Lymphovascular Invasion in Colorectal Cancer: An Interobserver Variability Study

Background: Lymphovascular invasion (LVI) in colorectal cancer (CRC) is considered a strong stage-independent prognostic factor and influences decisions regarding adjuvant chemotherapy in patients with Stage II tumors. However, the degree of interobserver agreement among pathologists for LVI in CRC is largely unknown. This study was undertaken to examine such interobserver variability, and we hypothesized that the use of immunohistochemical markers for vascular and lymphatic channels could improve interobserver agreement. Design: Fifty cases of AJCC stage II moderately differentiated CRC from 1990 to 2005 from the pathology archives were selected; mucinous, medullary, and other recognized special subtypes were excluded. Fifty H&E slides (one from each case) were circulated to 6 GI pathologists, who independently assessed small and large vessel invasion. No diagnostic guidelines were given to the participating pathologists; each was instructed to apply the criteria for LVI that he or she used in daily practice. Immunohistochemistry (IHC) for D2-40 and CD31 was performed on corresponding paraffin blocks. The IHC slides were randomized, recirculated, and rescored for LVI. Results were analyzed by kappa (κ)statistics, which correct for agreement by chance, and for percent agreement. Results: The average κ values were determined for the H&E slides (large and small vessel), CD31 (small vessel), and D2-40 (small vessel) (Figure 1). Agreement was fair for H&E small vessel invasion (κ = 0.28; 95%CI 0.22–0.34). The least agreement was seen in interpretation of H&E large vessel invasion (κ = 0.18; 95%CI 0.11–0.26). Agreement was not improved by use of immunohistochemical stains: CD31 (large vessel, κ = 0.42, 95%CI 0.20–0.63, small vessel, κ = 0.26, 95%CI 0.10–0.42) and D2-40 (κ = 0.32, 95%CI 0.21–0.42). Conclusions: Interobserver variability in diagnosis of LVI was substantial on H&E slides and did not improve upon use of IHC. Agreement in evaluation of large vessel invasion was only slightly higher than would be seen by chance alone. This study highlights the need for criteria in evaluation of lymphovascular invasion, as this assessment may impact patient prognosis and thus change the course of clinical treatment

MTG16 regulates colonic epithelial differentiation, colitis, and tumorigenesis by repressing E protein transcription factors

Aberrant epithelial differentiation and regeneration contribute to colon pathologies, including inflammatory bowel disease (iBD) and colitis-associated cancer (CAC). Myeloid translocation gene 16 (MTG16, also known as CBFA2T3) is a transcriptional corepressor expressed in the colonic epithelium. MTG16 deficiency in mice exacerbates colitis and increases tumor burden in CAC, though the underlying mechanisms remain unclear. Here, we identified MTG16 as a central mediator of epithelial differentiation, promoting goblet and restraining enteroendocrine cell development in homeostasis and enabling regeneration following dextran sulfate sodium-induced (DSS-induced) colitis. Transcriptomic analyses implicated increased Ephrussi box-binding transcription factor (E protein) activity in MTG16-deficient colon crypts. Using a mouse model with a point mutation that attenuates MTG16:E protein interactions (Mtg16(P20ST)), we showed that MTG16 exerts control over colonic epithelial differentiation and regeneration by repressing E protein-mediated transcription. Mimicking murine colitis, MTG16 expression was increased in biopsies from patients with active IBD compared with unaffected controls. Finally, uncoupling MTG16:E protein interactions partially phenocopied the enhanced tumorigenicity of Mtg16(-/)(-) colon in the azoxymethane/DSS-induced model of CAC, indicating that MTG16 protects from tumorigenesis through additional mechanisms. Collectively, our results demonstrate that MTG16, via its repression of E protein targets. is a key regulator of cell fate decisions during colon homeostasis, colitis, and cancer.Peer reviewe

Variable expression of somatostatin receptor type 2 in metastatic midgut carcinoid

189 Background: Somatostatin receptor type 2 (SSTR2) is frequently highly expressed in carcinoid tumors of the midgut. The presence of the receptor is the basis of imaging and therapeutics. Because of the high dependence of clinical care on SSTR2, we evaluated the immunohistochemical staining of specimens and correlated with clinical parameters. Methods: A tissue microarray (TMA) of 1 mm cores from paraffin embedded specimens of surgically resected midgut carcinoid were created with primary, lymph node, and liver metastatic lesions. These sections were stained by immunohistochemistry with anti-SSTR2 antibody, and scored on staining intensity. Sections were evaluated by membrane (0-none, 1 – weak, 2 – partial, 3- complete) and cytoplasmic (strong vs. weak) staining. Results: Thirty-five patient samples were included in the TMA. There were a total of 62 cores embedded: 21 primary lesions, 26 lymph node metastases, and 15 liver metastases. The total disease burden was captured in 17 patients, and specimens from all three sites were captured in 6 patients. Of the thirty-five patients, 6 had consistently weak SSTR2 staining (17% either 0 or 1). One of these cases had an intermediate grade tumor with Ki67 >20%. Of the patients where more than one location was captured, 19/21 (90%) had consistent SSTR2 staining at all sites. Most cases had strong staining in the membrane (69%) while a minority had strong cytoplasmic staining (31%). Of the patients with weak SSTR2 membrane staining, 2 still had positive octreoscans. Clinically, most patients had good outcomes and 94% are still alive. Eight cases had carcinoid carcinomatosis (23%). Of the six with weak membrane staining, three had carcinomatosis (50%), while only 5/30 (17%) with strong membrane staining had carcinomatosis. Conclusions: While the majority of small bowel carcinoids and their metastatic lesions have strong SSTR2 staining, not all do. However, even in cases with low amounts of SSTR2 on the membrane, the tumors can still be detected on octreoscan, demonstrating the effect of receptor turnover. The loss of SSTR2 may be associated with more advanced disease, but is not completely predictive

Abstract B43: Alisertib (MLN8237), an Aurora Kinase A inhibitor, enhances Docetaxel-induced cell death in Upper Gastrointestinal Adenocarcinoma cells

Abstract Upper gastrointestinal adenocarcinomas (UGCs) respond poorly to current chemotherapeutic regimes. We and others have previously reported frequent AURKA gene amplification and mRNA and protein overexpression in UGCs. It has been shown that AURKA can induce chemotherapeutic resistance and regulate several key signaling pathways in cancer cells, suggesting its role as a central node in cancer cell signaling. The DCF (docetaxel, cisplatin and 5-FU) regimen is the most promising chemotherapeutic regimen against UGC. However, AURKA overexpression can mediate resistance to both Taxol and cisplatin. These findings provide a credible rationale for targeting AURKA. In this study we have reported therapeutic response of the recently developed AURKA selective inhibitor MLN8237 as a single agent and in combination with docetaxel. Our findings provide a foundation for a promising new approach of future targeted therapy in UGCs. Using AGS, FLO-1 and OE33 UGC cell lines, with constitutive AURKA overexpression and variable-p53 status, we observed inhibition of cancer cell survival with 0.5μM MLN8237 (p<0.05) alone. This effect was significantly enhanced in combination with 1.0nM docetaxel (p<0.001). Cell cycle analyses after 48hr of treatment with MLN8237 demonstrated a significant increase in the percentage of polyploidy for AGS and OE33 UGC cells (p<0.01) which was further enhanced by docetaxel (p<0.001). Additionally, an increase in the percentage of cells in the sub-G1-phase at 24hrs and 48hrs was observed with MLN8237 in FLO-1 (p<0.01) cells which was significantly enhanced with MLN8237 and docetaxel combination (p<0.001). Western blot analysis demonstrated higher induction of cleaved caspase 3 and cleaved PARP with the combined treatment, as compared to a single agent treatment. Using xenograft models, we demonstrated an enhanced anti-tumor role for the MLN8237 and docetaxel combination, as compared to single agent treatments (p<0.001). In conclusion, this study demonstrates that MLN8237, combined with docetaxel, can mediate a better therapeutic outcome in UGCs

Diagnostic and therapeutic implications of a novel immunohistochemical panel detecting duodenal mucosal invasion by pancreatic ductal adenocarcinoma

Background: We investigated a series of pancreaticoduodenectomy and duodenal biopsies with a panel of immunohistochemical markers to identify duodenal mucosal invasion by pancreatic ductal adenocarcinoma (PDAC), including markers of poor prognosis and targets of promising novel immunotherapies. Materials and Methods: Eighteen consecutive pancreaticoduodenectomy specimens with duodenal mucosal invasion by PDAC were examined for expression of MUC1, MUC4, MUC5AC, MUC6, mesothelin, MUC2, CDX2, and DPC4 on formalin-fixed, paraffin-embedded sections of duodenal-ampullary-pancreatic junctions. Expression of all but MUC6 was also assessed in duodenal biopsies from 12 patients with duodenal mucosal invasion by PDAC. Results: The duodenal mucosa expressed MUC1 (crypts), MUC2 (goblet cells), MUC6 (Brunner glands), CDX2, and DPC4. PDACs in the duodenal mucosa from the resection (n=16-18) and biopsy (n=12) specimens were marked as follows: MUC1 100% (30/30), MUC4 83% (24/29), MUC5AC 83% (25/30), mesothelin 82% (23/28), MUC2 7% (2/30), and CDX2 36% (10/28). Loss of DPC4 expression was seen in 16 of 29 (55%) cases. Reactive mucosa adjacent to PDAC expressed MUC4, MUC5AC and mesothelin in 65% (17/26), 19% (5/27), and 19% (5/26) of cases, respectively. While MUC5AC and mesothelin had high diagnostic accuracy for detection of PDAC, MUC2, CDX2 and DPC4 expression demonstrated negative correlation with PDAC, with absent expression being highly specific for PDAC. Conclusion: Immunohistochemical labeling for PDAC biomarkers may aid the diagnosis of PDAC in duodenal biopsy, especially in situations where diagnosis of a pancreatic mass is challenging

Serine Threonine Kinase 17A Maintains the Epithelial State in Colorectal Cancer Cells

Serine threonine kinase 17A (STK17A) is a ubiquitously expressed kinase originally identified as a regulator of apoptosis; however, whether it functionally contributes to colorectal cancer has not been established. Here, we have analyzed STK17A in colorectal cancer and demonstrated decreased expression of STK17A in primary tumors, which is further reduced in metastatic lesions, indicating a potential role in regulating the metastatic cascade. Interestingly, changes in STK17A expression did not modify proliferation, apoptosis, or sensitivity of colorectal cancer cell lines to treatment with the chemotherapeutic 5-fluorouracil. Instead, knockdown induced a robust mesenchymal phenotype consistent with the epithelial-mesenchymal transition, including spindle-like cell morphology, decreased expression of adherens junction proteins, and increased migration and invasion. Additionally, overexpression of decreased cell size and induced widespread membrane blebbing, a phenotype often associated with activation of cell contractility. Indeed, STK17A-overexpressing cells displayed heightened phosphorylation of myosin light chain in a manner dependent on STK17A catalytic activity. Finally, patient-derived tumor organoid cultures were used to more accurately determine STK17A's effect in primary human tumor cells. Loss of STK17A induced morphologic changes, decreased E-cadherin, increased invasion, and augmented organoid attachment on 2D substrates, all together suggesting a more metastatic phenotype. Collectively, these data indicate a novel role for STK17A in the regulation of epithelial phenotypes and indicate its functional contribution to colorectal cancer invasion and metastasis. IMPLICATIONS: Loss of serine threonine kinase 17A occurs in colorectal cancer metastasis, induces mesenchymal morphologies, and contributes to tumor cell invasion and migration in colorectal cancer

Co-overexpression of AXL and c-ABL predicts a poor prognosis in esophageal adenocarcinoma and promotes cancer cell survival

Esophageal adenocarcinoma (EAC) is highly aggressive and characterized by poor prognosis. AXL expression has been linked to Barrett's tumorigenesis and resistance to chemotherapy, which is associated with c-ABL intracellular localization. However, the molecular and functional relationship between AXL and c-ABL and the clinical significance of the co-expression of these proteins in EAC remain unclear. We used immunohistochemical analysis (IHC) on tissue microarrays containing human EAC samples (n=53) and normal esophageal tissues (n=11) in combination with corresponding deidentified clinicopathological information to evaluate the expression and the prognostic significance of AXL and c-ABL in EAC. The data were statistically analyzed using Kruskal-Wallis, the chi-square, the Fisher's exact, and Pearson tests. The Kaplan-Meier method and Cox proportional hazards regression model were used to evaluate cancer patient survival. We used a serum deprivation EAC cell model to investigate the pro-survival function of AXL and c-ABL using cell viability, apoptosis, and lactate dehydrogenase activity assays. We performed assays, including Western blotting, quantitative real-time PCR, and translational chromatin immunoprecipitation (TrIP-Chip) to study the molecular relationship between AXL and c-ABL in EAC cells. IHC analysis revealed that AXL and c-ABL were overexpressed in 55% and 66% of EAC samples, respectively, as compared to normal tissues. Co-overexpression of the two proteins was observed in 49% of EAC samples. The chi-square test indicated a significant association between AXL and c-ABL expression in the EAC samples (χ = 6.873, 0.032), and the expression of these proteins was significantly associated with EAC patient age ( 0.001), tumor stage ( 0.01), and lymph node status ( 0.001). AXL and c-ABL protein expression data analysis exhibited an identical clinicopathological association profile. Additionally, we found a significant association between expression of AXL (χ = 16.7, 0.002) or c-ABL (χ = 13.4, 0.001) and survival of EAC patients. The Cox proportional hazards model and log rank test predicted a significant increase in mortality of patients with high expression of AXL [hazard ratio (HR): 2.86, 95% confidence interval (CI): 1.53 - 5.34, 0.003] or c-ABL [HR: 3.29, 95% CI: 1.35 - 8.03, 0.001] as compared to those patients with low expression of AXL or c-ABL proteins. Molecular investigations indicated that AXL positively regulates c-ABL protein expression through increased cap-dependent protein translation involving phosphorylation of EIF4E in EAC cells. Next, we investigated the functional relationship between AXL and c-ABL in EAC cells. We demonstrated that the pro-survival activity of AXL requires c-ABL expression in response to serum deprivation. This study highlights the importance of the co-overexpression of AXL and c-ABL proteins as a valuable prognostic biomarker and targeting these proteins could be an effective therapeutic approach in EAC or other solid tumors expressing high levels of AXL and c-ABL proteins