Endosomal protein expression of γ1-adaptin is associated with tumor growth activity and relapse-free survival in breast cancer.

BACKGROUND: γ1-Adaptin is a subunit of adaptor protein complex-1 (AP-1), which regulates intracellular transport between the trans-Golgi network (TGN) and endosomes. Since expression levels of AP-1 subunits have been reported to be associated with cell proliferation and cancer malignancy, we investigated the relationships between the immunohistochemical expression of γ1-adaptin and both clinicopathological factors and relapse-free survival (RFS) in breast cancer tissue. MATERIALS AND METHODS: SK-BR-3 cell line depleted of γ1-adaptin was used for cell proliferation, migration, and invasion assay. Intracellular localization of γ1-adaptin was examined with immunohistochemistry (IHC) using an antibody against γ1-adaptin, and with double immunohistofluorescence (IHF) microscopy using markers for the TGN and endosome. γ1-Adaptin intensities in IHC samples from 199 primary breast cancer patients were quantified and assessed in relation to clinicopathological factors and RFS. RESULTS: Cell growth, migration, and invasion of SK-BR-3 cells were significantly suppressed by the depletion of γ1-adaptin. Although the staining patterns in the cancer tissues varied among cases by IHC, double IHF demonstrated that γ1-adaptin was mainly localized in EEA1-positive endosomes, but not in the TGN. γ1-Adaptin intensity was significantly higher in the tumor regions than in non-tumor regions. It was also higher in patients with Ki-67 (high), ER (-), PgR (-), and HER2 (+). Among subtypes of breast cancer, γ1-adaptin intensity was higher in HER2 than in luminal A or luminal B. The results of the survival analysis indicated that high γ1-adaptin intensity was significantly associated with worse RFS, and this association was also observed in group with ER (+), PgR (+), HER2 (-), Ki-67 (high), or luminal B. In addition, the Cox proportional hazards model showed that high γ1-adaptin intensity was an independent prognostic factor. CONCLUSION: These results suggest that the endosomal expression of γ1-adaptin is positively correlated with breast cancer malignancy and could be a novel prognostic marker.

E3 Ubiquitin Ligase NEDD4 Affects Estrogen Receptor α Expression and the Prognosis of Patients with Hormone Receptor-Positive Breast Cancer.

Neural precursor cell-expressed developmentally downregulated 4-1 (NEDD4) is an E3 ligase that leads to the degradation of proteins, including estrogen receptor α. We evaluated whether the expression level of NEDD4 affected the outcome of breast cancer patients. We performed a retrospective cohort study enrolling 143 patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative early breast cancer. Of the 66 patients with high NEDD4 mRNA levels (high NEDD4 group) and 77 patients with low NEDD4 mRNA levels (low NEDD4 group), 98.4% and 96.1%, respectively, of the patients had received neoadjuvant/adjuvant hormone therapy. Disease-free survival and overall survival were significantly longer in the low NEDD4 group than in the high NEDD4 group (p = 0.048 and p = 0.022, respectively). Western blotting revealed a high expression of estrogen receptor α in the NEDD4-knockdown culture cells. The proliferation of NEDD4-knockdown cells treated with tamoxifen or estradiol deprivation was suppressed, compared with that of NEDD4-expressing cells. Knockdown of NEDD4 in breast cancer cells induced the accumulation of estrogen receptor α and increased sensitivity to hormone therapy. In summary, this mechanism may lead to a better prognosis in hormone receptor-positive breast cancer patients with a low expression of NEDD4.

[A Case Report of Uterine Metastasis Four Years after Breast Surgery for Breast Cancer].

We report a case of uterine metastasis of the breast cancer. The patient was diagnosed with invasive ductal carcinoma of the breast and underwent partial right mastectomy and sentinel lymph node biopsy. Tamoxifen was administered as adjuvant endocrine therapy. Four years after the surgery, she had irregular genital bleeding, and was referred to our hospital for cytological diagnosis of uterine cancer. Postoperative pathological diagnosis showed uterine metastasis of breast cancer, and it was decided to treat the recurrence of breast cancer with aromatase inhibitors and CDK4/6 inhibitors, a molecular targeted therapy. The patient has been progression-free for 5 months.

Prognostic impact of interleukin-6 and C-reactive protein on patients with breast cancer.

The prognostic impacts of preoperative C-reactive protein (CRP) and interleukin (IL)-6 expression levels in patients with breast cancer remain controversial. A total of 55 female patients with invasive breast cancer were enrolled, and preoperative prognostic parameters including IL-6 and CRP were analyzed. Overall survival (OS) and recurrence-free survival (RFS) were estimated using the Kaplan-Meier method, and candidates' prognostic factors were examined using a Cox proportional hazard model. Using receiver operating characteristic curve analysis, IL-6 at 10.0 pg/ml and CRP at 0.12 mg/dl were determined as threshold values to predict OS and RFS, respectively. Patients with IL-6 ≥10.0 pg/ml had poorer OS compared with those with IL-6 <10.0 pg/ml (P=0.003), and patients with CRP ≥0.12 mg/dl had poorer RFS compared with those with CRP <0.12 mg/dl (P<0.001). Serum IL-6 level (hazard ratio, 13.230; 95% confidence interval, 1.285-136.214; P=0.030) and triple-negative subtype (hazard ratio, 11.739; 95% confidence interval, 1.415-97.362; P=0.023) were independent prognostic factors for OS, and CRP expression level was an independent prognostic factor for RFS in patients with breast cancer (hazard ratio, 18.571; 95% confidence interval, 2.240-153.949; P=0.007). In patients with invasive breast cancer, preoperative serum IL-6 and triple-negative subtype may be independent prognostic factors for OS, while for RFS, preoperative CRP may be a more accurate prognostic factor compared with those currently established.

miRNA-148a-3p Regulates Immunosuppression in DNA Mismatch Repair-Deficient Colorectal Cancer by Targeting PD-L1.

Immunotherapy against the interaction between programmed cell death 1/programmed cell death ligand 1 (PD-L1) has emerged as a promising strategy for colorectal cancer with mismatch repair deficiency (dMMR) or microsatellite instability-high (MSI-H). The study aimed to identify miRNAs that posttranscriptionally control PD-L1 expression on tumor cells and also regulate immune evasion. A comprehensive miRNA screening using The Cancer Genome Atlas (TCGA) dataset (n = 260) combined with eight different miRNA target prediction programs resulted in the identification of a tumor suppressive miRNA, miR-148a-3p, as a potential negative regulator of PD-L1 expression, particularly in dMMR/MSI-H colorectal cancer. Using multiple cohorts of colorectal cancer, including TCGA data, a microarray dataset (n = 148), and formalin-fixed, paraffin-embedded samples (n = 395), we found that the expression of miR-148a-3p was decreased in dMMR/MSI-H tumors, correlating inversely with PD-L1 levels. We demonstrate that miR-148a-3p directly binds to the 3'-untranslated region of PD-L1, thereby reducing whole-cell and cell surface PD-L1 levels in HCT116 and SW837 cell lines. Overexpression of miR-148a-3p repressed IFNγ-induced PD-L1 expression on tumor cells and consequently diminished T-cell apoptosis in a coculture model of IL2-activated T cells and IFNγ-treated tumor cells. In conclusion, our data support a regulatory mechanism of PD-L1 expression on tumor cells and immune suppression via miR-148a-3p downregulation in colorectal cancer. IMPLICATIONS: This study provides novel evidence that miR-148a-3p negatively regulates tumor cell PD-L1 expression and decreased levels of miR-148a-3p contributes to the immunosuppressive tumor microenvironment.

[A Case of Synchronous Bilateral Neuroendocrine Ductal Carcinoma In Situ].

Neuroendocrine ductal carcinoma in situ(NE-DCIS)is a unique subtype of ductal carcinoma in situ(DCIS)that is not described in the general rules for clinical and pathological recording of breast cancer. NE-DCIS is described as an unusual variant of DCIS in the 2012 World Health Organization(WHO)classification. The chief complaint in NE-DCIS is hemorrhagic nipple discharge. The histological characteristics of NE-DCIS are solid growth of cancer cells with granular and spindle-shaped nuclei. Histologically, NE-DCIS is suggestive of low malignancy but a poor prognosis of neuroendocrine carcinoma of the breast has been reported. The report by Honami et al was the only other report of synchronous bilateral neuroendocrine ductal carcinoma in situ. We report the second case of NE-DCIS diagnosed synchronously in both breasts in a patient who had visited our outpatient clinic with hemorrhagic nipple discharge.

Prognostic role of FUT8 expression in relation to p53 status in stage II and III colorectal cancer.

The expression of fucosyltransferase 8, an enzyme responsible for core fucosylation encoded by FUT8, influences tumor biology and correlates with patient prognosis in several solid cancers. We hypothesized that p53 alteration modifies prognostic associations of FUT8 expression in colorectal cancer (CRC), since FUT8 has recently been identified as a direct transcriptional target of wild-type p53. Utilizing multiple datasets of microarray and RNA sequence of CRC, FUT8 mRNA was found to be highly expressed in wild-type p53 tumors (n = 382) compared to those of mutant p53 (n = 437). Prognostic values of FUT8 expression in conjunction with the p53 status for disease-free survival (DFS) were analyzed using two independent cohorts of stage II and III CRC after curative surgery, including the immunohistochemistry (IHC) cohort (n = 123) and the microarray cohort (n = 357). In both cohorts, neither FUT8 expression nor the p53 status was associated with DFS. Strikingly, positive expression of FUT8 protein was significantly associated with better DFS only in tumors with negative p53, while it had no prognostic impact in tumors with positive p53 in the IHC cohort. Although not statistically significant, a similar prognostic trend was observed in the microarray cohort when patients were stratified by the p53 status. Our results suggest that the prognostic values of FUT8 expression on DFS may be modified by the p53 status, and the expression of FUT8 protein can be a prognostic biomarker for patients with stage II and III CRC.

Glycosyltransferase Gene Expression Identifies a Poor Prognostic Colorectal Cancer Subtype Associated with Mismatch Repair Deficiency and Incomplete Glycan Synthesis.

Purpose: We aimed to discover glycosyltransferase gene (glycogene)-derived molecular subtypes of colorectal cancer associated with patient outcomes.Experimental Design: Transcriptomic and epigenomic datasets of nontumor, precancerous, cancerous tissues, and cell lines with somatic mutations, mismatch repair status, clinicopathologic and survival information were assembled (n = 4,223) and glycogene profiles were analyzed. IHC for a glycogene, GALNT6, was conducted in adenoma and carcinoma specimens (n = 403). The functional role and cell surface glycan profiles were further investigated by in vitro loss-of-function assays and lectin microarray analysis.Results: We initially developed and validated a 15-glycogene signature that can identify a poor-prognostic subtype, which closely related to deficient mismatch repair (dMMR) and GALNT6 downregulation. The association of decreased GALNT6 with dMMR was confirmed in multiple datasets of tumors and cell lines, and was further recapitulated by IHC, where approximately 15% tumors exhibited loss of GALNT6 protein. GALNT6 mRNA and protein was expressed in premalignant/preinvasive lesions but was subsequently downregulated in a subset of carcinomas, possibly through epigenetic silencing. Decreased GALNT6 was independently associated with poor prognosis in the IHC cohort and an additional microarray meta-cohort, by multivariate analyses, and its discriminative power of survival was particularly remarkable in stage III patients. GALNT6 silencing in SW480 cells promoted invasion, migration, chemoresistance, and increased cell surface expression of a cancer-associated truncated O-glycan, Tn-antigen.Conclusions: The 15-glycogene signature and the expression levels of GALNT6 mRNA and protein each serve as a novel prognostic biomarker, highlighting the role of dysregulated glycogenes in cancer-associated glycan synthesis and poor prognosis. Clin Cancer Res; 24(18); 4468-81. ©2018 AACR.

Augmentation of antibody-dependent cellular cytotoxicity with defucosylated monoclonal antibodies in patients with GI-tract cancer.

Enhancement of antibody-dependent cellular cytotoxicity (ADCC) with some modalities may be a promising approach to enhance the efficacy of therapeutic monoclonal antibodies (mAbs). It has previously been demonstrated that the removal of fucose from antibody oligosaccharides (defucosylation) leads to augmentation of ADCC activity. To establish clinically relevant evidence of this procedure, the present study evaluated trastuzumab- and cetuximab-mediated ADCC by comparing defucosylated mAbs with conventional mAbs using peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from 20 patients with gastrointestinal tract cancer and 10 healthy volunteers. ADCCs were measured using PBMCs as effector cells and two gastric cancer cell lines as target cells. ADCCs were significantly enhanced with defucosylated mAbs compared with conventional mAbs using PBMC from the healthy donors and patients with cancer. The results confirmed that the cetuximab- and trastuzumab-mediated ADCCs in advanced disease were impaired in comparison to those in early disease or healthy individuals. However, when the defucosylated mAbs were used instead of the conventional mAbs, the ADCC activities in the advanced cases were almost comparable with those in early disease or healthy individuals. Furthermore, the expression of ADCC associated molecules were modified toward immunosuppressive status with a mitogen-activated protein kinase inhibitor in vitro, the conventional cetuximab- and trastuzumab-mediated ADCC was downregulated, and the defucosylated mAbs overcome the downregulation of ADCC. In conclusion, defucosylated therapeutic mAbs may enhance ADCC activities in patients with cancer, which may lead to more effective anti-cancer treatments.

[A Case of Encapsulated Papillary Carcinoma of the Breast].

An 84-year-old woman was revealed to have focal asymmetric density(FAD)based on mammography, and ultrasonography showed a 0.5 cm sized cyst in the left breast. It gradually increased in size and contained solid components. A core needle biopsy revealed an intracystic papillary carcinoma of the breast. Partial mastectomy and sentinel lymph node biopsy were performed. Histopathological examination revealed an encapsulated papillary carcinoma and a papillary lesion surrounded by a thick fibrous capsule. Myoepithelial cells were not found at the periphery of the lesion or within fibrovascular cores. Currently, it is classified as non-invasive carcinoma, and the patient has a good prognosis.

Immunogenic tumor cell death induced by chemotherapy in patients with breast cancer and esophageal squamous cell carcinoma.

It has been reported that chemo-radiotherapy can induce immunogenic tumor cell death (ICD), which triggers T-cell immunity mainly mediated by high-mobility group box 1 protein (HMGB1) and calreticulin. However, there is still limited information to support this theory relating to chemotherapy alone. In the present study, the expression of HMGB1 and calreticulin was evaluated by immunohistochemistry in pre-treatment biopsy specimens and surgically resected specimens, which were obtained from patients with breast cancer (n=52) and esophageal squamous cell carcinoma (ESCC) (n=8) who had been treated with neoadjuvant chemotherapy (NAC). We also analyzed HMGB1 and calreticulin expression in breast cancer cell lines treated with chemotherapeutic drugs. As a result, both HMGB1 and calreticulin expression levels were significantly upregulated after NAC in both breast cancer and ESCC tissues. However, no significant correlation was observed between HMGB1 expression and pathological response after NAC or between HMGB1 expression and patient survival. Furthermore, although overall survival in the high infiltration group of CD8-positive T cells was significantly superior to that in the low infiltration group in breast cancer patients, there were no correlations between the number of CD8-positive T cells and HMGB1 or calreticulin expression levels. In addition, chemotherapeutic drugs induced upregulation of HMGB1 and calreticulin in all tested cell lines. Our findings indicate that chemotherapy alone can significantly induce ICD regardless of the degree of pathological response after chemotherapy.

[Regulation of PD-L1 by MicroRNA in Mismatch Repair Deficient-Colorectal Cancer].

Programmed cell death 1(PD-1)/PD-ligand 1(PD-L1)immune checkpoint blockade has emerged as a promising therapeutic strategy in various types of cancer. In a recent phase II clinical trial, treatment with the anti-PD-1 agent, pembrolizumab, resulted in considerable clinical benefit in patients with mismatch repair(MMR)-deficient colorectal cancer(CRC). Upregulation of PD-1on T-cells and PD-L1 on tumor cells induces inhibitory signals to suppress T-cell activation, leading to an immune-suppressive microenvironment particularly in MMR-deficient tumors. However, the regulation of PD-L1 expression on CRC cells is poorly understood. We hypothesized that certain microRNAs(miRNAs)are involved in the immunosuppressive microenvironment by directly targeting PD-L1. We identified candidate miRNAs by RNA-sequence analyses for mRNA and miRNA expression obtained from the TCGA colon adenocarcinoma database combined with miRNA target prediction programs. We found that forced miRNA expression could decrease PD-L1 expression on cancer cell lines. Our findings may facilitate an understanding of the role of miRNAs in PD-L1 regulation and also suggest potential miRNAs to serve as biomarkers and therapeutic targets for cancer immunotherapy.

[A Case of Occult Breast Cancer].

We report a case of occult breast cancer. A 61-years-old woman underwent tumorectomy of right axillary mass. Pathological diagnosis was adenocarcinoma. Two years after, right axillary mass was discovered again. Wide local excision, axillary lymph node dissection and radiation therapy of the breast was performed. Pathological findings showed lymph node metastasis of breast cancer, or primary cancer of the axially tail of the breast. Ten months after second operation, she presented an axillary mass again. She underwent resection of the axillary tumor. The pathological findings showed lymph node metastasis of breast cancer. There was no evidence of primary tumor of the breast during the period. We suspected lymph node metastasis of occult breast cancer. Irradiation was administered to the right axilla, and she is receiving endocrine therapy.

[A Case of Breast Carcinoma Associated with Osteoclast-Like Giant Cells].

Mammary carcinoma with osteoclast-like giant cells is uncommon, and its onset mechanism and malignancy are unknown. We report a case of mammary carcinoma with osteoclast-like giant cells. A 41-year-old woman noticed a lump in her left breast. Ultrasound sonography findings suggested breast cancer. A core needle biopsy revealed invasive ductal carcinoma of the breast. Modified radicalmastectomy and sentinell ymph node biopsy were performed. Histopathologicalexamination revealed papillotubular carcinoma with osteoclast-like giant cells. Cells were positive for estrogen receptor and progesterone, and negative for HER2. MIB-1 index was under 5%. The giant cells were generally associated with an inflammatory, fibroblastic, hyper-vascular stroma. The carcinomatous part of the lesion was most frequently a well-to moderately differentiated invasive ductalcarcinoma. Immunohistochemicaland ultrastructuralstudies suggested that the osteoclast-like giant cells were of stromalhistiocytic origin. To understand biochemicalfindings of this carcinoma, more case studies are required to be reported.

Association between circulating galectin-3 levels and the immunological, inflammatory and nutritional parameters in patients with colorectal cancer.

Galectin-3, a ß-galactoside-binding lectin, exhibits pleiotropic biological functions and has a role as one of the immunological modulators. However, the associations between circulating galectin-3 and immunological, inflammatory and nutritional parameters have not yet been fully elucidated. The serum concentration of galectin-3 was examined in association with interleukin-10 (IL-10), IL-12 and IL17 production, lymphocyte stimulation, neutrophil/lymphocyte ratio (NLR), white blood cell count (WBC), C-reactive protein (CRP) and rapid turnover proteins, including retinol-binding protein (RBP), prealbumin (PA) and transferrin (TF) in 50 patients with untreated colorectal cancers. Significant increases (P<0.05) were observed in the serum galectin-3 levels in patients with untreated colorectal cancer (9.6±4.5 ng/ml) compared with the normal controls (3.2±1.6 ng/ml). Higher serum galectin-3 concentrations were observed in patients with colon cancer (11.5±4.4 ng/ml) compared to in patients with rectal cancer (8.0±4.0 ng/ml) (P=0.005). The levels of circulating galectin-3 inversely correlated with the production of IL-10 (r=-0.59, P<0.001), and IL-12 (r=-0.69, P<0.001). Galectin-3 concentration also inversely correlated with the lymphocyte stimulation assay stimulation index (r=-0.42, P=0.021). However, the level of serum galectin-3 correlated with IL-17 production (r=0.67, P<0.001). Serum galectin-3 levels exhibited significant correlations with NLR (r=0.41, P=0.009), WBC (r=0.32, P=0.035), and CRP (r=0.63, P<0.001), and statistically significant inverse correlations with RBP (r=-0.45, P=0.002), PA (r=-0.46, P=0.001) and TF (r=-0.72, P<0.001). Galectin-3 may be one of the key factors in the regulation of immunological, inflammatory and nutritional conditions.

Stromal VCAN expression as a potential prognostic biomarker for disease recurrence in stage II-III colon cancer.

To develop prognostic biomarkers that can discriminate stage II-III colorectal cancer patients with high risk of postoperative recurrence, we conducted a genome-wide screening of relapse-related genes utilizing multiple microarray cohorts. Among differentially expressed genes between tumor and nontumor, we identified eight candidate genes associated with relapse in two datasets of stage II-III patients (n = 94 and 145, respectively, P < 0.05). Using datasets of laser-microdissected samples and FACS-purified cell populations, the localization of candidate genes, including COL4A2, COL4A1, VCAN and SERPINE1, were found predominantly in cancer stroma rather than epithelial components. Among those relapse-related stromal genes, VCAN mRNA, specifically expressed in cancer-associated fibroblasts, was further validated to be a prognostic factor in two additional independent datasets, consisting of 453 (P = 0.0334) and 89 (P = 0.0041) stage II-III patients. Furthermore, in our large set of formalin-fixed paraffin-embedded cohort (n = 338), VCAN protein was detected exclusively in cancer stroma by immunohistochemistry, demonstrating a stepwise increase of stromal VCAN from normal tissues through stage 0 to stage IV tumors. Stromal VCAN protein was associated with shorter relapse-free survival (RFS) in stage II-III colon cancer, independent of other clinical factors by multivariate analysis (P = 0.004). Stratified analyses revealed that stromal VCAN was a strong prognostic indicator particularly in stage II colon cancer (P = 0.0029). In all five analyzed cohorts, the expression of VCAN, in transcript or protein levels, was associated with poor RFS in stage II-III patients. We conclude that VCAN is a promising biomarker to identify stage II-III patients at high risk of relapse who may benefit from intensive postoperative management.