Esophageal cancer (EC) is a highly aggressive disease with a poor prognosis. Therapy resistance and early recurrences are major obstacles in reaching a better outcome. Esophageal cancer stem-like cells (CSCs) seem tightly related with chemoradiation resistance, initiating new tumors and metastases. Several oncogenic pathways seem to be involved in the regulation of esophageal CSCs and might harbor novel therapeutic targets to eliminate CSCs. Previously, we identified a subpopulation of EC cells that express high levels of CD44 and low levels of CD24 (CD44+/CD24−), show CSC characteristics and reside in hypoxic niches. Here, we aim to clarify the role of the hypoxia-responding mammalian target of the rapamycin (mTOR) pathway in esophageal CSCs. We showed that under a low-oxygen culture condition and nutrient deprivation, the CD44+/CD24− population is enriched. Since both low oxygen and nutrient deprivation may inhibit the mTOR pathway, we next chemically inhibited the mTOR pathway using Torin-1. Torin-1 upregulated SOX2 resulted in an enrichment of the CD44+/CD24− population and increased sphere formation potential. In contrast, stimulation of the mTOR pathway using MHY1485 induced the opposite effects. In addition, Torin-1 increased autophagic activity, while MHY1485 suppressed autophagy. Torin-1-mediated CSCs upregulation was significantly reduced in cells treated with autophagy inhibitor, hydroxychloroquine (HCQ). Finally, a clearly defined CD44+/CD24− CSC population was detected in EC patients-derived organoids (ec-PDOs) and here, MHY1485 also reduced this population. These data suggest that autophagy may play a crucial role in mTOR-mediated CSCs repression. Stimulation of the mTOR pathway might aid in the elimination of putative esophageal CSCs.

Objectives To assess the complementary value of human epidermal growth factor receptor 2 (HER2)-related biological tumor markers to clinico-radiomic models in predicting complete response to neoadjuvant chemoradiotherapy (NCRT) in esophageal cancer patients. Methods Expression of HER2 was assessed by immunohistochemistry in pre-treatment tumor biopsies of 96 patients with locally advanced esophageal cancer. Five other potentially active HER2-related biological tumor markers in esophageal cancer were examined in a sub-analysis on 43 patients. Patients received at least four of the five cycles of chemotherapy and full radiotherapy regimen followed by esophagectomy. Three reference clinico-radiomic models based on 18 F-FDG PET were constructed to predict pathologic response, which was categorized into complete versus incomplete (Mandard tumor regression grade 1 vs. 2–5). The complementary value of the biological tumor markers was evaluated by internal validation through bootstrapping. Results Pathologic examination revealed 21 (22%) complete and 75 (78%) incomplete responders. HER2 and cluster of differentiation 44 (CD44), analyzed in the sub-analysis, were univariably associated with pathologic response. Incorporation of HER2 and CD44 into the reference models improved the overall performance ( R 2 s of 0.221, 0.270, and 0.225) and discrimination AUCs of 0.759, 0.857, and 0.816. All models exhibited moderate to good calibration. The remaining studied biological tumor markers did not yield model improvement. Conclusions Incorporation of HER2 and CD44 into clinico-radiomic prediction models improved NCRT response prediction in esophageal cancer. These biological tumor markers are promising in initial response evaluation. Key Points • A multimodality approach, integrating independent genomic and radiomic information, is promising to improve prediction of γpCR in patients with esophageal cancer. • HER2 and CD44 are potential biological tumor markers in the initial work-up of patients with esophageal cancer. • Prediction models combining 18 F-FDG PET radiomic features with HER2 and CD44 may be useful in the decision to omit surgery after neoadjuvant chemoradiotherapy in patients with esophageal cancer.

Esophageal cancer (EC) is an aggressive disease with a poor prognosis. Treatment resistance is a major challenge in successful anti-cancer therapy. Pathological complete response after neoadjuvant chemoradiation (nCRT) is low, thus requiring therapy optimization. The Hedgehog (HH) pathway has been implicated in therapy resistance, as well as in cancer stemness. This article focusses on the HH pathway as a putative target in the treatment of EC. Immunohistochemistry on HH members was applied to EC patient material followed by modulation of 3D-EC cell cultures, fluorescence-activated cell sorting (FACS), and gene expression analysis after HH pathway modulation. Sonic Hedgehog (SHH) and its receptor Patched1 (PTCH1) were significantly enriched in EC resection material of patients with microresidual disease (mRD) after receiving nCRT, compared to the control group. Stimulation with SHH resulted in an up-regulation of cancer stemness in EC sphere cultures, as indicated by increased sphere formation after sorting for CD44+/CD24− EC cancer stem-like cell (CSC) population. On the contrary, inhibiting this pathway with vismodegib led to a decrease in cancer stemness and both radiation and carboplatin resistance. Our results strengthen the role of the HH pathway in chemoradiotherapy resistance. These findings suggest that targeting the HH pathway could be an attractive approach to control CSCs.

Slokdarmkanker is een van de meest agressieve kankersoorten wereldwijd met een slechte overlevingskans. Gezien de relatieve ‘stille’ ontwikkeling van deze aandoening, komt slechts de helft van de nieuw gediagnosticeerde patienten in aanmerking voor behandeling waarbij genezing het einddoel is. Deze standaard behandeling is volgens het CROSS schema; wekelijkse chemotherapie (carboplatin en paclitaxel) gedurende 5 weken in combinatie met radiotherapie (41.4 Gy, in 23 fracties van 1.8 Gy) gevolgd door een operatie waarbij de tumor met ruime marges verwijderd wordt. Een 5-jaars overlevingskans van 47% wordt met deze behandeling bereikt in vergelijking met 33% met enkel een chirurgische aanpak. Helaas, ligt het percentage complete respons na chemoradiatie op 29%. Het merendeel, 71%, reageert onvoldoende, waarvan 18% geen enkele respons laat zien. Deze laatste groep patienten zullen helaas wel de nadelige bijwerkingen van chemoradiatie ondervinden zonder enige verkleining van de tumor. Opmerkelijk is dat vroege terugkeer van de tumor ook kan voorkomen bij patienten met een complete respons. Deze observaties wijzen op een uiterst agressieve ziekte met hoge resistentie tegen de huidige chemoradiatie. Van een populatie tumorcellen is bekend dat het resistent is voor conventionele chemo- en radiotherapie, de zogenoemde kankerstamcellen. Het elimineren van deze populatie cellen zou potentieel de therapierespons en hiermee ook de overleving kunnen verbeteren. Dit proefschrift zal zich richten op het identificeren van predictieve factoren van de huidige chemoradiatie en daarnaast het opsporen van doelgerichte therapieen die de therapierespons verbeteren met focus op de kankerstamcelpopulatie. Hiermee kan een behandeling op maat worden gecreeerd.

Esophageal cancers (EC) are highly aggressive tumors, commonly presented in a locally advanced stage with a poor prognosis and survival. Up to 50% of the patients are eligible for treatment with curative intent and receive the standard treatment with neoadjuvant chemoradiotherapy (nCRT) and surgery. Currently, pathologic complete response to nCRT is 20-30%, with a partial or no response in about 50% and 20%, respectively. EC recurrences occur frequently even after successful anti-cancer treatment, suggesting high aggressiveness with increased metastatic potential. A tumor sub-population of so-called cancer stem cells (CSCs), is known to display a high metastatic potential and resistance to conventional anti-cancer therapy, hereby being responsible for the unbeneficial clinical features. In this review, a concise overview will be given of the current literature on esophageal CSCs and related metastases. Esophageal CSC markers will be discussed followed by the pathways that initiate and sustain these cells. In addition, the cellular processes, epithelial-mesenchymal transition (EMT), hypoxia and autophagy, known to contribute to cancer stemness and metastasis will be explained. Finally, potential options for treatment also related to cancer genome atlas (TCGA) data on EC will be discussed.

BACKGROUND: The purpose of this study was to provide more insight in the course of cytokine concentrations related to pathologic response (pR) and complications after neoadjuvant chemoradiotherapy (NCRT) and esophagectomy in esophageal cancer patients.METHODS: Patients treated with NCRT followed by transthoracic esophagectomy (n = 35) or transthoracic esophagectomy alone (n = 8) were included. Eight different cytokine concentrations were determined during NCRT, esophagectomy, and the first postoperative week.RESULTS: Platelet-activating factor before NCRT was associated with pR (P = .011) and remained elevated in patients with a better response. Concentrations of intestinal fatty acid-binding protein and angiopoietin 1 (Ang-1) were different between patients with and without NCRT. Decreased concentrations of Ang-1 on the third postoperative day were associated with postoperative complications (P = .046).CONCLUSIONS: In this observational study, elevated platelet-activating factor concentrations before NCRT were associated with pR. NCRT is associated with decreased Ang-1 concentrations, whereas reduced Ang-1 concentrations were associated with postoperative complications. (C) 2016 Elsevier Inc. All rights reserved.

43 Background: Despite modern advances in the treatment of esophageal cancer (EC), using neoadjuvant chemoradiotherapy (CRT) and esophagectomy, most patients face poor outcome. Growing evidence indicates that cancer stem cells (CSCs) might contribute to the poor prospects. CSCs are usually resistant to CRT and ultimately can generate a new tumor. The mammalian target of rapamycin (mTOR) pathway is associated with cancer stemness. However, its role in EC CSC-like populations needs to be elucidated. Here, we investigate the role of mTOR pathway on the stemness of a putative CSC-like population. Methods: Previously, we identified a putative CSC-like population (CD44+/CD24-) in EC cell lines and in tumor biopsy from EC patients. qPCR was used to measure the expression of mTOR in CD44+/CD24- CSC-like population of OE21 squamous cell carcinoma and OE33 adenocarcinoma cell lines compared to controls, that consisted of solid tumors generated from the same cell lines obtained from xenografts. mTOR inhibitors rapamycin and torin-1 were used to see their effect on CD44+/CD24- expression and sphere formation. Results: mTOR expression was 2-fold up-regulated in the OE33 CD44+/CD24- CSC-like population compared to control. Furthermore, in OE21 this up-regulation was 1.9-fold. Surprisingly, inhibiting the mTOR pathway with rapamycin enhanced OE33 CD44+/CD24- expression compared to its control (p = 0.01). In pilot experiments this effect was dose dependent and cells treated with rapamycin formed more spheres than control. Rapamycin did not alter the expression of CD44+/CD24- in OE21. Inhibiting the mTOR pathway with Torin-1 enhanced OE21 CD44+/CD24- expression by 1.2-fold compared to control (N = 2). In another pilot experiment Torin-1 treated cells were able to form more spheres compared to control. Torin-1 did not have an effect on the expression of CD44+/CD24- in OE33. Conclusions: These findings indicate that inhibiting the mTOR pathway may enhance CSC-like properties in EC. Additional research needs to be done to further support this hypothesis and elucidate the mechanism in this process. Furthermore, the effect of mTOR pathway inducers in EC needs to be explored.

77 Background: Despite advances in the treatment of esophageal cancer (EC), most patients face poor outcome. Mounting evidence indicates that cancer stem cells (CSCs) might contribute to the poor prospects. Whereas most cancer cells are sensitive to chemo- and/or radiotherapy (CRT), CSCs are generally resistant and ultimately have the potential to generate a new tumor. Mechanisms regulating EC CSCs are poorly understood and need to be elucidated. Previously, we identified a putative CSC-like population (CD24-/CD44+) in EC cell lines and in esophageal adenocarcinoma (EAC). Here we show that the Hedgehog signaling pathway may regulate CSC-like populations in EC. Methods: To identify up-regulated CSC related genes, qPCR arrays were performed on CD24-/CD44+ OE21 esophageal squamous cell carcinoma (ESCC) and OE33 (EAC). CD24-/CD44+ was compared to CD24+/CD44+ and solid tumors generated from the same cell lines obtained from xenografts. Immunohistochemical (IHC) staining from tumor material of microscopical residual disease (mRD) patients after CRT was compared to surgery alone patients (S). Results: From a panel of 84 CSC related genes, Ptch1 was found to be up-regulated in the OE21 CD24-/CD44+ putative CSC-like population when compared to CD24+/CD44+ population and the solid tumor (2.5 and 3.7 fold, respectively). Furthermore, in OE33 this up-regulation was 1.4 and 1.7 fold. From 6 preselected candidate CRT response markers, CD44 and Sonic Hedgehog (SHH) expression was 55% (p<0.05) and 64% (P<0.005) enhanced in mRD compared to the S group in IHC material. Moreover, SHH expression showed a positive correlation coefficient of 0.551 (P<0.005) with CD44 and 0.512 (P<0.05) with Excision Repair Cross-Complementation group 1 (ERCC1, a nucleotide excision repair gene) in mRD tissue. Conclusions: These findings indicate that the Hedgehog pathway might be involved in regulating EC CSC-like properties. Although requiring further research, an exciting prospect would be the modulation of the Hedgehog pathway in combating CSC-like populations and the potential predictive value of SHH expression on treatment outcome.

Objectives: To evaluate the effectiveness of clinical pathway on shortening average length of stay and reducing hospitalization cost in 5 kinds of cancers. Method: 1575 patients treated with clinical pathway and 806 patients receiving the routine treatment in 5 kinds of cancers during Jan to Dec in 2012 were selected for retrospective study. The average length of stay and average hospitalization cost were compared.Results: There were significant difference in average length of stay between breast carcinoma and gastric tumor and average hospitalization cost among thyroid tumor, breast carcinoma, gastric tumor and colon tumor. Conclusions: Clinical pathway could shorten the average length of stay and reduce average hospitalization cost.

BACKGROUND: We evaluated the impact of neoadjuvant chemoradiotherapy (CRT) on nodal micro-metastases (NMMs) in esophageal adenocarcinoma (EAC) patients with histologically negative nodes ([y]pN0). METHODS: Of 48 consecutively treated patients with neoadjuvant CRT, we selected 20 EAC ypN0 patients (group 1). These patients were matched with 20 pN0 EAC patients who had surgery alone (group 2). Harvested (y) pN0 lymph nodes were examined immunohistochemically (anti-CK8/18 [CAM 5.2]) according to a validated sentinel node protocol. A 3rd group (n = 11) staged as ypN1 after neoadjuvant CRT was used as the control group. RESULTS: Upstaging to NMM+ occurred in 2 patients (10%) in group 1 and in 8 patients (40%) in group 2 (P = .028). Disease-free and overall survival rates in NMM+ patients in group 1 were worse compared with NMM- patients (P = .014 and P = .003, respectively) but comparable with ypN1 patients (n = 11). CONCLUSIONS: A 30% reduction of NMM+ was obtained after neoadjuvant treatment in (y) pN0 patients. NMM+ after CRT had a negative impact on survival in ypN1 patients. These data warrant further investigation in larger prospective datasets. (C) 2013 Elsevier Inc. All rights reserved.

Objective To investigate the expression of β-catenin and Wnt-1 in esophageal carcinoma and their correlations with the pathologic parameter of cancer and prognosis of esophageal carcinoma. Methods This study was performed at the Pathological Department of Jiangsu People′s Hospital in 2012. 40 esophageal cancer sections and 10 surrounding tissues embedded in paraffin was enrolled. The β-catenin and Wnt-1 expression by immunohistochemistry were detected. Quantification was performed by measuring the intensity of the signals with JD-801 Pathological image analysis system. All the esophageal cancer sections were divided into the high expression group and low expression group. Statistical analysis of the β-catenin and Wnt-1 expression between the two groups were performed using Stata 9.0(USA). Univariate Kaplan-Meier analysis was used to estimate the survival rate between the two groups. Results Wnt-1 expression was associated with a significantly higher survival rate by Kaplan-Meier analysis(P = 0.0177). β-catenin high expression group had a high survival rate,while there was not statistically significant(P = 0.1010). Conclusion As the correlated proteins of WNT signal pathway,the expression level of Wnt-1 and β-catenin are close relationship with progression,invasion and the prognosis of esophageal carcinoma.

Objective:To investigate the mechanisms of anti-cancer drug,SN38,on Fas-mediated apoptosis and to study the role of lipid raft in such progress.Methods:WR/FAS-SM(-) cells and WR/FAS-SMS1 cells were treated by SN38 and/or CH11.DNA damage-associated moleculars and caspase were studied for further investigation for activation level and characteristics of such moleculars by Western blot.Results:Combination of SN38 and CH11 could induce the activation of ATM-Chk1-p53 pathway and caspase-3,8 in WR/Fas-SMS1 cells and the inactivation of p21.Whereas the activation of Chk1-p53 and caspase-3,8 did not induce the inactivation of p21 in WR/Fas-SM(-) cells.Conclusion:The stronger apoptosis in WR/Fas-SMS1 cells induced by the combination of SN38 and CH11 was caused by the activation of ATM-Chk1-p53 pathway and caspase-3,8 and the inactivation of p21.Lipid raft induced stronger apoptosis in WR/Fas-SMS1 cells than in WR/Fas-SM(-) cells by decreasing the expression of phospho-p21 with the treatment of SN38 and CH11.

73 Background: Neoadjuvant chemoradiation (CRT) is important in the treatment ofesophageal cancer. Rationales are tumor-downstaging and elimination of micrometastases improving resectability and curability rate. In this study we evaluated the effect of neo-adjuvant CRT on nodal micrometastases (NMM) in pN0 esophageal cancer patients. METHODS From a prospective database, we selected a matched group of patients on cT-stage, histological type and treatment without (group 1) or with neo-adjuvant CRT (group 2). Patients were staged by EUS, PET-scan, CT-scan and EUS guided cytology. CRT was given in a randomized clinical trial and consisted of paclitaxel 50 mg/m2 and carboplatin AUC = 2 for 5 weeks and concurrent radiotherapy (41.4 Gy/23 frs). All patients (n = 19) underwent a curative intended transthoracic esophagectomy with extended 2-field nodal dissection. After reviewing routine pathological examination all tumors were confirmed as stage pN0. Four sections of every examined lymph node (n = 261) were made at different levels according to a sentinel node protocol and further analyzed immunohistochemically with anti-CK8/18 (CAM 5.2) to detect NMM. RESULTS Patients characteristics were equally distributed. All patients had a microscopically radical (R0) resection. In group 2 (n = 9) the response rate was 55% which was complete (CR) in 33%. The median number of resected nodes was comparable: 15 ± 6.5 and 15 ± 5.6 in both groups. Thirtheen of the 261 (5%) pN0 nodes in these 19 pts contained NMM. In group 1 (n = 10); 12 of the 136 (9%) examined pN0 lymph nodes were positive and in group 2 only one of the 125 (0.8%) examined pN0 nodes was positive (p = 0.003). NMM were found in 7 pts (70%) in group 1 and in only one patient (11%) in group 2 (p = 0.02). Interestingly, this last patient had responded well to CRT. CONCLUSIONS Neo-adjuvant CRT seems to have a significant impact on the numbers of NMMs in esophageal cancer. Based on the presence of NMM even in patients with good responses, we still advocate a curatively intended nodal dissection. No significant financial relationships to disclose.