism of breast cancer growth regulated by specific microRNAs.

Oncological safety of immediate breast reconstruction with skin- or nipple-sparing mastectomy: the value of tumor-to-dermis distance measured by preoperative ultrasonography.

BACKGROUND: Immediate breast reconstruction with skin-sparing (SSM) or nipple-sparing mastectomy (NSM) has become a common procedure. In this study, we evaluated the distance between breast tumor and skin in a series of patients undergoing IBR as it relates to oncologic safety, namely, the incidence of recurrence. METHODS: The distance of the tumor to the dermis, rather than the outer layer of skin, was the key parameter of our preoperative ultrasound measurements. Our data set comprised the cases of 171 patients and 181 breasts with breast cancer that had undergone two-stage breast reconstruction by expander. The median age of the patients was 47 years (25-75 years). The overall median follow-up period was 47.1 months (8.8-125.3 months). Eighty-five breasts underwent IBR with SSM/NSM; the others underwent conventional mastectomy. RESULTS: Among the total of 181 reconstructed breast mounds, the locoregional recurrence rate was 1.1% (2 breasts) with no cases of skin flap recurrence or skin flap necrosis. The tumor-to-dermis distance of cases with skin preservation (NSM/SSM) was significantly less than that of cases with conventional mastectomy (3.8 ± 2.7 mm vs 5.2 ± 2.4 mm). In cases with invasive carcinoma, all cases whose tumor-to-dermis distance was less than 2 mm underwent resection of the skin immediately overlying the tumor. CONCLUSIONS: Our results suggested that a 2-mm distance between the dermis and tumor on ultrasound evaluation is sufficient for the use of this tissue as a skin flap in SSM/NSM procedures. Our study indicated that immediate breast reconstruction with SSM/NSM can be an oncologically safe surgical option for breast cancer. However, we recommend that resection of the skin overlying the tumor be performed in cases with invasive breast cancer in which the tumor-to-dermis distance is less than 2 mm. TRIAL REGISTRATION: Patients in this study were retrospectively registered. This study design was approved by our Clinical Ethics Committee (No 1297) ( http://ciru.dept.showa.gunma-u.ac.jp/guidance/storage-sample/list.html ).

Computer-assisted image analysis of cytological specimens clarify the correlation between nuclear size and intranuclear cytoplasmic inclusions regardless of BRAFV600E mutation in papillary thyroid carcinoma.

OBJECTIVE: The morphological features of nuclei in cytological and histological specimens were compared and examined for the presence of BRAFV600E mutation and the appearance rate of intranuclear cytoplasmic inclusions (NI). METHODS: BRAFV600E mutation was identified using a mutation-specific antibody (clone; VE1) in 103 thyroid papillary carcinoma cases at Gunma University Hospital. The nuclear area, perimeter, and roundness of the corresponding cytological specimens and haematoxylin and eosin-stained specimens were analysed using image analysis software, and the appearance rate of NI was calculated and compared. RESULTS: BRAFV600E mutation was detected in 71 (69%) cases. The appearance rate of NI was significantly higher in the BRAFV600E mutation-positive group in cytological and histological specimens (P = .0070 and .0184, respectively). Significant differences were observed between the BRAFV600E mutation-negative and -positive groups in the average nuclear area and average nuclear perimeter in cytological specimens (P = .0137 and .0152, respectively). In addition, nuclear enlargement was correlated with the appearance rate of NI regardless of the presence of BRAFV600E mutation in cytological specimens. In the BRAFV600E mutation-negative group, the nuclear area and perimeter were significantly smaller in the lymph node metastasis-positive cases (P = .0182 and .0260, respectively). CONCLUSION: This study found that the appearance rate of NI was positively correlated with the nuclear area and perimeter and negatively correlated with nuclear roundness in cytological specimens. Furthermore, these results were observed regardless of the existence of BRAFV600E mutation. These results have never been previously reported and clearly demonstrate the usefulness of cytological specimens in computer-assisted image analysis.

Targetable ERBB2 mutation status is an independent marker of adverse prognosis in estrogen receptor positive, ERBB2 non-amplified primary lobular breast carcinoma: a retrospective in silico analysis of public datasets.

BACKGROUND: Invasive lobular carcinoma (ILC) accounts for 10-15% of primary breast cancers and is typically estrogen receptor alpha positive (ER+) and ERBB2 non-amplified. Somatic mutations in ERBB2/3 are emerging as a tractable mechanism underlying enhanced human epidermal growth factor 2 (HER2) activity. We tested the hypothesis that therapeutically targetable ERBB2/3 mutations in primary ILC of the breast associate with poor survival outcome in large public datasets. METHODS: We performed in silico comparison of ERBB2 non-amplified cases of ER+ stage I-III primary ILC (N = 279) and invasive ductal carcinoma (IDC, N = 1301) using METABRIC, TCGA, and MSK-IMPACT information. Activating mutations amenable to HER2-directed therapy with neratinib were identified using existing functional data from in vitro cell line and xenograft experiments. Multivariate analysis of 10-year overall survival (OS) with tumor size, grade, and lymph node status was performed using a Cox regression model. Differential gene expression analyses by ERBB2 mutation and amplification status was performed using weighted average differences and an in silico model of response to neratinib derived from breast cancer cell lines. RESULTS: ILC tumors comprised 17.7% of all cases in the dataset but accounted for 47.1% of ERBB2-mutated cases. Mutations in ERBB2 were enriched in ILC vs. IDC cases (5.7%, N = 16 vs. 1.4%, N = 18, p < 0.0001) and clustered in the tyrosine kinase domain of HER2. ERBB3 mutations were not enriched in ILC (1.1%, N = 3 vs. 1.8%, N = 23; p = 0.604). Median OS for patients with ERBB2-mutant ILC tumors was 66 months vs. 211 months for ERBB2 wild-type (p = 0.0001), and 159 vs. 166 months (p = 0.733) for IDC tumors. Targetable ERBB2 mutational status was an independent prognostic marker of 10-year OS-but only in ILC (hazard ratio, HR = 3.7, 95% CI 1.2-11.0; p = 0.021). Findings were validated using a novel ERBB2 mutation gene enrichment score (HR for 10-year OS in ILC = 2.3, 95% CI 1.04-5.05; p = 0.040). CONCLUSIONS: Targetable ERBB2 mutations are enriched in primary ILC and their detection represents an actionable strategy with the potential to improve patient outcomes. Biomarker-led clinical trials of adjuvant HER-targeted therapy are warranted for patients with ERBB2-mutated primary ILC.

FDG uptake reflects breast cancer immunological features: the PD-L1 expression and degree of TILs in primary breast cancer.

BACKGROUND: High F18-fluorodeoxyglucose (FDG) uptake has been reported to be a predictor of poor prognosis in patients with breast cancer. We investigated the relationship between FDG uptake and immunological factors, including the data of programmed cell death-ligand 1 (PD-L1), CD8, and tumor-infiltrating lymphocytes (TILs). METHODS: Breast cancer tissues of 97 patients who underwent surgery without preoperative therapy were examined. The grade of stromal TILs was immunohistochemically evaluated using the criteria of the International TILs Working Group in breast cancer. PD-L1 positivity and CD8 positivity were immunohistochemically evaluated. The FDG uptakes were evaluated based on the standardized uptake value max (SUVmax). The relationships between SUVmax and TIL grade and expression of PD-L1 and CD8 were investigated. RESULTS: Among the 97 patients, 41 (42.3%) had a high SUVmax in their primary tumor, based on the SUVmax cut-off value 3 yielded by receiver operating characteristic curves. PD-L1 was positive in 17 patients (17.5%). Our analyses revealed that large tumor size, high nuclear grade, high degree of TILs and positive expression of PD-L1 were significantly associated with high SUVmax in the primary tumor. There were significant associations between SUVmax and the degree of TILs (r = 0.428, p < 0.001) and between SUVmax and the PD-L1 positivity (r = 0.413, p < 0.001). All cases with a high degree of TILs showed high CD8 expression. CONCLUSION: Our results indicate that the FDG uptake may be predictive of immunological features including TILs and PD-L1 expression in breast cancer patients. Additional research is necessary to further evaluate FDG-PET as a biomarker of immune checkpoint therapy in breast cancer.

Clinicopathological significance of lipocalin 2 nuclear expression in invasive breast cancer.

PURPOSE: The epithelial-mesenchymal transition (EMT) plays a key role in breast cancer progression and metastasis. Lipocalin 2 (LCN2) is involved in the regulation of EMT. The aim of this study was to investigate the clinicopathological significance of LCN2 expression in breast cancer. METHODS: The expression of LCN2 protein was immunohistochemically assessed in two well-characterised annotated cohorts of breast cancer (discovery cohort, n = 612; validation cohort, n = 1363). The relationship of LCN2 expression and subcellular location with the clinicopathological factors and outcomes of patients was analysed. RESULTS: Absent or reduced nuclear LCN2 expression was associated with features of aggressive behaviour, including high histological grade, high Nottingham Prognostic Index, high Ki67 labelling index, hormone receptor negativity and human epidermal growth factor receptor 2 positivity. The high cytoplasmic expression of LCN2 was correlated with lymph node positivity. The nuclear downregulation of LCN2 was correlated with the overexpression of EMT associated proteins (N-cadherin and Twist-related protein 2) and basal biomarkers (cytokeratin 5/6 and epidermal growth factor receptor). Unlike the cytoplasmic expression of LCN2, the loss of nuclear expression was a significant predictor of poor outcome. The combinatorial expression tumours with high cytoplasmic and low nuclear expression were associated with the worst prognosis. CONCLUSIONS: Tumour cell expression of LCN2 plays a role in breast cancer progression with loss of its nuclear expression which is associated with aggressive features and poor outcome. Further functional analysis is warranted to confirm the relationship between the subcellular localisation LCN2 and behaviour of breast cancer.

Phase II study of sequential S-1 and cyclophosphamide therapy in patients with metastatic breast cancer.

BACKGROUND: S-1 and cyclophosphamide (CPA) can be given orally, and their combination may have great potential for treating metastatic breast cancer (MBC). A phase I study of sequential S-1 and CPA therapy was conducted in patients with MBC; the recommended doses that were determined for this regimen were 80 mg/m2/day for S-1 and 100 mg/m2/day for CPA. We then conducted a phase II study of this oral S-1 and CPA regimen. METHODS: This was a single-arm, open-label, single-center prospective phase II study to evaluate the efficacy of a sequential S-1 and CPA regimen for MBC. S-1 was administered orally 2×/day for 14 consecutive days, and then CPA was administered orally 2×/day for 14 consecutive days in a repeating 4-week cycle (S-1 for 2 weeks, CPA for 2 weeks). The primary endpoint was the overall response rate (ORR). Secondary endpoints included the overall survival (OS), progression-free survival (PFS), clinical benefit rate (CBR) and safety. RESULTS: Thirty-six patients were enrolled in this study. The overall response was complete response in 0 (0%), partial response in 12 (33.3%), stable disease in 12 (33.3%), and progressive disease in 11 (30.1%) patients. The ORR was 33.3% (12/36). The CBR was 66.7% (24/36). The median PFS was 9.5 months (95%CI: 7.8-12.6 months). The median OS was 20.2 months (95%CI: 15.0-25.4 months) Grade 3/4 adverse events included leukopenia in seven patients (19.4%). Dose reductions because of adverse events occurred in 12 patients (33.3%). There was no treatment-related mortality. CONCLUSION: The combination of sequential therapy with S-1 and CPA was tolerable and had efficacy with good disease control. Sequential therapy with S-1 and CPA may be a feasible new treatment option for patients with MBC; however, further study is warranted to explore the efficacy of this therapy. TRIAL REGISTRATION: JRCT, JRCTs031180296 . Registered 2 December 2019 - Retrospectively registered.

Difference in LET-based biological doses between IMPT optimization techniques: Robust and PTV-based optimizations.

PURPOSE: While a large amount of experimental data suggest that the proton relative biological effectiveness (RBE) varies with both physical and biological parameters, current commercial treatment planning systems (TPS) use the constant RBE instead of variable RBE models, neglecting the dependence of RBE on the linear energy transfer (LET). To conduct as accurate a clinical evaluation as possible in this circumstance, it is desirable that the dosimetric parameters derived by TPS ( D RBE = 1.1 ) are close to the "true" values derived with the variable RBE models ( D v RBE ). As such, in this study, the closeness of D RBE = 1.1 to D v RBE was compared between planning target volume (PTV)-based and robust plans. METHODS: Intensity-modulated proton therapy (IMPT) treatment plans for two Radiation Therapy Oncology Group (RTOG) phantom cases and four nasopharyngeal cases were created using the PTV-based and robust optimizations, under the assumption of a constant RBE of 1.1. First, the physical dose and dose-averaged LET (LETd ) distributions were obtained using the analytical calculation method, based on the pencil beam algorithm. Next, D v RBE was calculated using three different RBE models. The deviation of D v RBE from D RBE = 1.1 was evaluated with D99 and Dmax , which have been used as the evaluation indices for clinical target volume (CTV) and organs at risk (OARs), respectively. The influence of the distance between the OAR and CTV on the results was also investigated. As a measure of distance, the closest distance and the overlapped volume histogram were used for the RTOG phantom and nasopharyngeal cases, respectively. RESULTS: As for the OAR, the deviations of D max v RBE from D max RBE = 1.1 were always smaller in robust plans than in PTV-based plans in all RBE models. The deviation would tend to increase as the OAR was located closer to the CTV in both optimization techniques. As for the CTV, the deviations of D 99 v RBE from D 99 RBE = 1.1 were comparable between the two optimization techniques, regardless of the distance between the CTV and the OAR. CONCLUSION: Robust optimization was found to be more favorable than PTV-based optimization in that the results presented by TPS were closer to the "true" values and that the clinical evaluation based on TPS was more reliable.

A key genomic subtype associated with lymphovascular invasion in invasive breast cancer.

BACKGROUND: Lymphovascular invasion (LVI) is associated with the development of metastasis in invasive breast cancer (BC). However, the complex molecular mechanisms of LVI, which overlap with other oncogenic pathways, remain unclear. This study, using available large transcriptomic datasets, aims to identify genes associated with LVI in early-stage BC patients. METHODS: Gene expression data from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort (n = 1565) was used as a discovery dataset, and The Cancer Genome Atlas (TCGA; n = 854) cohort was used as a validation dataset. Key genes were identified on the basis of differential mRNA expression with respect to LVI status as characterised by histological review. The relationships among LVI-associated genomic subtype, clinicopathological features and patient outcomes were explored. RESULTS: A 99-gene set was identified that demonstrated significantly different expression between LVI-positive and LVI-negative cases. Clustering analysis with this gene set further divided cases into two molecular subtypes (subtypes 1 and 2), which were significantly associated with pathology-determined LVI status in both cohorts. The 10-year overall survival of subtype 2 was significantly worse than that of subtype 1. CONCLUSION: This study demonstrates that LVI in BC is associated with a specific transcriptomic profile with potential prognostic value.

ß2-Adrenergic receptor expression is associated with biomarkers of tumor immunity and predicts poor prognosis in estrogen receptor-negative breast cancer.

PURPOSE: Antitumor immunity plays an important role in the progression of breast cancer. ß2-adrenergic receptor (ß2AR) was found to regulate the antitumor immune response and breast cancer progression in preclinical studies. To understand the clinical role of ß2AR in cancer progression, we investigated the clinicopathological and prognostic significance of ß2AR expression in invasive breast cancer. METHODS: ß2AR levels in breast tumors were evaluated by immunohistochemistry in a well-characterized patient cohort with long-term follow-up (n = 278). We evaluated the relationship of ß2AR expression to patient survival and clinicopathological factors, including immune biomarkers such as tumor-infiltrating lymphocytes (TILs) and programmed death ligand 1 (PD-L1) expression. Breast cancer-specific survival was compared between high- and low-ß2AR expression groups. RESULTS: Although ß2AR was not related to clinicopathological factors across the whole cohort, high ß2AR was significantly related to PD-L1 negativity in estrogen receptor (ER)-negative patients. Tumors with high ß2AR tended to have low TIL grade, and high ß2AR was an independent prognostic factor for reduced survival in ER-negative patients. CONCLUSIONS: ß2AR is an independent poor prognostic factor in ER-negative breast cancer. The findings suggest that tumor ß2AR regulates immune checkpoint activity, which may have therapeutic implications for patients with ER-negative breast cancer.

Utility of ankyrin 3 as a prognostic marker in androgen-receptor-positive breast cancer.

PURPOSE: Androgen receptor (AR) and AR signaling pathways are thought to play a role in breast cancer (BC) and are potentially related to treatment responses and outcomes. Ankyrin 3 (ANK3) is associated with AR stability in cancer cells. In the present study, we investigated the clinicopathological utility of ANK3 expression with emphasis on AR and its associated signalling pathway at transcriptomic and proteomic phases. PATIENTS AND METHODS: The Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort (n = 1980) and The Cancer Genome Atlas (TCGA) dataset (n = 1039) were used to assess the expression and significance of ANK3 mRNA and other AR signalling pathway-associated gene signature. Using immunohistochemistry, ANK3 protein expression was evaluated in large (n = 982) cohort of early-stage BC with long-term follow-up and compared with clinicopathological characteristics and its prognostic value in the whole cohort and the subgroups stratified by AR protein expression. RESULTS: An AR-related gene signature was developed, comprising 20 genes, which included ANK3. This AR-related gene signature was significantly associated with AR mRNA expression, oestrogen receptor, human epidermal growth factor receptor 2 (HER2) status and the patients' outcomes. In tumours with high AR protein expression (n = 614), high ANK3 protein expression was significantly associated with progesterone receptor positivity and it was independently associated with the good outcomes (p = 0.025). CONCLUSIONS: This study indicates that ANK3 is related to AR signalling pathway and is associated with BC prognosis.

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.

Clinicopathological and prognostic significance of Ras association and pleckstrin homology domains 1 (RAPH1) in breast cancer.

BACKGROUND: Ras association and pleckstrin homology domains 1 (RAPH1) is involved in cytoskeleton regulation and re-epithelialisation in invasive carcinoma and, therefore, may play a key role in carcinogenesis and metastasis. We, herein, investigated the biological and clinical significance of RAPH1 in breast cancer using large annotated cohorts. METHODS: The clinicopathological and prognostic significance of RAPH1 was assessed at the genomic and transcriptomic levels using The Cancer Genome Atlas (TCGA) dataset (n = 1039) and the results were validated using the Molecular taxonomy of breast cancer international consortium (METABRIC) cohort (n = 1980). RAPH1 protein expression was evaluated by immunohistochemistry in a large, well-characterised cohort of early-stage breast cancer (n = 1040). RESULTS: In both the TCGA and METABRIC cohorts, RAPH1 mRNA expression and RAPH1 copy number alteration were strongly correlated. RAPH1 mRNA overexpression was significantly correlated with high expression of adhesion and EMT markers including CDH1, TGFß1 and CD44. RAPH1 mRNA overexpression was a significant predictor of a poor prognosis (Hazard ratio 3.88; p = 0.049). High RAPH1 protein expression was associated with higher grade tumours with high proliferation index, triple negative phenotype and high E-cadherin expression. High RAPH1 protein expression was an independent predictor of shorter survival (Hazard ratio 4.37; p = 0.037). CONCLUSIONS: High RAPH1 expression is correlated with aggressive breast cancer phenotypes and provides independent prognostic value in invasive breast cancer.

Clinical and biological roles of Kelch-like family member 7 in breast cancer: a marker of poor prognosis.

BACKGROUND: The functions of many proteins are tightly regulated with a complex array of cellular functions including ubiquitination. In cancer cells, aberrant ubiquitination may promote the activity of oncogenic pathways with subsequent tumour progression. Kelch-like family member 7 (KLHL7) is involved in the regulation of ubiquitination and may play a role in breast cancer (BC). Present study aims to evaluate the biological and clinical usefulness of KLHL7 in BC utilising large well-characterised cohorts with long-term follow-up. METHODS: The relationships between KLHL7 gene copy number alteration (CNA) and mRNA expression and clinicopathological variables and clinical outcomes were evaluated in 1980 patients from the METABRIC BC cohort. Prognostic significance of KLHL7 mRNA was validated using the Breast Cancer Gene-Expression Miner v4.0 datasets (n = 5206). KLHL7 protein expression was assessed using immunohistochemistry in a large annotated series of early-stage BC (n = 917) with long-term follow-up. RESULTS: KLHL7 CNA was significantly correlated with its mRNA expression. KLHL7 mRNA expression was higher in luminal B and basal-like molecular subtypes and in higher grade tumours. Increased KLHL7 protein expression was significantly correlated with features of aggressive phenotype including lymphovascular invasion, high histological grade, hormonal receptor negativity, high PIK3CA and p53 expression. Outcome analysis showed that high KLHL7 expression is an independent predictor of shorter survival (p = 0.0011). CONCLUSIONS: KLHL7 appears to play an important role in BC progression. High KLHL7 protein expression identified a subgroup of BC with aggressive behaviour and provided independent prognostic information.

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.

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.

Establishment of a novel method to evaluate peritoneal microdissemination and therapeutic effect using luciferase assay.

Peritoneal dissemination is a major cause of recurrence in patients with malignant tumors in the peritoneal cavity. Effective anticancer agents and treatment protocols are necessary to improve outcomes in these patients. However, previous studies using mouse models of peritoneal dissemination have not detected any drug effect against peritoneal micrometastasis. Here we used the luciferase assay to evaluate peritoneal micrometastasis in living animals and established an accurate mouse model of early peritoneal microdissemination to evaluate tumorigenesis and drug efficacy. There was a positive correlation between luminescence intensity in in vivo luciferase assay and the extent of tumor dissemination evaluated by ex vivo luciferase assay and mesenteric weight. This model has advantages over previous models because optimal luciferin concentration without cell damage was validated and peritoneal microdissemination could be quantitatively evaluated. Therefore, it is a useful model to validate peritoneal micrometastasis formation and to evaluate drug efficacy without killing mice.

Control of primary lesions using resection or radiotherapy can improve the prognosis of metastatic colorectal cancer patients.

BACKGROUND: Control of the primary lesions in metastatic colorectal cancer (mCRC) is still controversial. For rectal cancer patients, not only resection but also irradiation is expected to provide palliative effects. We investigated the effects of resection and irradiation of primary lesions (local control) on the prognosis of mCRC patients. PATIENTS: Forty-seven patients with mCRC at our institute were examined, with 34 in the local controlled group and 13 in the uncontrolled group. RESULTS: The median survival time (MST) of the local controlled and uncontrolled groups were 2.90 and 1.39 years (P = 0.028). Cox proportional hazard regression analysis showed that local control was an independent prognostic factor (P < 0.05). The patients who underwent primary lesion resection had significantly longer MST (2.90 vs. 1.39 years, P = 0.032) than those in the uncontrolled group. In rectal cancer patients, the patients who underwent irradiation to control the primary lesions had a significantly longer MST than the uncontrolled patient group (1.97 vs. 1.39 years, P = 0.019). CONCLUSIONS: Local control of primary lesions may improve the prognosis in mCRC patients. In rectal cancer patients with metastasis, not only resection but also irradiation of the primary lesions may be a useful therapeutic strategy. J. Surg. Oncol. 2016;114:75-79. © 2016 Wiley Periodicals, Inc.

KPNA2 over-expression is a potential marker of prognosis and therapeutic sensitivity in colorectal cancer patients.

BACKGROUND: Karyopherin α 2 (KPNA2) is a member of the Karyopherin α family and has recently been reported to play an important role in tumor progression. The aim of the current study was to elucidate the clinicopathological significance of KPNA2 over-expression in colorectal cancer (CRC). PATIENTS AND METHODS: KPNA2 expression was evaluated by immunohistochemistry in 122 surgically resected CRC and 13 biopsy specimens obtained at colonoscopy during screening for preoperative hyperthermochemoradiation therapy (HCRT). The association between KPNA2 expression and clinicopathological features and preoperative HCRT efficacy were examined. RESULTS: The high and low KNPA2 expression groups were comprised of 91 (74.6%) and 31 CRC patients, respectively. A significant association was observed between high expression and lymphatic invasion (P = 0.0245). KPNA2 high expression group had decreased overall survival (P = 0.00374). Multivariate analysis demonstrated high KPNA2 expression was independently associated with poor prognosis. Histological examinations revealed 11 (84.6%) and 2 (15.4%) of cases were KPNA2 positive and negative, respectively. Pathological complete response (pCR) was observed in 9.1% of KPNA2-positive cases and 100% of KPNA2-negative cases. CONCLUSION: High KPNA2 expression was found to be associated with poor prognosis and resistance to HCRT.