Correlation of Vein-Rich Tumor Microenvironment of Intrahepatic Cholangiocarcinoma With Tertiary Lymphoid Structures and Patient Outcome.

Intrahepatic cholangiocarcinoma (iCCA) is an aggressive cancer composed of large-duct and small-duct types. Understanding the tumor immune microenvironment and its related vascular system is important for developing novel and efficient therapies. We focused on tertiary lymphoid structure (TLS) as a hallmark of antitumor immunity and investigated the clinicopathologic significance of TLSs and the influence of vascular microenvironment on TLS formation in iCCAs. We examined 261 iCCA cases clinicopathologically and analyzed the vascular system using immunohistochemistry. Single-cell (102,685 cells) and bulk RNA (33 iCCA cases) sequencing analyses were performed using data sets downloaded from public databases, and endothelial cell characteristics in iCCA tissues and functional networks related to the tumor microenvironment were bioinformatically examined. High densities of both intratumoral and peritumoral TLSs were significantly associated with prolonged survival only in large-duct-type iCCA. Multivariate analyses showed that peritumoral TLS was a prognostic factor for the large-duct type. TLS-rich iCCA had a significantly higher vein density and tumor-infiltrating T-cell count than TLS-poor iCCA. Both the presence of TLSs and high vein endothelial cells in iCCA tissues were significantly associated with molecular networks representing active immune responses in transcriptomic analysis. Vein density was a prognostic factor in patients with large-duct and small-duct types. This suggests that TLS formation is involved in a microenvironment with high vein density, which represents an antitumor-directed immune microenvironment.

Novel Insights Into Immunohistochemical Analysis For Acinar Cell Neoplasm of The Pancreas: Carboxypeptidase A2, Carboxypeptidase A1, and Glycoprotein 2.

Acinar cell carcinoma (ACC) is a rare and highly malignant pancreatic tumor. Owing to histologic similarity, ACC is often difficult to distinguish from other solid medullary pancreatic tumors, particularly neuroendocrine neoplasm (NEN) and intraductal tubulopapillary neoplasm (ITPN). We aimed to identify new immunohistochemical markers commonly expressed in tumor cells with acinar cell differentiation and useful for both surgical and small biopsy specimens. Candidate molecules exclusively expressed in neoplastic or non-neoplastic acinar cells in pancreatic tissues with specific and available antibodies suitable for immunohistochemistry were selected. We selected carboxypeptidase A1 (CPA1), carboxypeptidase A2 (CPA2), and glycoprotein 2 (GP2), which were expressed in 100%, 100%, and 96% of cases, respectively, in ACC (n=27) or neoplasia with acinar cell differentiation, including mixed acinar-neuroendocrine carcinoma (n=9), mixed acinar-ductal carcinoma (n=3), pancreatoblastoma (n=4), and acinar cystic transformation (n=2), in the cytoplasm of tumor cells with a granular pattern. Both CPA2 and CPA1 were not expressed in any other tumors without acinar cell differentiation, including NEN (n=44), pancreatic ductal adenocarcinoma (n=44), and ITPN (n=4). GP2 was not expressed in these tumors except in rare cases, including 14% of NEN, 15% of intraductal papillary-mucinous neoplasm, 25% of intraductal oncocytic papillary neoplasm, 25% of ITPN, and 7% of pancreatic ductal adenocarcinoma, wherein a small proportion of tumor cells expressed GP2 in their apical cell membrane. NEN cases also showed cytoplasmic GP2 expression. Therefore, CPA2, CPA1, and potentially GP2 may act as ACC markers.

Neoadjuvant therapy alters the collagen architecture of pancreatic cancer tissue via Ephrin-A5.

BACKGROUND: The treatment of pancreatic cancer (PDAC) remains clinically challenging, and neoadjuvant therapy (NAT) offers down staging and improved surgical resectability. Abundant fibrous stroma is involved in malignant characteristic of PDAC. We aimed to investigate tissue remodelling, particularly the alteration of the collagen architecture of the PDAC microenvironment by NAT. METHODS: We analysed the alteration of collagen and gene expression profiles in PDAC tissues after NAT. Additionally, we examined the biological role of Ephrin-A5 using primary cultured cancer-associated fibroblasts (CAFs). RESULTS: The expression of type I, III, IV, and V collagen was reduced in PDAC tissues after effective NAT. The bioinformatics approach provided comprehensive insights into NAT-induced matrix remodelling, which showed Ephrin-A signalling as a likely pathway and Ephrin-A5 (encoded by EFNA5) as a crucial ligand. Effective NAT reduced the number of Ephrin-A5+ cells, which were mainly CAFs; this inversely correlated with the clinical tumour shrinkage rate. Experimental exposure to radiation and chemotherapeutic agents suppressed proliferation, EFNA5 expression, and collagen synthesis in CAFs. Forced EFNA5 expression altered CAF collagen gene profiles similar to those found in PDAC tissues after NAT. CONCLUSION: These results suggest that effective NAT changes the extracellular matrix with collagen profiles through CAFs and their Ephrin-A5 expression.

Association between the expression of core 3 synthase and survival outcomes of patients with cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a highly aggressive and metastatic type of malignant carcinoma that is associated with high mortality rates and is difficult to detect at early stages. Core 3 structure is a mucin-type O-glycans synthesized by ß1,3-N-acetylglucosaminyltransferase 6 (core 3 synthase), which plays an important role in the digestive system, in particular gastrointestinal goblet cells. It has been reported that core 3 synthase-expressing cells show lower migratory and invasive rates, and lower metastatic activity. A immunohistochemical study also showed that this enzyme was expressed in normal epithelial cells of the colon, but completely disappeared in colorectal cancer cells. The present study aimed to identify biomarkers that could be used to predict the prognosis of patients with CCA. Pathological specimens of 185 CCA tissues were immunohistochemically stained with two antibodies, G8-144 and MECA-79, which recognize core 3 synthase and 6-sulfated N-acetyllactosamine on the extended core-1 O-glycans, respectively. The association between G8-144 or MECA-79 positivity and patient prognosis was statistically analyzed. Positive expression of G8-144 was associated with improved prognosis in patients with distal CCA (dCCA). Patients with dCCA positive for G8-144 showed lower mortality rates than those with negative expression. However, the positive expression of MECA-79 was associated with CCA progression and metastasis, indicating that it is a poor prognostic marker for CCA. In conclusion, as both antibodies resulted in mirror-image staining, the involvement of G8-144 and MECA-79 in O-glycan synthesis could be considered as potential favorable and unfavorable biomarkers, respectively, for CCA prognosis.

Novel insights into immunohistochemical analysis for diagnosing serous neoplasm of the pancreas: aquaporin 1, stereocilin, and transmembrane protein 255B.

AIMS: Serous (cystic) neoplasm (SCN) of the pancreas is generally benign, and surgical treatment is recommended in only a limited number of cases. To avoid unnecessary surgery, an accurate diagnosis of SCN is essential. In the present study, we aimed to identify new immunohistochemical markers with which to distinguish SCN from other tumours. METHODS AND RESULTS: We compared the comprehensive gene expression profiles of SCN with those of normal pancreas and pancreatic ductal adenocarcinoma (PDAC). We selected the candidate molecules that were up-regulated in SCN, were minimally expressed or unexpressed in PDAC, and had specific and available antibodies suitable for immunohistochemistry, and then analysed their immunohistochemical expression in various tumours. We selected aquaporin 1 (AQP1), stereocilin (STRC), fibroblast growth factor receptor 3 (FGFR3), and transmembrane protein 255B (TMEM255B), which were diffusely expressed in SCN cells in 79%, 100%, 100% and 100% of SCN cases. AQP1 was not expressed in other tumours, except in 20% of mucinous cystic neoplasms (MCNs) and 19% of PDACs. STRC was rarely expressed in MCNs, neuroendocrine neoplasms (NENs), and PDACs. FGFR3 was expressed in 31% of intraductal papillary mucinous neoplasms (IPMNs), 50% of intraductal oncocytic papillary neoplasms, 40% of NENs, 30% of acinar cell carcinomas, 40% of solid pseudopapillary neoplasms, and 52% of PDACs. TMEM255B was not expressed in the other tumours, except in 50% of MCNs, 80% of gastric-subtype IPMNs, and 29% of PDACs. All antigens were usually expressed in a small proportion of cells when they were positive in tumours other than SCN. CONCLUSIONS: These findings indicate that AQP1 and STRC, and potentially TMEM255B, may act as SCN markers.

On-tissue polysulfide visualization by surface-enhanced Raman spectroscopy benefits patients with ovarian cancer to predict post-operative chemosensitivity.

Chemosensitivity to cisplatin derivatives varies among individual patients with intractable malignancies including ovarian cancer, while how to unlock the resistance remain unknown. Ovarian cancer tissues were collected the debulking surgery in discovery- (n = 135) and validation- (n = 47) cohorts, to be analyzed with high-throughput automated immunohistochemistry which identified cystathionine γ-lyase (CSE) as an independent marker distinguishing non-responders from responders to post-operative platinum-based chemotherapy. We aimed to identify CSE-derived metabolites responsible for chemoresistant mechanisms: gold-nanoparticle (AuN)-based surface-enhanced Raman spectroscopy (SERS) was used to enhance electromagnetic fields which enabled to visualize multiple sulfur-containing metabolites through detecting scattering light from Au-S vibration two-dimensionally. Clear cell carcinoma (CCC) who turned out less sensitive to cisplatin than serous adenocarcinoma was classified into two groups by the intensities of SERS intensities at 480 cm-1; patients with greater intensities displayed the shorter overall survival after the debulking surgery. The SERS signals were eliminated by topically applied monobromobimane that breaks sulfane-sulfur bonds of polysulfides to result in formation of sulfodibimane which was detected at 580 cm-1, manifesting the presence of polysulfides in cancer tissues. CCC-derived cancer cell lines in culture were resistant against cisplatin, but treatment with ambroxol, an expectorant degrading polysulfides, renders the cells CDDP-susceptible. Co-administration of ambroxol with cisplatin significantly suppressed growth of cancer xenografts in nude mice. Furthermore, polysulfides, but neither glutathione nor hypotaurine, attenuated cisplatin-induced disturbance of DNA supercoiling. Polysulfide detection by on-tissue SERS thus enables to predict prognosis of cisplatin-based chemotherapy. The current findings suggest polysulfide degradation as a stratagem unlocking cisplatin chemoresistance.

BP180 Is a Prognostic Factor in Head and Neck Squamous Cell Carcinoma.

BACKGROUND/AIM: Prognosis plays a vital role in head and neck squamous cell carcinoma (HNSCC) patient management and decision-making. This study aimed to identify the role of BP180 as a prognostic factor in HNSCC. PATIENTS AND METHODS: Protein expression of bullous pemphigoid antigen II (BP180) was verified by immunohistochemistry (IHC) in a tissue microarray study of 202 cases. RESULTS: IHC analysis revealed that protein expression of BP180 among HNSCC patients differed significantly in the presence and absence of neural invasion, and according to T status in laryngeal and pharyngeal cancer subgroups. Overall survival and multivariate analysis showed that positive BP180-IHC and advanced clinical stage were significant independent positive predictors of mortality in HNSCC patients. In addition, in the oral cancer subgroup, independent positive predictors were positive BP180-IHC, advanced N status and neural invasion. In laryngeal and pharyngeal cancer subgroups, predictors were positive BP180-IHC and advanced clinical stage. CONCLUSION: BP180 is a prognostic factor in head and neck squamous cell carcinoma.

IAP inhibitor, Embelin increases VCAM-1 levels on the endothelium, producing lymphocytic infiltration and antitumor immunity.

There is an increasing unmet need for successful immunotherapeutic interventions. Lymphocyte extravasation via tumor tissue endothelial cells (TECs) is required for lymphocyte infiltration into tumor sites. This study aimed to investigate the clinical significance of dysfunctional TECs in pancreatic ductal adenocarcinoma (PDAC) and identify chemical compounds that boost tumor-infiltrating lymphocyte (TIL) numbers. We performed immunohistochemical detection and clinicopathological analysis of VCAM-1 on TECs, which is essential for lymphocyte trafficking. We characterized the gene expression profiles of TECs from fresh PDAC tissues. We isolated compounds that upregulated VCAM-1 and E-selectin expression in TECs and examined their biological activities. Compared to endothelial cells from chronic pancreatitis tissue, TECs showed significantly lower VCAM-1 and E-selectin expression and significant weaknesses in lymphocyte adhesion and transmigration, resulting in decreased T cell infiltration around vessels. Patients with a relatively high percentage of VCAM-1+ vessels among all vessels in PDAC tissue had an improved prognosis. A bioinformatics survey demonstrated that TNFR1 signaling was involved in abnormal VCAM-1 and E-selectin expression in TECs. We screened compounds affecting TNFR1 signaling, and the IAP inhibitor, Embelin, induced these molecules on TECs and enhanced T cell adhesion to TECs and transmigration. Furthermore, in vivo, Embelin enhanced tumor-infiltrating T cell numbers, leading to an antitumor immune response. Embelin accelerates TIL infiltration and the antitumor immune response by recovering VCAM-1 expression in TECs. Our strategy may be a therapeutic approach for accelerating the immunotherapeutic response in immune-quiescent tumors, leading to clinical trials' success.

Clinicopathological significance of core 3 O-glycan synthetic enzyme, ß1,3-N-acetylglucosaminyltransferase 6 in pancreatic ductal adenocarcinoma.

Mucin-type O-glycans are involved in cancer initiation and progression, although details of their biological and clinicopathological roles remain unclear. The aim of this study was to investigate the clinicopathological significance of ß1,3-N-acetylglucosaminyltransferase 6 (ß3Gn-T6), an essential enzyme for the synthesis of core 3 O-glycan and several other O-glycans in pancreatic ductal adenocarcinoma (PDAC). We performed immunohistochemical and lectin-histochemical analyses to detect the expression of ß3Gn-T6 and several O-glycans in 156 cases of PDAC with pancreatic intraepithelial neoplasias (PanINs) and corresponding normal tissue samples. The T antigen, Tn antigen, sialyl Lewis X (sLeX) antigen, and sLeX on core 2 O-glycan were more highly expressed in PDAC cells than in normal pancreatic duct epithelial cells (NPDEs). Conversely, the expression of 6-sulfo N-acetyllactosamine on extended core 1 O-glycan was found in NPDEs and was low in PDAC cells. These glycan expression levels were not associated with patient outcomes. ß3Gn-T6 was expressed in ~20% of PDAC cases and 30-40% of PanINs but not in NPDEs. Higher expression of ß3Gn-T6 was found in PDAC cells in more differentiated adenocarcinoma cases showing significantly longer disease-free survival in both univariate and multivariate analyses. In addition, the expression of ß3Gn-T6 in PDAC cells and PanINs significantly correlated with the expression of MUC5AC in these cells, suggesting that ß3Gn-T6 expression is related to cellular differentiation status of the gastric foveolar phenotype. Thus, it is likely that ß3Gn-T6 expression in PDAC cells is a favorable prognostic factor in PDAC patients, and that the expression of ß3Gn-T6 correlates with the gastric foveolar phenotype in pancreatic carcinogenesis.

Expression of classical human leukocyte antigen class I antigens, HLA-E and HLA-G, is adversely prognostic in pancreatic cancer patients.

The expression of classical human leukocyte antigen class I antigens (HLA-I) on the surfaces of cancer cells allows cytotoxic T cells to recognize and eliminate these cells. Reduction or loss of HLA-I is a mechanism of escape from antitumor immunity. The present study aimed to investigate the clinicopathological impacts of HLA-I and non-classical HLA-I antigens expressed on pancreatic ductal adenocarcinoma (PDAC) cells. We performed immunohistochemistry to detect expression of HLA-I antigens in PDAC using 243 PDAC cases and examined their clinicopathological influences. We also investigated the expression of immune-related genes to characterize PDAC tumor microenvironments. Lower expression of HLA-I, found in 33% of PDAC cases, was significantly associated with longer overall survival. Higher expression of both HLA-E and HLA-G was significantly associated with shorter survival. Multivariate analyses revealed that higher expression of these three HLA-I antigens was significantly correlated with shorter survival. Higher HLA-I expression on PDAC cells was significantly correlated with higher expression of IFNG, which also correlated with PD1, PD-L1 and PD-L2 expression. In vitro assay revealed that interferon gamma (IFNγ) stimulation increased surface expression of HLA-I in three PDAC cell lines. It also upregulated surface expression of HLA-E, HLA-G and immune checkpoint molecules, including PD-L1 and PD-L2. These results suggest that the higher expression of HLA-I, HLA-E and HLA-G on PDAC cells is an unfavorable prognosticator. It is possible that IFNγ promotes a tolerant microenvironment by inducing immune checkpoint molecules in PDAC tissues with higher HLA-I expression on PDAC cells.

A Novel Role of Interleukin 13 Receptor alpha2 in Perineural Invasion and its Association with Poor Prognosis of Patients with Pancreatic Ductal Adenocarcinoma.

Perineural invasion (PNI) is one of the major pathological characteristics of pancreatic ductal adeno-carcinoma (PDAC), which is mediated by invading cancer cells into nerve cells. Herein, we identify the overexpression of Interleukin-13 Receptor alpha2 (IL-13Rα2) in the PNI from 236 PDAC samples by studying its expression at the protein levels by immunohistochemistry (IHC) and the RNA level by in situ hybridization (ISH). We observe that ≥75% samples overexpressed IL-13Rα2 by IHC and ISH in grade 2 and 3 tumors, while ≥64% stage II and III tumors overexpressed IL-13Rα2 (≥2+). Interestingly, ≥36 % peripancreatic neural plexus (PL) and ≥70% nerve endings (Ne) among PNI in PDAC samples showed higher levels of IL-13Rα2 (≥2+). IL-13Rα2 +ve PL and Ne subjects survived significantly less than IL-13Rα2 -ve subjects, suggesting that IL-13Rα2 may have a unique role as a biomarker of PNI-aggressiveness. Importantly, IL-13Rα2 may be a therapeutic target for intervention, which might not only prolong patient survival but also help alleviate pain attributed to perineural invasion. Our study uncovers a novel role of IL-13Rα2 in PNI as a key factor of the disease severity, thus revealing a therapeutically targetable option for PDAC and to facilitate PNI-associated pain management.

CD20+ tumor-infiltrating immune cells and CD204+ M2 macrophages are associated with prognosis in thymic carcinoma.

Thymic carcinoma is a rare malignant disease with no standard systemic chemotherapy. The purpose of the present study was to investigate tumor-infiltrating immune cells (TIIC) in the tumor microenvironment (TME), focusing on the impact of TIIC and program death-ligand 1 (PD-L1) expression on clinical outcomes in thymic cancer. Patients with thymic carcinoma resected between 1973 and 2017 were investigated. The tissue specimens were analyzed through immunohistochemical staining to elucidate the prognostic effects of TIIC, their ratios and PD-L1 in a preliminary cohort (n = 10). The density of TIIC as well as PD-L1 expression was evaluated in intraepithelial and tumor-stromal areas on the representative whole section of tumors. The immune factors showing significant association with disease-free survival (DFS) were evaluated in the total cohort (n = 42). TIIC in the preliminary population showed no significant difference between the two groups. However, CD8, CD20, CD204, FOXP3 and CD20/CD204 ratio demonstrated a tendency to act as predictive markers for recurrence. In the total cohort, significant differences were observed for CD8+ , CD20+ and CD204+ cells in tumor islets, and for CD8+ , CD20+ and FOXP3+ cells as well as the CD8/CD204 and CD20/CD204 ratios in the stroma, indicating their prognostic effect. The prognostic effect of the PD-L1 expression in tumor cells could not be established, possibly because of intratumoral heterogeneity. CD8, CD20 and CD204 positive TIIC in stroma were identified as possible better prognostic biomarkers, considering the heterogeneity of other biomarkers. The present study paves the way for exploring strategies of combination immunotherapy targeting B cell immunity in thymic carcinoma.

Tissue amino acid profiles are characteristic of tumor type, malignant phenotype, and tumor progression in pancreatic tumors.

Tissue amino acid profiles depend on the cell types and extracellular components that constitute the tissue, and their functions and activities. We aimed to characterize the tissue amino acid profiles in several types of pancreatic tumors and lesions. We examined tissue amino acid profiles in 311 patients with pancreatic tumors or lesions. We used newly developed LC-MS/MS methods to obtain the profiles, which were compared with clinicopathological data. Each tumor or lesion presented a characteristic tissue amino acid profile. Certain amino acids were markedly altered during the multistep pancreatic carcinogenesis and pancreatic ductal adenocarcinoma (PDAC) progression. A tissue amino acid index (TAAI) was developed based on the amino acids that were notably changed during both carcinogenesis and cancer progression. Univariate and multivariate survival analyses revealed that PDAC patients with a high TAAI exhibited a significantly shorter survival rate, and these findings were validated using a second cohort. We suggest that tissue amino acid profiles are characteristic for normal tissue type, tumor histological type, and pathological lesion, and are representative of the cancer grade or progression stage in multistep carcinogenesis and of malignant characteristics. The TAAI could serve as an independent prognosticator for patients with PDAC.

Reliable evaluation of tumor-infiltrating lymphocytes in pancreatic cancer tissue biopsies.

Tumor-infiltrating lymphocytes (TILs) represent cancer microenvironment. We previously reported TILs was prognosticators in pancreatic ductal adenocarcinoma (PDAC) patients by immunohistochemically measuring them in surgically-resected tissues. The aim of this study was to assess how best to evaluate TILs in PDAC tissue biopsies. First, we showed expression of CD3, CD4, or CD8 genes in PDAC tissue measured by quantitative RT-PCR (RT-qPCR) was prognostic using 241 surgically-resected specimens. We assessed whether the TILs in biopsied tissues can be effectively evaluated by comparing between immunohistochemistry and RT-qPCR. As a study model, we sampled twenty biopsies from surgically-resected PDAC specimen (n = 17). We investigated the variation levels of TILs in the different biopsies from the same specimen and evaluated using the intraclass correlation coefficient (ICC). The ICC value was 0.58 for CD3, 0.61 for CD4, and 0.46 for CD8, respectively; these ICC values meant correlations of "moderate" to "substantial" levels. To reach "near perfect", 3, 3, and 5 times biopsies were necessary for CD3, CD4, and CD8, respectively. When ICC values of immunolabeled TILs were of "low", ≥6 times biopsies were necessary to reach "moderate" levels. We found that TILs measured by RT-qPCR and repeated sampling increased reliability in TILs detected from biopsied PDAC tissues.

Reduction of intrapancreatic neural density in cancer tissue predicts poorer outcome in pancreatic ductal carcinoma.

Neural invasion is one of the malignant features contributing to locally advanced and/or metastatic disease progression in patients with pancreatic ductal adenocarcinoma (PDAC). Few studies exist on the distribution and state of nerve fibers in PDAC tissue and their clinicopathological impacts. The aim of the present study was to investigate the clinicopathological characteristics and prognostic value of intrapancreatic neural alterations in patients with PDAC. We retrospectively analyzed 256 patients with PDAC who underwent macroscopic curative surgery. Nerve fibers, immunolabeled with a specific neural marker GAP-43, were digitally counted and compared among PDAC, chronic pancreatitis (CP) and normal pancreatic tissues. Interlobular nerve fibers were apparently hypertrophic in both CP and PDAC, although intrapancreatic neural density and nerve number decreased characteristically in PDAC. They tended to decrease toward the center of the tumor. Kaplan-Meier survival analyses revealed a statistically significant correlation between low neural density and shorter overall survival (OS) (P = 0.014), and between high neural invasion and shorter OS (P = 0.017). Neural density (P = 0.04; HR = 1.496; 95% CI 1.018-2.199) and neural invasion ratio (P = 0.064; HR = 1.439; 95% CI .980-2.114) were prognostic factors of shorter OS in the multivariate analysis. These findings suggest low intrapancreatic neural density in patients with PDAC as an independent prognosticator, which may represent aggressive tumor behavior. Furthermore, we propose a simple, practical and reproducible method (to measure neural density and the neural invasion ratio during conventional histopathological diagnosis of PDAC), which has been validated using another cohort (n = 81).

Tumor-associated CD204+ M2 macrophages are unfavorable prognostic indicators in uterine cervical adenocarcinoma.

Uterine cervical adenocarcinoma is rare, but its prevalence has increased. To improve outcomes and ensure the suitability of recent immunotherapies, the aim of this study was to evaluate the clinicopathological impact of the tumor immune microenvironment of uterine cervical adenocarcinoma. We investigated 148 adenocarcinoma cases, including 21 cases of adenocarcinoma in situ (AIS) and 127 cases of invasive adenocarcinoma, using immunohistochemistry to detect tumor-infiltrating immune cells and the expression of programmed cell death 1 ligand-1 (PD-L1) and p16 on tumor cells. We then carried out correlation and survival analyses. The density of immune cells and expression levels were compared between the tumor cell nest and stroma and between AIS and invasive adenocarcinoma using digital image analysis. A higher density of tumor-infiltrating CD204+ M2 macrophages was significantly associated with shorter disease-free survival, although no other tumor-infiltrating immune cells were prognostic, including CD4+ , CD8+ , FOXP3+ , and PD-1+ lymphocytes and CD68+ macrophages. The density of stroma-infiltrating lymphocytes and macrophages was significantly higher in invasive adenocarcinoma than in AIS. The density of tumor-infiltrating lymphocytes in p16-expressing human papillomavirus (HPV)-positive tumors was significantly higher than that in HPV-negative tumors. The HPV status was not associated with patient outcome. Expression of PD-L1 on tumor cells was found only in invasive adenocarcinoma cases (17.3%). A higher density of stroma-infiltrating lymphocytes and macrophages was found in PD-L1-positive tumors than in negative tumors. Patients with PD-L1-positive tumors tended to experience longer survival. It is suggested that tumor-infiltrating CD204+ M2 macrophages may predict poor prognoses in patients with cervical adenocarcinoma.

Strong antitumor efficacy of a pancreatic tumor-targeting oncolytic adenovirus for neuroendocrine tumors.

Although oncolytic adenoviruses are promising cancer therapy agents, for effective oncolytic activity, viruses need to specifically infect and effectively replicate in cancer cells but not in normal cells. We have previously identified a pancreatic cancer-targeting ligand, SYENFSA (SYE), by screening an adenovirus library displaying random peptides against human pancreatic cancer cells and reported that a survivin promoter-regulated adenovirus, displaying the SYE ligand (AdSur-SYE), provided effective oncolysis of pancreatic ductal adenocarcinoma (PDAC) in a preclinical study. As we examined the infectivity of AdSur-SYE in human surgical specimens of various pancreatic tumors, we unexpectedly found that AdSur-SYE showed high gene transduction efficiency for pancreatic neuroendocrine tumors (PNETs) as well as for PDAC, 9.1- and 6.2-fold, respectively, compared to that of the nontargeting virus (AdSur). The infectivity of both vectors was almost the same in other cancers and organs such as the pancreas. Immunostaining indicated that the cells infected with AdSur-SYE were PNET cells but not stromal cells. AdSur-SYE showed a significantly higher oncolytic potency than that of AdSur in human PNET cell lines, and intratumoral infection with AdSur-SYE completely diminished subcutaneous tumors in a murine model, in which AdSur-SYE effectively proliferated and spread. AdSur-SYE exerted a stronger oncolytic effect in primary PNET cells cocultured with mouse embryonic fibroblasts than AdSur did. Thus, AdSur-SYE shows promise as a next-generation therapy for PNET.

Clinicopathologic Association and Prognostic Value of Microcystic, Elongated, and Fragmented (MELF) Pattern in Endometrial Endometrioid Carcinoma.

Microcystic, elongated, and fragmented (MELF) pattern is seen in the invasive front of some endometrial endometrioid carcinomas. Although MELF pattern can be expected as an indicator of patient outcomes, its prognostic significance remains unclear. This study was conducted to elucidate clinicopathologic features and the prognostic impact of MELF pattern in patients with endometrial endometrioid carcinoma. We retrospectively analyzed data of 479 consecutive patients with endometrial endometrioid carcinoma that had been surgically resected. In 45 of 427 patients (11%) with low-grade endometrioid carcinoma, MELF pattern was found, but it was found in none of the 52 patients with high-grade endometrioid carcinoma. Among the patients with low-grade endometrioid carcinoma, MELF pattern was associated significantly with larger tumor size, myometrial invasion of more than 50%, advanced International Federation of Gynecology and Obstetrics stages, lymphovascular space invasion, lymph node metastasis, papillary architecture, and mucinous differentiation. However, survival analysis revealed that the patients with MELF pattern showed no significantly worse prognosis than those without MELF pattern either in disease-specific survival or in recurrence-free survival. MELF was not a significant prognosticator after adjustment for International Federation of Gynecology and Obstetrics stage (disease-specific survival [hazard ratio, 1.47; 95% confidence interval, 0.28-7.67; P=0.64], recurrence-free survival [hazard ratio, 0.98, 95% confidence interval, 0.32-2.99, P=0.98]). Immunohistochemical analysis revealed that MELF pattern was positive for p16 and p21 and almost negative for Ki-67 labeling, which suggested that tumor cells in MELF pattern were involved in growth arrest or cellular senescence. We conclude that MELF pattern could have little impact on outcomes of patients with low-grade endometrial endometrioid carcinoma.

An Oncogenic ALK Fusion and an RRAS Mutation in KRAS Mutation-Negative Pancreatic Ductal Adenocarcinoma.

PURPOSE: Oncogenic mutations in the KRAS gene are a well-known driver event, occurring in >95% of pancreatic cancers. The objective of this study was to identify driver oncogene aberrations in pancreatic cancers without the KRAS mutation. METHODS: Whole-exome and transcriptome sequencing was performed on four cases of KRAS mutation-negative pancreatic ductal adenocarcinoma, which were identified in a cohort of 100 cases. RESULTS: One case harbored an oncogenic DCTN1-ALK fusion. The fusion gene enabled interleukin-3-independent growth of Ba/F3 cells and rendered them susceptible to the anaplastic lymphoma kinase tyrosine kinase inhibitors crizotinib and alectinib. The structure of the breakpoint junction indicated that the fusion was generated by nonhomologous end joining between a segment of DCTN1 exon DNA and a segment of ALK intron DNA, resulting in the generation of a cryptic splicing site. Another case harbored an oncogenic RRAS mutation that activated the GTPase of the RRAS protein. CONCLUSION: Rare oncogenic aberrations, such as the ALK fusion and RRAS mutation, may drive pancreatic carcinogenesis independent of the KRAS mutation. The Oncologist 2017;22:158-164Implications for Practice: The oncogenic DCTN1-ALK fusion and the RRAS mutation were associated with the development of pancreatic ductal adenocarcinoma (PDAC) in the absence of the KRAS mutation. Constitutional activation of DCTN1-ALK fusion protein was suppressed by the anaplastic lymphoma kinase tyrosine kinase inhibitors crizotinib and alectinib. Thus, a small subset of PDAC patients might benefit from therapy using these inhibitors.