Identification of TRPV4 as a novel target in invasiveness of colorectal cancer.

BACKGROUND: Emerging evidence has indicated the critical role of TRPV4 in diverse human cancers. However, the underlying molecular mechanism of TRPV4 in colon cancer invasiveness is still unknown. METHODS: Immunohistochemistry staining was used to analyze the expression of TRPV4 and ZEB1 in clinical tissues; Wound healing and transwell assays were applied to determine the cell invasiveness; Western blot was used to explore the relation between TRPV4 and ZEB1. RESULTS: Colon cancer cells were transfected with siRNA against TRPV4 or HC067047 (a selective TRPV4 antagonist), TRPV4 full-length plasmid or siRNA against ZEB1, or both, in order to measure cell migration and invasion. And we found that TRPV4 silencing or inhibition exhibited an inhibitory role in colon cancer cell migration and invasion, coupled with compromised EMT process, and suppressed AKT activity. TRPV4 stimulated expression of ZEB1 and consequently contributed to EMT process and invasiveness. It was also revealed that overexpression of TRPV4 and ZEB1 in clinical patients with local metastasis, and positive correlation between TRPV4 and ZEB1. CONCLUSIONS: Our results uncovered the role of TRPV4 in tumor metastasis and highlighted the potential mechanism of TRPV4-ZEB1 axis in indicating EMT.

A Genotype Signature for Predicting Pathologic Complete Response in Locally Advanced Rectal Cancer.

PURPOSE: To construct and validate a predicting genotype signature for pathologic complete response (pCR) in locally advanced rectal cancer (PGS-LARC) after neoadjuvant chemoradiation. METHODS AND MATERIALS: Whole exome sequencing was performed in 15 LARC tissues. Mutation sites were selected according to the whole exome sequencing data and literature. Target sequencing was performed in a training cohort (n = 202) to build the PGS-LARC model using regression analysis, and internal (n = 76) and external validation cohorts (n = 69) were used for validating the results. Predictive performance of the PGS-LARC model was compared with clinical factors and between subgroups. The PGS-LARC model comprised 15 genes. RESULTS: The area under the curve (AUC) of the PGS model in the training, internal, and external validation cohorts was 0.776 (0.697-0.849), 0.760 (0.644-0.867), and 0.812 (0.690-0.915), respectively, and demonstrated higher AUC, accuracy, sensitivity, and specificity than cT stage, cN stage, carcinoembryonic antigen level, and CA19-9 level for pCR prediction. The predictive performance of the model was superior to clinical factors in all subgroups. For patients with clinical complete response (cCR), the positive prediction value was 94.7%. CONCLUSIONS: The PGS-LARC is a reliable predictive tool for pCR in patients with LARC and might be helpful to enable nonoperative management strategy in those patients who refuse surgery. It has the potential to guide treatment decisions for patients with different probability of tumor regression after neoadjuvant therapy, especially when combining cCR criteria and PGS-LARC.

Well-differentiated rectal neuroendocrine tumors: analysis of histology, including insulinoma-associated protein 1 expression, and biologic behavior, involving a large cohort of 94 cases.

Well-differentiated rectal neuroendocrine tumors (R-NETs) are increasingly being detected by screening colonoscopy, commonly manifesting as polyps. Chromogranin A is frequently negative in R-NETs. Insulinoma-associated protein 1 (INSM1) is a novel transcription factor that has recently shown excellent sensitivity and specificity for neuroendocrine (NE) differentiation in various anatomic sites but has not been systematically evaluated in R-NET. A retrospective histologic review of all available R-NETs was performed and stained for INSM1 immunohistochemistry, as well as for Ki-67 and chromogranin A, if not already available. Clinical and follow-up information was obtained from the medical chart. A total of 94 R-NETs were included in our cohort. Of these, 82 (87%) were <10 mm in greatest dimension, and submucosal involvement was noted in 70 patients (74%). The tumors displayed a variety of histologic patterns, and the majority of the cases had intratumoral fibrosis (61%). Synaptophysin and INSM1 were reactive in 100% cases, whereas chromogranin A was reactive in 45% cases. The mean Ki-67 proliferative index was 1.6% (range: 0.5-5%). The median follow-up of the cohort was 30 months (80 cases, range: 3-226 months). Only three patients were identified with regional lymph node metastasis, all of which showed a tumor size ≥10 mm and had lymphovascular invasion (LVI). R-NETs in our fairly large cohort display an indolent biologic behavior without distant metastasis. Metastatic disease in lymph nodes was associated with tumor size and the presence of LVI, but not with the Ki-67 proliferative index. This is also the first systematic study documenting INSM1 as a highly sensitive NE marker in R-NET.

Collision of Malignant Melanoma and Squamous Cell Carcinoma in Rectum: First Report of a Rare Tumor.

The term collision tumor is used to describe two neoplasms occurring in the same anatomic location with juxtaposition of different tumor elements. Such a coexistence of tumors anywhere in the body is relatively rare. We report a case of 32-year-old female with collision tumor of rectum. The tumor showed two distinct histological patterns with predominant component consisting of malignant melanoma and a minor component of squamous cell carcinoma. The morphological picture of collision was further confirmed by specific immunohistochemical profile of the two tumors. Collision tumors of rectum are uncommon with most of the reported cases comprising adenocarcinoma and neuroendocrine tumors. To the best of our knowledge this is the first case of collision tumor of malignant melanoma and squamous cell carcinoma in the rectum.

Non-operative management of rectal cancer: understanding tumor biology.

The management of locally-advanced rectal cancer involves a combination of chemotherapy, chemoradiation, and surgical resection to provide excellent local tumor control and overall survival. However, aspects of this multimodality approach are associated with significant morbidity and long-term sequelae. In addition, there is growing evidence that patients with a clinical complete response to chemotherapy and chemoradiation treatments may be safely offered initial non-operative management in a rigorous surveillance program. Weighed against the morbidity and significant sequelae of rectal resection, recognizing how to best optimize non-operative strategies without compromising oncologic outcomes is critical to our understanding and treatment of this disease.

Cervical metastases originating from a primary rectal adenocarcinoma due to a pagetoid spread.

Vulvar Paget disease is a rare skin disorder, considered an in situ adenocarcinoma. It is characterized by intraepithelial Paget cells, of which the origin is unclear. About 75% of cases have a cutaneous origin; the other 25% originate from an intestinal or urological malignancy. We report the first case of retrograde pagetoid spread from a rectal adenocarcinoma to the vulva and cervix. A 66-year-old woman presented with postmenopausal bleeding and a history of Crohn disease. Gynecological workup revealed vulvar and endocervical lesions consisting of Paget cells and adenocarcinoma, respectively. A rectal adenocarcinoma with in situ adenocarcinoma was diagnosed. The surgical specimen demonstrated Paget cells in the squamous epithelium of the anus and vulva. Immunohistochemistry demonstrated an intestinal phenotype of these cells. Genetic testing revealed the same TP53 mutation in tumor cells of the rectal adenocarcinoma and vulvar and endocervical lesions, demonstrating that the Paget cells originated from the same intestinal tumor.

[Hypoxia-inducible factor-1α and CD133 predicts pathological complete response and survival for locally advanced rectal cancer patients after neoadjuvant chemoradiotherapy].

Objective: To investigate the expression of hypoxia-inducible factor 1α (HIF-1α) and CD133 in predicting pathologic remission and survival of patients with locally advanced rectal cancer undergoing neoadjuvant chemoradiotherapy. Methods: One hundred and fourteen patients with locally advanced rectal cancer undergoing neoadjuvant chemoradiotherapy from January 2010 to December 2015 in Jinhua Municipal Central Hospital were enrolled in the study. RT-PCR and immunohistochemistry methods were used to detect the mRNA and protein expression of HIF-1α and CD133 before and after chemoradiotherapy. Spearman rank correlation was used to analyze the correlation between HIF-1α and CD133 mRNA expression. Univariate and logistic multivariate analyses were used to determine the factors related to pathological complete response (pCR). Logistic regression analysis and Cox's proportional hazard model were used to determine factors related to overall survival and recurrence-free survival. Results: The expression of HIF-1α and CD133 mRNA was correlated with pT, ypTNM, pCR, recurrence and metastasis of rectal cancer, while not correlated with sex, age and BMI of patients. HIF-1α mRNA expression was positively correlated with CD133 mRNA expression ( α=0.579, P=0.000). Immunohistochemistry analysis showed that residual cancer cells strongly expressing HIF-1α also expressed CD133 strongly. Univariate analysis showed that HIF-1α mRNA and CD133 mRNA were significantly correlated with pCR ( P=0.001, P=0.022, respectively). Multivariate analysis showed that HIF-1α and CD133 mRNA expression were independent prognostic factors of pCR ( P=0.012, P=0.047, respectively). Cox regression analysis showed that the expression of HIF-1α mRNA and CD133 mRNA were independent predictors of recurrence-free survival and overall survival ( P=0.025, P=0.033, respectively). Conclusion: The study indicates that HIF-1α and CD133 can predict pathological complete remission and survival of patients with locally advanced rectal cancer.

Lymph Node Micrometastasis Cannot Be Considered as Positive Lymph Node in Nonmetastatic Colorectal Cancer.

The prognostic value of micrometastasis in colorectal cancer (CRC) remains controversial. The study investigated whether lymph node (LN) micrometastasis can have prognostic value in CRC as compared with macrometastasis. The study included 488 patients with curatively resected stage I, II, or III CRC treated between 2004 and 2011. Immuohistochemical staining with monoclonal antibody CAM 5.2 was performed on negative LNs by hematoxylin-eosin staining. The prognostic value of LN micrometastasis was investigated in multivariate analysis. Regression analysis was performed to identify a causal relationship between micro- and macrometastasis. Survival differences were compared between conventional N staging and hypothetic N staging taking micrometastasis in the positive node. A total of 93 patients (19.1%) showed LN micrometastasis. Patients with micrometastasis had more advanced tumor characteristics in terms of tumor size, grade, T stage, N stage, lymphatic invasion, and vascular invasion. In multivariate analysis, micrometastasis was not related with recurrence. Preoperative carcinoembryonic antigen level, neural invasion, and macrometastasis were independent risk factors in the analysis. Regression analysis showed that there was not a causal relationship between micro- and macrometastasis (R2 = 0.004, P = 0.153). When the cumulative numbers of micro- and macrometastatic LNs were calculated together, the discriminative power of survival difference between each node stage became less prominent, compared with conventional N staging. LN micrometastasis is related with advanced tumor characteristics, but does not reflect poor prognosis in nonmetastatic CRC. Micrometastasis cannot be considered as positive LN to predict poor prognosis.

EWSR1 Fusions With CREB Family Transcription Factors Define a Novel Myxoid Mesenchymal Tumor With Predilection for Intracranial Location.

Recurrent gene fusions involving EWSR1 with members of the cAMP response element binding protein (CREB) family (ATF1 and CREB1) have been reported in a diverse group of tumors including angiomatoid fibrous histiocytoma (AFH), soft tissue and gastrointestinal clear cell sarcoma, primary pulmonary myxoid sarcoma, and hyalinizing clear cell carcinoma of salivary gland. We have recently encountered a group of 5 myxoid mesenchymal tumors positive for EWSR1 fusions with one of the CREB family member (ATF1, CREB1, and CREM), with histologic features distinct from any of the previously described pathologic entities. Tumors occurred in children or young adults (12 to 23 y; mean, 18 y), with equal sex distribution. All except 1 were intracranial (intra-axial, 2; meningeal, 2), whereas 1 was perirectal. Histologically, the tumors were well circumscribed, often lobulated, composed of uniform ovoid to round cells, and arranged in cord-like or reticular structures in a myxoid background. All except 1 displayed unique sunburst amianthoid fibers. Immunohistochemically, tumors were positive for epithelial membrane antigen (5/5; 4 focal, 1 diffuse) and desmin (3/5). A novel EWSR1-CREM fusion was identified by RNA sequencing in the perirectal tumor, which was further confirmed by fluorescence in situ hybridization (FISH) and reverse transcription-polymerase chain reaction (RT-PCR). A second case with similar EWSR1-CREM fusion was identified by RT-PCR and FISH in a meningeal tumor. The remaining cases studied by FISH showed the presence of EWSR1-CREB1 fusion in 2 cases and EWSR1-ATF1 in 1. In conclusion, we report a distinct group of myxoid mesenchymal neoplasms occurring in children or young adults with a predilection for intracranial locations. Although the immunoprofile [epithelial membrane antigen (EMA), desmin] and the fusion type raise the possibility of a myxoid AFH, none of the typical histologic findings of AFH were present, suggesting a novel entity.

Tumour CD274 (PD-L1) expression and T cells in colorectal cancer.

OBJECTIVE: Evidence suggests that CD274 (programmed death-ligand 1, B7-H1) immune checkpoint ligand repress antitumour immunity through its interaction with the PDCD1 (programmed cell death 1, PD-1) receptor of T lymphocytes in various tumours. We hypothesised that tumour CD274 expression levels might be inversely associated with T-cell densities in colorectal carcinoma tissue. DESIGN: We evaluated tumour CD274 expression by immunohistochemistry in 823 rectal and colon cancer cases within the Nurses' Health Study and Health Professionals Follow-up Study. We conducted multivariable ordinal logistic regression analyses to examine the association of tumour CD274 expression with CD3+, CD8+, CD45RO (PTPRC)+ or FOXP3+ cell density in tumour tissue, controlling for potential confounders including tumour status of microsatellite instability (MSI), CpG island methylator phenotype, long interspersed nucleotide element-1 methylation level and KRAS, BRAF and PIK3CA mutations. RESULTS: CD274 expression in tumour cells or stromal cells (including immune cells) was detected in 731 (89%) or 44 (5%) cases, respectively. Tumour CD274 expression level correlated inversely with FOXP3+ cell density in colorectal cancer tissue (outcome) (ptrend=0.0002). For a unit increase in outcome quartile categories, multivariable OR in the highest (vs lowest) CD274 expression score was 0.22 (95% CI 0.10 to 0.47). Tumour CD274 expression was inversely associated with MSI-high status (p=0.001). CD274 expression was not significantly associated with CD3+, CD8+ or CD45RO+ cell density, pathological lymphocytic reactions or patient survival prognosis. CONCLUSIONS: Tumour CD274 expression is inversely associated with FOXP3+ cell density in colorectal cancer tissue, suggesting a possible influence of CD274-expressing carcinoma cells on regulatory T cells in the tumour microenvironment.

Lymphovascular invasion in more than one-quarter of small rectal neuroendocrine tumors.

AIM: To identify the frequency, clinicopathological risk factors, and prognostic significance of lymphovascular invasion (LVI) in endoscopically resected small rectal neuroendocrine tumors (NETs). METHODS: Between June 2005 and December 2015, 104 cases of endoscopically resected small (≤ 1 cm) rectal NET specimens at Hallym University Sacred Heart Hospital in Korea were retrospectively evaluated. We compared the detected rate of LVI in small rectal NET specimens by two methods: hematoxylin and eosin (H&E) and ancillary immunohistochemical staining (D2-40 and Elastica van Gieson); in addition, LVI detection rate difference between endoscopic procedures were also evaluated. Patient characteristics, prognosis and endoscopic resection results were reviewed by medical charts. RESULTS: We observed LVI rates of 25.0% and 27.9% through H&E and ancillary immunohistochemical staining. The concordance rate between H&E and ancillary studies was 81.7% for detection of LVI, which showed statistically strong agreement between two methods (κ = 0.531, P < 0.001). Two endoscopic methods were studied, including endoscopic submucosal resection with a ligation device and endoscopic submucosal dissection, and no statistically significant difference in the LVI detection rate was detected between the two (26.3% and 26.8%, P = 0.955). LVI was associated with large tumor size (> 5 mm, P = 0.007), tumor grade 2 (P = 0.006). Among those factors, tumor grade 2 was the only independent predictive factor for the presence of LVI (HR = 4.195, 95%CI: 1.321-12.692, P = 0.015). No recurrence was observed over 28.8 mo regardless of the presence of LVI. CONCLUSION: LVI may be present in a high percentage of small rectal NETs, which may not be associated with short-term prognosis.

[Rectal gastrointestinal stromal tumor: a clinical and pathologic analysis].

OBJECTIVE: To investigate the clinicopathologic characteristics and therapy of rectal gastrointestinal stromal tumors (GISTs). METHODS: Clinical findings, morphologic features, immunophenotype and prognosis of 53 cases (58 samples) of rectal GISTs were investigated. RESULTS: Thirty-three patients were male and 20 were female. The age of patients ranged from 19 to 81 years, with an average of 49.7 years. The main symptoms included rectal disorders in 29 patients and vaginal mass in 2 patients, while the tumors in 22 patients were found by routine physical examination. Thirty-five primary GISTs were resected completely without preoperative therapy, and thirteen tumors were resected after therapy of imatinib. Five tumors were recurrent. Imatinib therapy in 13 patients led to smaller and softer tumor mass grossly and decreased cellularity and marked degeneration histologically. Of the 35 primary rectal GISTs, there were 17 (48.6%), 6 (17.1%), 0(0), and 12 (34.3%) cases diagnosed as very low risk, low risk, medium risk, and high risk respectively. Eight cases had tumor of 1 cm or less in diameter. In the five recurrent cases, the tumors showed increased cellularity, mitotic figures, and Ki-67 index. Imatinib therapy led to smaller and softer tumor mass grossly and decreased cellularity and marked degeneration histologically. Immunohistochemical stains showed CD117, DOG1, and CD34 positivity, S-100 protein negativity and indefinite SMA stain. CONCLUSIONS: Rectal GISTs are rare tumors with a male predominance.Patients without obvious sypmtoms are found by themselves and by routine physical examination. The tumor diameter less than 2 cm is common while larger than 5 cm is few. Diagnosis of rectal GISTs is easily made by biopsy and patients often acquire preoperative therapy for preserving anal sphincter function.

Implication of programmed cell death ligand 1 expression in tumor recurrence and prognosis in rectal cancer with neoadjuvant chemoradiotherapy.

BACKGROUND: Programmed cell death ligand 1 (PD-L1) regulates immune responses through interaction with its receptor. PD-L1 is not only a predictor of poor prognosis but also a new therapeutic target in several malignancies. Neoadjuvant chemoradiotherapy (CRT) is an effective tool for local control of rectal cancer, but the disease recurrence rate remains high. The aim of this study was to retrospectively evaluate the correlation between PD-L1 expression and clinicopathological variables in rectal cancer after neoadjuvant CRT. MATERIALS AND METHODS: A total of 90 rectal cancer patients who underwent neoadjuvant CRT were enrolled in this study. We evaluated PD-L1 expression using immunohistochemistry. Moreover, we investigated the correlation between PD-L1 expression and tumor-infiltrating T cells, and between CD8- and Foxp3-positive cells. RESULTS: Patients with high PD-L1 expression more frequently had vascular invasion and tumor recurrence compared to patients with low PD-L1 expression (P = 0.0225 and P = 0.0051). High PD-L1 expression was significantly associated with poor recurrence-free and overall survival (P = 0.0027 and P = 0.0357). Multivariate analysis revealed lymph node metastasis and high PD-L1 expression as independent risk factors for tumor recurrence (P = 0.0102 and P = 0.0374). Numbers of infiltrating CD8-positive cells in patients with high PD-L1 expression were significantly lower than in patients with low PD-L1 expression (P = 0.0322). CONCLUSION: Our data suggest that inhibition of PD-L1 may be a new immunotherapeutic strategy to reduce tumor recurrence and improve prognosis in patients with rectal cancer after neoadjuvant CRT.

Intra-tumor genetic heterogeneity in rectal cancer.

Colorectal cancer arises in part from the cumulative effects of multiple gene lesions. Recent studies in selected cancer types have revealed significant intra-tumor genetic heterogeneity and highlighted its potential role in disease progression and resistance to therapy. We hypothesized the existence of significant intra-tumor genetic heterogeneity in rectal cancers involving variations in localized somatic mutations and copy number abnormalities. Two or three spatially disparate regions from each of six rectal tumors were dissected and subjected to the next-generation whole-exome DNA sequencing, Oncoscan SNP arrays, and targeted confirmatory sequencing and analysis. The resulting data were integrated to define subclones using SciClone. Mutant-allele tumor heterogeneity (MATH) scores, mutant allele frequency correlation, and mutation percent concordance were calculated, and copy number analysis including measurement of correlation between samples was performed. Somatic mutations profiles in individual cancers were similar to prior studies, with some variants found in previously reported significantly mutated genes and many patient-specific mutations in each tumor. Significant intra-tumor heterogeneity was identified in the spatially disparate regions of individual cancers. All tumors had some heterogeneity but the degree of heterogeneity was quite variable in the samples studied. We found that 67-97% of exonic somatic mutations were shared among all regions of an individual's tumor. The SciClone computational method identified 2-8 shared and unshared subclones in the spatially disparate areas in each tumor. MATH scores ranged from 7 to 41. Allele frequency correlation scores ranged from R(2)=0.69-0.96. Measurements of correlation between samples for copy number changes varied from R(2)=0.74-0.93. All tumors had some heterogeneity, but the degree was highly variable in the samples studied. The occurrence of significant intra-tumor heterogeneity may allow selected tumors to have a genetic reservoir to draw from in their evolutionary response to therapy and other challenges.

Clinical impact of atypical endoscopic features in rectal neuroendocrine tumors.

AIM: To validate the association between atypical endoscopic features and lymph node metastasis (LNM). METHODS: A total of 247 patients with rectal neuroendocrine tumors (NETs) were analyzed. Endoscopic images were reviewed independently by two endoscopists, each of whom classified tumors by sized and endoscopic features, such as shape, color, and surface change (kappa coefficient 0.76 for inter-observer agreement). All of patients underwent computed tomography scans of abdomen and pelvis for evaluation of LNM. Univariate and multivariate analyses were performed to identify the factors associated with LNM. Additionally, the association between endoscopic atypical features and immunohistochemical staining of tumors was analyzed. RESULTS: Of 247 patients, 156 (63.2%) were male and 15 (6.1%) were showed positive for LNM. On univariate analysis, tumor size (P < 0.001), shape (P < 0.001), color (P < 0.001) and surface changes (P < 0.001) were significantly associated with LNM. On multivariate analysis, tumor size (OR = 11.53, 95%CI: 2.51-52.93, P = 0.002) and atypical surface (OR = 27.44, 95%CI: 5.96-126.34, P < 0.001) changes were independent risk factors for LNM. The likelihood of atypical endoscopic features increased as tumor size increased. Atypical endoscopic features were associated with LNM in rectal NETs < 10 mm (P = 0.005) and 10-19 mm (P = 0.041) in diameter. Immunohistochemical staining showed that the rate of atypical endoscopic features was higher in non L-cell tumors. CONCLUSION: Atypical endoscopic features as well as tumor size are predictive factors of LNM in patients with rectal NETs.