Circulating miRNAs in patients with Barrett's esophagus, high-grade dysplasia and esophageal adenocarcinoma.

BACKGROUND: Diagnostic screening of premalignant esophageal lesions is hampered by the absence of biomarkers indicative of metaplastic and/or malignant transformation. The aim of this exploratory study was to investigate the potential use of miRNAs as biomarkers capable of identifying patients with (pre)malignant lesions: Barrett's esophagus (BE) metaplasia, high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC). METHODS: A total of 69 patients were included in the study. Six serum samples from each of four study groups, i.e., patients with normal squamous epithelium (SE), BE, HGD and EAC, were profiled using the Nanostring miRNA analysis platform. Differential miRNA expression patterns then were validated in 69 patient samples using qRT-PCR. RESULTS: miRNA expression profiling revealed seven miRNAs with a 2-fold change in expression level. Validation by qRT-PCR confirmed that serum miR-320e levels were significantly decreased in the BE group compared to the SE (P≤0.001, AUC 0.790) and HGD groups (P≤0.005, AUC 0.786). Serum miR-199a-3p levels were significantly decreased in the BE group compared to the SE group (P≤0.001), area under the curve (AUC) of 0.813. CONCLUSIONS: The results of this study suggest that decreased serum miRNA levels of miR-199a-3p and miR-320e could help to identify patients with BE and HGD.

Cancer Stem Cells, Epithelial to Mesenchymal Markers, and Circulating Tumor Cells in Small Cell Lung Cancer.

BACKGROUND: Small cell lung cancer (SCLC) has a poor prognosis, and even with localized (limited) disease, the 5-year survival has only been around 20%. Elevated levels of circulating tumor cells (CTCs) have been associated with a worse prognosis, and markers of cancer stem cells (CSCs) and epithelial to mesenchymal transition have been associated with increased chemoresistance and metastatic spread in SCLC. PATIENTS AND METHODS: The biopsy specimens of 38 SCLC patients were used for marker evaluation by immunohistochemistry. The markers for CSCs were CD44 and SOX2. The markers for epithelial to mesenchymal transition were E-cadherin, epithelial cell adhesion molecule, cytokeratins 8, 18, and 19, vimentin, and c-MET. Staining was scored as low (weak) or high (strong) intensity for SOX2, epithelial cell adhesion molecule, cytokeratins 8, 18, and 19, and c-MET and using the immunoreactive score for CD44, E-cadherin, and vimentin, expressed as low or high expression. RESULTS: High expression of c-MET (c-METH) and low expression of E-cadherin (E-cadL) showed a trend toward a better prognosis (P = .07 and P = .09, respectively). The combination of c-METH and E-cadL resulted in significantly better survival (P = .007). The tested markers were not associated with CTCs, although a trend was seen for c-METHE-cadL (P = .09) with low CTCs. The CSC markers SOX2 and CD44 were not associated with overall survival in this patient cohort. CONCLUSION: SCLC with a mesenchymal-like phenotype (c-METHE-cadL) is associated with longer survival and showed a trend toward lower CTCs.

Molecular Fluorescence Endoscopy Targeting Vascular Endothelial Growth Factor A for Improved Colorectal Polyp Detection.

UNLABELLED: Small and flat adenomas are known to carry a high miss-rate during standard white-light endoscopy. Increased detection rate may be achieved by molecular fluorescence endoscopy with targeted near-infrared (NIR) fluorescent tracers. The aim of this study was to validate vascular endothelial growth factor A (VEGF-A) and epidermal growth factor receptor (EGFR)-targeted fluorescent tracers during ex vivo colonoscopy with an NIR endoscopy platform. METHODS: VEGF-A and EGFR expression was determined by immunohistochemistry on a large subset of human colorectal tissue samples--48 sessile serrated adenomas/polyps, 70 sporadic high-grade dysplastic adenomas, and 19 hyperplastic polyps--and tissue derived from patients with Lynch syndrome--78 low-grade dysplastic adenomas, 57 high-grade dysplastic adenomas, and 31 colon cancer samples. To perform an ex vivo colonoscopy procedure, 14 mice with small intraperitoneal EGFR-positive HCT116(luc) tumors received intravenous bevacizumab-800CW (anti-VEGF-A), cetuximab-800CW (anti-EGFR), control tracer IgG-800CW, or sodium chloride. Three days later, 8 resected HCT116(luc) tumors (2-5 mm) were stitched into 1 freshly resected human colon specimen and followed by an ex vivo molecular fluorescence colonoscopy procedure. RESULTS: Immunohistochemistry showed high VEGF-A expression in 79%-96% and high EGFR expression in 51%-69% of the colorectal lesions. Both targets were significantly overexpressed in the colorectal lesions, compared with the adjacent normal colon crypts. During ex vivo molecular fluorescence endoscopy, all tumors could clearly be delineated for both bevacizumab-800CW and cetuximab-800CW tracers. Specific tumor uptake was confirmed with fluorescent microscopy showing, respectively, stromal and cell membrane fluorescence. CONCLUSION: VEGF-A is a promising target for molecular fluorescence endoscopy because it showed a high protein expression, especially in sessile serrated adenomas/polyps and Lynch syndrome. We demonstrated the feasibility to visualize small tumors in real time during colonoscopy using a NIR fluorescence endoscopy platform, providing the endoscopist a wide-field red-flag technique for adenoma detection. Clinical studies are currently being performed in order to provide in-human evaluation of our approach.

GATA6 expression in Barrett's oesophagus and oesophageal adenocarcinoma.

BACKGROUND: Barrett's oesophagus can progress towards oesophageal adenocarcinoma through a metaplasia-dysplasia-carcinoma sequence, but the underlying mechanisms are poorly understood. The transcription factor GATA6 is known to be involved in columnar differentiation and proliferation, and GATA6 gene amplification was recently linked with poor survival in oesophageal adenocarcinoma. AIM: To study the expression of GATA6 during Barrett's oesophagus development and malignant transformation. To determine the prognostic value of GATA6 in oesophageal adenocarcinoma. METHODS: Two retrospective cohorts were derived from the pathological archive of the University Medical Center Groningen. The first cohort contained 130 tissue samples of normal squamous epithelium, metaplasia, dysplasia and oesophageal adenocarcinoma. The second cohort consisted of a tissue microarray containing tissue from 92 oesophageal adenocarcinoma patients. Immunohistochemistry was used to examine GATA6 protein expression and to correlate GATA6 expression in oesophageal adenocarcinoma with overall and disease-free survival. RESULTS: The percentage of GATA6-positive cells was low in squamous epithelium (10%) but increased progressively in Barrett's oesophagus (30%, P < 0.001) and high-grade dysplasia (82%, P = 0.005). GATA6 expression was not associated with overall or disease-free survival in oesophageal adenocarcinoma patients (P = 0.599 and P = 0.700 respectively). CONCLUSION: GATA6 expression is progressively increased during Barrett's oesophagus development and its malignant transformation. However, no prognostic value of GATA6 expression could be found in oesophageal adenocarcinoma.

Loss of CD44 and SOX2 expression is correlated with a poor prognosis in esophageal adenocarcinoma patients.

BACKGROUND: It has been suggested that markers associated with cancer stem cells (CSC) may play a role in esophageal cancer. Our aim was to investigate the expression pattern of proposed CSC markers ALDH1, Axin2, BMI1, CD44, and SOX2 in esophageal adenocarcinoma (EAC) and to relate their expression to survival. METHODS: In this study we included 94 EAC patients and examined the expression of the above-mentioned markers by using immunohistochemistry on tissue microarrays. Expression was scored as positive or negative or categorized as low or high in terms of an immunoreactivity score (IRS). Expression rates were related to clinicopathologic characteristics and overall and disease-free survival (DFS). RESULTS: In a multivariate analysis, negative expression of CD44 and of SOX2 were both significant prognostic factors for DFS [hazard ratio (HR), 1.73; 95 % confidence interval (CI), 1.00-2.96; P = 0.046 and HR, 2.06; 95 % CI 1.14-3.70 P = 0.016). When CD44 and SOX2 expression were analyzed together, negative SOX2 expression was an independent prognostic factor for DFS (HR, 1.91; 95 % CI 1.05-3.46; P = 0.034). Low IRS scores for ALDH1 or Axin2 were associated with a reduced median survival (12.8 vs. 28.7 and 12.1 vs. 25.5 months, respectively). However, these markers and BMI1 were not prognostic factors for survival. CONCLUSIONS: Loss of CD44 expression and loss of SOX2 expression are prognostic factors of poor survival in EAC patients. This suggests a role of these proteins in EAC that requires further investigation.

Targeting FLIP and Mcl-1 using a combination of aspirin and sorafenib sensitizes colon cancer cells to TRAIL.

The multikinase inhibitor sorafenib is highly effective against certain types of cancer in the clinic and prevents colon cancer cell proliferation in vitro. Non-steroidal anti-inflammatory drugs, such as acetylsalicylic acid (aspirin), have shown activity against colon cancer cells. The aims of this study were to determine whether the combination of aspirin with sorafenib has enhanced anti-proliferative effects and increases recombinant human tumour necrosis factor-related apoptosis-inducing ligand (rhTRAIL)-induced apoptosis in the human SW948, Lovo, Colo205, Colo320, Caco-2 and HCT116 colon cancer cell lines. In four cell lines, aspirin strongly stimulated the anti-proliferative effects of sorafenib (∼four-fold enhancement) by inducing cell cycle arrest. Furthermore, combining low doses of aspirin (≤ 5 mm) and sorafenib (≤ 2.5 µm) greatly sensitized TRAIL-sensitive and TRAIL-resistant colon cancer cells to rhTRAIL, much more potently than either drug combined with rhTRAIL. The increase in rhTRAIL sensitivity was due to inhibition of FLIP and Mcl-1 protein expression following aspirin and sorafenib co-treatment, as confirmed by knock-down studies. Next, the clinical relevance of targeting FLIP and Mcl-1 in colon cancer was examined. Using immunohistochemistry, we found that Mcl-1 expression was significantly increased in colon adenoma and carcinoma patient material compared to healthy colonic epithelium, similar to the enhanced FLIP expression we recently observed in colon cancer. These results underscore the potential of combining low doses of aspirin with sorafenib to inhibit proliferation and target the anti-apoptotic proteins FLIP and Mcl-1 in colon cancer cells.

The extrinsic apoptosis pathway and its prognostic impact in ovarian cancer.

OBJECTIVE: Death ligand FasL, its agonistic receptor Fas, tumor necrosis factor related apoptosis inducing ligand (TRAIL) and its agonistic death receptors DR4 and DR5 are implied in carcinogenesis, tumor immune surveillance and response to chemotherapy. TRAIL receptor agonists are evaluated as anti-cancer agents. This study aimed to relate expression of death ligands/receptors and downstream initiator caspase 8 and its anti-apoptotic homologue FLICE like inhibitory protein (c-FLIP) in ovarian cancers to chemotherapy response and survival. METHODS: Fas, FasL, TRAIL, DR4, DR5, caspase 8 and c-FLIP were determined immunohistochemically on a tissue microarray containing 382 ovarian cancers. Protein expression profiles were correlated with clinicopathologic variables, chemotherapy response and survival. RESULTS: Most tumors expressed DR4, DR5, caspase 8 and c-FLIP. High c-FLIP expression was associated with expression of caspase 8 and both TRAIL receptors. TRAIL and Fas were associated with low tumor grade and better progression-free survival (HR 0.63, p=.018 and HR 0.54, p=.012), respectively, and Fas with disease-specific survival (HR 0.49, p=0.009) in univariate analysis. CONCLUSIONS: Fas and TRAIL loss is associated with dedifferentiation and worse prognosis. Expression of DR4, DR5, caspase 8 and c-FLIP by most ovarian cancers does not correlate with survival. High c-FLIP expression should be taken into account for death receptor targeted therapies.

Enhanced antitumor efficacy of a DR5-specific TRAIL variant over recombinant human TRAIL in a bioluminescent ovarian cancer xenograft model.

PURPOSE: Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) is clinically evaluated as novel anticancer drug. rhTRAIL-DR5, a rhTRAIL variant that specifically binds to DR5 receptor, has recently been developed. We investigated whether rhTRAIL-DR5 is more efficient than rhTRAIL in combination with cisplatin in DR5-expressing human A2780 ovarian cancer cells. DESIGN: Effect of cisplatin alone or in combination with rhTRAIL or rhTRAIL-DR5 on DR5 surface expression, apoptosis, and cell survival of A2780 was measured. Biodistribution analysis was done in mice with (125)I-rhTRAIL administered intravenously versus intraperitoneally. Antitumor efficacy of rhTRAIL-DR5 versus rhTRAIL was determined in an intraperitoneally growing bioluminescent A2780 xenograft model. RESULTS: Cisplatin strongly enhanced DR5 surface expression. Both rhTRAIL and rhTRAIL-DR5 in combination with cisplatin induced high levels of caspase-3 activation, apoptosis, and cell kill, with rhTRAIL-DR5 being most potent. Intraperitoneal administration of (125)I-rhTRAIL resulted in a 1.7-fold higher area under the curve in serum, increased tumor exposure, and more caspase-3 activation in the tumor than intravenous administration. Intraperitoneal administration of rhTRAIL-DR5 delayed A2780 tumor progression, reflected in a mean light reduction of 68.3% (P = 0.015), whereas rhTRAIL or rhTRAIL-DR5 plus cisplatin resulted in 85% (P = 0.003) and 97% (P = 0.002) reduction compared with A2780 tumor progression in vehicle-treated animals. Combination of rhTRAIL-DR5 with cisplatin was more effective than cisplatin alone (P = 0.027). CONCLUSION: rhTRAIL-DR5 was superior over rhTRAIL in vitro and in vivo against DR5-expressing ovarian cancer also in combination with cisplatin. Intraperitoneal administration of rhTRAIL-DR5 warrants further exploration in ovarian cancer.

PMS2 involvement in patients suspected of Lynch syndrome.

It is well-established that germline mutations in the mismatch repair genes MLH1, MSH2, and MSH6 cause Lynch syndrome. However, mutations in these three genes do not account for all Lynch syndrome (suspected) families. Recently, it was shown that germline mutations in another mismatch repair gene, PMS2, play a far more important role in Lynch syndrome than initially thought. To explore this further, we determined the prevalence of pathogenic germline PMS2 mutations in a series of Lynch syndrome-suspected patients. Ninety-seven patients who had early-onset microsatellite instable colorectal or endometrial cancer, or multiple Lynch syndrome-associated tumors and/or were from an Amsterdam Criteria II-positive family were selected for this study. These patients carried no pathogenic germline mutation in MLH1, MSH2, or MSH6. When available, tumors were investigated for immunohistochemical staining (IHC) for PMS2. PMS2 was screened in all patients by exon-by-exon sequencing. We identified four patients with a pathogenic PMS2 mutation (4%) among the 97 patients we selected. IHC of PMS2 was informative in one of the mutation carriers, and in this case, the tumor showed loss of PMS2 expression. In conclusion, our study confirms the finding of previous studies that PMS2 is more frequently involved in Lynch syndrome than originally expected.

JC virus infection in colorectal neoplasia that develops after liver transplantation.

PURPOSE: Liver transplant recepients (LTRs) have an increased risk of colorectal neoplasia. The mechanism responsible for this is unknown. JCV encodes for TAg and has been implicated in colorectal carcinogenesis. We hypothesized that the use of immunosuppression in LTRs facilitates activation of JCV and is responsible for the increased risk of neoplasia. EXPERIMENTAL DESIGN: JCV TAg DNA and protein expression were determined in normal colonic epithelium (n = 15) and adenomatous polyps (n = 26) from LTRs and compared with tissue samples from control patients (normal colon, n = 21; adenomas, n = 40). Apoptosis and proliferation were determined by M30 and Ki-67 immunoreactivity, respectively. RESULTS: JCV TAg DNA was found in 10 of 15 (67%) of normal colonic mucosa from LTRs compared with 5 of 21 (24%) of control normal mucosa (P = 0.025). JCV TAg DNA was detected in 16 of 26 (62%) of the adenomas from LTRs and in 20 of 40 (50%) of control adenomas. JCV TAg protein was expressed in 13 of 26 (50%) adenomas from LTRs versus 2 of 40 (5%) of adenomas from controls (P < 0.001). In adenomas from LTRs, the mean proliferative activity was higher compared with controls (60.3 +/- 3.2% versus 42.7 +/- 2.8%, P < 0.001), whereas mean apoptotic indices were lower in LTRs (0.29 +/- 0.08% versus 0.39 +/- 0.06%, P = 0.05). CONCLUSIONS: The presence of JCV in the colorectal mucosa and adenomas from LTRs, in concert with the use of immunosuppressive agents, suggests that JCV may undergo reactivation, and the subsequent TAg protein expression might explain the increased risk of colorectal neoplasia in LTRs.

Early carcinogenic events in HNPCC adenomas: differences with sporadic adenomas.

BACKGROUND: Tumorigenesis in hereditary nonpolyposis colorectal cancer (HNPCC) differs from that in sporadic colorectal cancer during the early stage. We examined the expression of proliferation- and apoptosis-regulating proteins in relation to proliferation and apoptosis in HNPCC and sporadic adenomas. METHODS: Proliferation and apoptosis were quantified, and the expression of cyclin B1, D3 and E, p21, p27, bcl-2, bax, p53 and cox-2 was determined by immunohistochemistry in 100 patients (42 with HNPCC and 48 with sporadic adenomas). RESULTS: No differences between the two groups of patients in terms of proliferation and apoptosis were detected. Low-grade dysplastic HNPCC adenomas differed from sporadic ones by expressing bcl-2 more often (69 vs. 42%) and bax less often (50 vs. 73%). In comparison to sporadic adenomas, fewer high-grade dysplastic HNPCC expressed cyclin B1 and E (50 and 38% vs. 87 and 87%, respectively), p21 (6% vs. 53%) and bax (31% vs. 80%). In addition, HNPCC adenomas had a lower overexpression of p53 (5 vs. 19%). CONCLUSION: The expression of cell cycle- and apoptosis-related proteins differs between HNPCC and sporadic adenomas from early through to advanced stages although proliferation and apoptosis are not different. These differences may contribute to the different clinical behavior of HNPCC and sporadic adenomas.

Sulindac treatment in hereditary non-polyposis colorectal cancer.

UNLABELLED: Non-steroidal anti-inflammatory drugs, e.g. sulindac have been extensively studied for chemoprevention in familial adenomatous polyposis, but not in hereditary non-polyposis colorectal cancer (HNPCC). We evaluated these effects in HNPCC using surrogate end-points for cancer risk. In a randomised double-blind cross-over study, 22 subjects (9 female; age 30-66 years, mean 44), all ascertained or probable mutation carriers for HNPCC, were included. Sulindac 150 mg b.i.d. and placebo were given for 4 weeks each, with 4 weeks in between, with biopsies taken from ascending, transverse and sigmoid colon and rectum by colonoscopy after both periods. Proliferation was determined by Ki-67 staining and apoptosis by staining of cytokeratin 18 cleavage products. Expression of cyclins B1, D3 and E and p21, p27, bax, bcl2 and cox-2 was studied immunohistochemically. Proliferation was higher during sulindac treatment than drug placebo treatment in ascending and transverse colon, but not in sigmoid and rectum. Apoptosis was not affected. Besides an increase in cyclin D3, no differences were found in expression of regulating proteins in the proximal colon. CONCLUSION: Sulindac induces an increase in epithelial cell proliferation in the proximal colon of subjects with HNPCC. Since colorectal cancer predominantly arises in the proximal colon in HNPCC, these results cast doubts on the potential chemopreventive effects of sulindac in HNPCC.

Prognostic significance of tumor necrosis factor-related apoptosis-inducing ligand and its receptors in adjuvantly treated stage III colon cancer patients.

PURPOSE: In preclinical models, there is synergism between chemotherapy and recombinant human tumor necrosis factor (TNF) -related apoptosis-inducing ligand (TRAIL) on apoptosis induction in tumor cells. Therefore, the prognostic relevance was analyzed of the expression of TRAIL and its death receptors DR4 and DR5 on disease-free survival and overall survival in stage III colon cancer patients treated with adjuvant chemotherapy. METHODS: Tissue microarrays were constructed of primary tumor tissue from 376 stage III colon cancer patients treated in a randomized adjuvant chemotherapy study (fluorouracil/levamisole v fluorouracil/levamisole/leucovorin) and stained immunohistochemically for TRAIL, DR4, and DR5. Log-rank tests and Cox proportional hazard analysis, with adjustment for treatment arm, sex, age, N stage, microsatellite instability status, and p53 mutation status, were performed. RESULTS: The majority of tumors showed high expression of TRAIL (83%), DR4 (92%), and DR5 (87%). Median follow-up was 43 months. High DR4 expression was associated with worse disease-free survival (odds ratio [OR] = 2.19; 95% CI, 1.06 to 4.53; P = .03), worse overall survival (OR = 2.22; 95% CI,1.03 to 4.81; P = .04) and shorter time to recurrence (P = .02) compared with those with low DR4 expression. TRAIL or DR5 expression had no prognostic value. CONCLUSION: High DR4 expression is associated with worse disease-free and overall survival in stage III adjuvant-treated colon cancer patients. Evaluation of DR4 expression in stage III colon cancer patients may identify a subset requiring more aggressive adjuvant treatment.

TRAIL induces apoptosis in human colorectal adenoma cell lines and human colorectal adenomas.

PURPOSE: Recombinant human (rh) tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential new anticancer drug which can induce apoptosis in colorectal cancer cell lines. The aim of this study was to investigate whether it is possible to induce apoptosis in human adenoma cell lines and human adenomas using rhTRAIL. EXPERIMENTAL DESIGN: Two human adenoma cell lines were exposed to 0.1 microg/mL of rhTRAIL for 5 hours. Apoptosis and caspase activation in cell lines were evaluated using immunocytochemistry, fluorimetric caspase assays, and Western blotting. Short-term explant cultures were established from freshly removed human adenomas (n = 38) and biopsies of normal colon epithelium (n = 15), and these were incubated for 5 hours in the presence or absence of 1 microg/mL of rhTRAIL. Apoptosis was determined in paraffin-embedded tissue using morphologic criteria and cleaved caspase-3 staining. RESULTS: In the adenoma cell lines, rhTRAIL induced up to 55% apoptosis. This coincided with caspase-8 and caspase-3 activation and could be inhibited by a pan-caspase inhibitor. rhTRAIL induced caspase-dependent apoptosis in adenomas with high-grade dysplasia (n = 21) compared with the paired untreated counterparts (apoptotic index, 34 +/- 5% versus 17 +/- 2%, mean +/- SE; P = 0.002), but not in adenomas with low-grade dysplasia (n = 17) or in normal colon epithelium (n = 15). CONCLUSIONS: Colorectal adenoma cell lines and adenomas with high-grade dysplasia are sensitive to rhTRAIL-induced apoptosis, whereas normal colon epithelium is not. This suggests the potential application of rhTRAIL in the treatment of adenomas with high-grade dysplasia.