Survival after secondary liver resection in metastatic colorectal cancer: Comparing data of three prospective randomized European trials (LICC, CELIM, FIRE-3).

Metastatic colorectal cancer (mCRC) patients with liver-limited disease (LLD) have a chance of long-term survival and potential cure after hepatic metastasectomy. However, the appropriate postoperative treatment strategy is still controversial. The CELIM and FIRE-3 studies demonstrated that secondary hepatic resection significantly improved overall survival (OS). The objective of this analysis was to compare these favorable outcome data with recent results from the LICC trial investigating the antigen-specific cancer vaccine tecemotide (L-BLP25) as adjuvant therapy in mCRC patients with LLD after R0/R1 resection. Data from mCRC patients with LLD and secondary hepatic resection from each study were analyzed for efficacy outcomes based on patient characteristics, treatment and surveillance after surgery. In LICC, 40/121 (33%) patients, in CELIM 36/111 (32%) and in FIRE-3-LLD 29/133 (22%) patients were secondarily resected, respectively. Of those, 31 (77.5%) patients in LICC and all patients in CELIM were R0 resected. Median disease-free survival after resection was 8.9 months in LICC, 9.9 months in CELIM. Median OS in secondarily resected patients was 66.1 months in LICC, 53.9 months in CELIM and 56.2 months in FIRE-3-LLD. Median age was about 5 years less in LICC compared to CELIM and FIRE-3. Secondarily resected patients of LICC, CELIM and FIRE-3 showed an impressive median survival with a tendency for improved survival for patients in the LICC trial. A younger patient cohort but also more selective surgery, improved resection techniques, deep responses and a close surveillance program after surgery in the LICC trial may have had a positive impact on survival.

Comparative analysis of nuclear and mitochondrial DNA from tissue and liquid biopsies of colorectal cancer patients.

The current standard for molecular profiling of colorectal cancer (CRC) is using resected or biopsied tissue specimens. However, they are limited regarding sampling frequency, representation of tumor heterogeneity, and sampling can expose patients to adverse side effects. The analysis of cell-free DNA (cfDNA) from blood plasma, which is part of a liquid biopsy, is minimally invasive and in principle enables detection of all tumor-specific mutations. Here, we analyzed cfDNA originating from nucleus and mitochondria and investigated their characteristics and mutation status in a cohort of 18 CRC patients and 10 healthy controls using targeted next-generation sequencing (NGS) and digital PCR. Longitudinal analyses of nuclear cfDNA level and size during chemotherapy revealed a decreasing cfDNA content and a shift from short to long fragments, indicating an appropriate therapy response, while shortened cfDNAs and increased cfDNA content corresponded with tumor recurrence. Comparative NGS analysis of nuclear tissue and plasma DNA demonstrated a good patient-level concordance and cfDNA revealed additional variants in three of the cases. Analysis of mitochondrial cfDNA surprisingly revealed a higher plasma copy number in healthy subjects than in CRC patients. These results highlight the potential clinical utility of liquid biopsies in routine diagnostics and surveillance of CRC patients as complementation to tissue biopsies or as an attractive alternative in cases where tissue biopsies are risky or the quantity/quality does not allow testing.

The Role of p53 Dysfunction in Colorectal Cancer and Its Implication for Therapy.

Colorectal cancer (CRC) is one of the most common and fatal cancers worldwide. The carcinogenesis of CRC is based on a stepwise accumulation of mutations, leading either to an activation of oncogenes or a deactivation of suppressor genes. The loss of genetic stability triggers activation of proto-oncogenes (e.g., KRAS) and inactivation of tumor suppression genes, namely TP53 and APC, which together drive the transition from adenoma to adenocarcinoma. On the one hand, p53 mutations confer resistance to classical chemotherapy but, on the other hand, they open the door for immunotherapy, as p53-mutated tumors are rich in neoantigens. Aberrant function of the TP53 gene product, p53, also affects stromal and non-stromal cells in the tumor microenvironment. Cancer-associated fibroblasts together with other immunosuppressive cells become valuable assets for the tumor by p53-mediated tumor signaling. In this review, we address the manifold implications of p53 mutations in CRC regarding therapy, treatment response and personalized medicine.

Radiochemotherapy with or without cetuximab for unresectable esophageal cancer: final results of a randomized phase 2 trial (LEOPARD-2).

PURPOSE: To investigate the efficacy and toxicity of cetuximab when added to radiochemotherapy for unresectable esophageal cancer. METHODS: This randomized phase 2 trial (clinicaltrials.gov, identifier NCT01787006) compared radiochemotherapy plus cetuximab (arm A) to radiochemotherapy (arm B) for unresectable esophageal cancer. Primary objective was 2­year overall survival (OS). Arm A was considered insufficiently active if 2­year OS was ≤40% (null hypothesis = H0), and promising if the lower limit of the 95% confidence interval was >45%. If that lower limit was >40%, H0 was rejected. Secondary objectives included progression-free survival (PFS), locoregional control (LC), metastases-free survival (MFS), response, and toxicity. The study was terminated early after 74 patients; 68 patients were evaluable. RESULTS: Two-year OS was 71% in arm A (95% CI: 55-87%) vs. 53% in arm B (95% CI: 36-71%); H0 was rejected. Median OS was 49.1 vs. 24.1 months (p = 0.147). Hazard ratio (HR) for death was 0.60 (95% CI: 0.30-1.21). At 2 years, PFS was 56% vs. 44%, LC 84% vs. 72%, and MFS 74% vs. 54%. HRs were 0.51 (0.25-1.04) for progression, 0.43 (0.13-1.40) for locoregional failure, and 0.43 (0.17-1.05) for distant metastasis. Overall response was 81% vs. 69% (p = 0.262). Twenty-six and 27 patients, respectively, experienced at least one toxicity grade ≥3 (p = 0.573). A significant difference was found for grade ≥3 allergic reactions (12.5% vs. 0%, p = 0.044). CONCLUSION: Given the limitations of this trial, radiochemotherapy plus cetuximab was feasible. There was a trend towards improved PFS and MFS. Larger studies are required to better define the role of cetuximab for unresectable esophageal cancer.

Young-onset gastric cancer and Epstein-Barr Virus (EBV) - a major player in the pathogenesis?

OBJECTIVE: Gastric cancer (GC) is a leading cause of cancer death, occurs predominantly in older age, with increasing incidence in young patients. The Cancer Genome Atlas indicates four subtypes for GC among which Epstein-Barr virus (EBV) subtype is estimated at 8.7%. We aim to determine the prevalence of EBV subtype in young GC patients (≤45 years) compared with an average-onset cohort (≥55 years) and characterize the clinicopathologic pattern of young-onset GC. METHODS: Gastric cancer samples of patients of both cohorts were screened for EBV by qPCR. Additional staining was done for Human epidermal growth factor receptor 2 (HER2), microsatellite instability (MSI) status and Programmed death-ligand 1 (PD-L1). Demographics and clinical data were retrieved from the medical records. RESULTS: Thirty-nine young-onset and 35 average-onset GC patients were reviewed. There was no apparent difference in tumor location, family history, histology and HER2 status between the cohorts. More young-onset patients were diagnosed with metastatic disease (27% vs 9%, p = 0.0498). EBV was significantly more prevalent in the young-onset cohort (33% vs 11%, p = 0.025). 15/17 EBV positive patients were under the median age of diagnosis for GC in the US (68 years). MSI-H was found only in the average-onset cohort [0% vs 27%, p = 0.001). PD-L1 positivity was higher in the young-onset cohort (31% vs 3%, p = 0.002). CONCLUSION: Our study indicates that EBV subtype is more prevalent in young-onset GC and may play a key role in the pathogenesis. Higher rate of PD-L1 positivity in young-onset GC could change treatment strategies. We are currently evaluating these findings in a prospective trial.

Extended Molecular Profiling Improves Stratification and Prediction of Survival After Resection of Colorectal Liver Metastases.

OBJECTIVE: The aim of this study was to assess the effect of cancer-related genes and their mutations analyzed by next-generation sequencing (NGS) on the oncological outcome after resection of colorectal liver metastases (CRLM). BACKGROUND: Traditional prognostic scores include clinical and pathological parameters of primary tumor and metastases. The modified clinical risk score (m-CS), based on size of metastases, primary tumor nodal status, and RAS mutation status outperformed traditional scores. We hypothesized to further improve the scoring system based on the results of NGS. METHODS: Cancer tissues of 139 patients with CRLM were used for NGS. The work-up included the analysis of recurrent somatic mutations and copy number changes of 720 genes. Clinical data were extracted from a prospectively collected institutional liver database. RESULTS: Depending on significance, the following cancer-related genes and their alterations (%) were further investigated: APC (86%), TP53 (78%), KRAS (29%), SMAD4 (15%), PIK3CA (14%), BRAF (8%), ERBB2 (6%), SMAD3 (5%), SMAD2 (4%), and NRAS (4%). The most predictive parameters for poor oncological outcome were alterations in the SMAD family (P = 0.0186) and RAS-RAF pathway (P = 0.032). Refining the m-CS by replacing RAS with RAS-RAF pathway and adding SMAD family resulted in an extended clinical risk score which is highly predictive for oncological outcome (P < 0.0001). CONCLUSION: In conclusion, mutations of the SMAD family revealed a strong prognostic effect after surgery for CRLM. Integration of alterations of the SMAD family as well as the RAS/RAF pathway resulted in a new, still simple but highly prognostic score.

Loss of LLGL1 Expression Correlates with Diffuse Gastric Cancer and Distant Peritoneal Metastases.

Background: Loss of LLGL1 has been associated with loss of cellular adhesion and dissemination of cells from colorectal cancer and malignant melanoma. Regulation and relevance of LLGL1 were analyzed in gastric cancer patients with lymphatic and distant dissemination. Furthermore, LLGL1 expression was analyzed in relation to the cellular adhesion protein E-cadherin. Methods: LLGL1 and E-cadherin transcription levels were evaluated in 56 gastric cancer patients and five gastric cancer cell lines. IHC staining for LLGL1 was performed on 39 gastric cancer specimens. LLGL1 was stably transfected into LLGL1 negative gastric cancer cell line SNU16 (del(17) (p11.2)) for functional in vitro assays and a xenograft bioassay. Results: Gastric cancer specimens and cell lines displayed LLGL1 and E-cadherin expression levels with variable intensity. In gastric mucosa, LLGL1 exhibited weak cytoplasmic and strong cortical staining. Loss of LLGL1 expression occurred in 65% of gastric cancers and significantly correlated with loss of E-cadherin expression (P=0.00009). Loss of LLGL1 expression was associated with the diffuse type of gastric cancer (P=0.029) with peritoneal carcinomatosis (M1; P=0.006) and with female gender (P=0.017). Stable reexpression of LLGL1 in SNU16 cells significantly increased both plastic surface adhesion and extracellular matrix proteins laminin and fibronectin, but had no impact on in vitro proliferation, apoptosis, or invasion or on in vivo proliferation or differentiation in our xenograft bioassay. Conclusion: LLGL1 is coexpressed with E-cadherin. Loss of expression of either protein is associated with diffuse gastric cancer and peritoneal metastases. LLGL1 does not impact on proliferation or epithelial-mesenchymal transition (EMT) rather increasing cellular adhesion.

Safety and efficacy of afatinib as add-on to standard therapy of gemcitabine/cisplatin in chemotherapy-naive patients with advanced biliary tract cancer: an open-label, phase I trial with an extensive biomarker program.

BACKGROUND: To date, the cornerstone of treatment in patients with advanced or metastatic cholangiocarcinoma (CCA) is systemic chemotherapy based on a combination of gemcitabine and a platinum derivative. Other therapeutic approaches including targeted agents and tyrosine kinase inhibitors (TKI) have demonstrated disappointing results, highlighting the complexity of CCA. Recently, drugs aiming at the inhibition of HER-receptors have shown first therapeutic benefit in patients with late stage disease. The aim of this phase I study was to test the dose level toxicities (DLTs), safety and efficacy of afatinib, a highly specific panErbB family receptor TKI, in chemotherapy naive patients with advanced CCA in conjunction with an extensive biomarker program. METHODS: Afatinib was administered continuously p. o. as add-on in patients with advanced CCA who received conventional chemotherapy with gemcitabine/cisplatin. A classical 3 + 3 phase I study was employed, while the maximum tolerated dose (MTD) of oral afatinib was determined in a 2 step dose escalation. Safety, overall survival (OS) and progression free survival (PFS) were evaluated for all patients. Finally, a translational biomarker analysis was conducted for the EGFR and VEGF signalling cascades. RESULTS: Overall, 9 patients were enrolled. Further recruitment was discontinued due to lack of efficacy results of the tested drug in other indications. 30 mg afatinib could be safely administered as add-on to 80% of standard dose gemcitabine/cisplatin. The mOS and mPFS were 7.7 and 6.0 months, respectively. Diarrhoea and haematological disorders were the most common observed AEs. Almost all patients overexpressed EGFR on their tumour tissues, whereas none of them expressed mutations in Exons 18, 19 and 21. Non-responders showed a higher variation of VEGF-C, -D, leptin and sEGFR in their sera. CONCLUSIONS: Afatinib failed to show survival benefits in combination with gemcitabine/cisplatin in patients with advanced CCA. Mutational analysis of EGFR and pathways associated with VEGF-C, -D and leptin might show promising results in future studies. CLINICAL TRIALS REGISTRATION: NCT01679405 August, 2012.

Lapatinib with ECF/X in the first-line treatment of metastatic gastric cancer according to HER2neu and EGFR status: a randomized placebo-controlled phase II study (EORTC 40071).

PURPOSE: HER2-targeted therapy with trastuzumab and (CF/X) prolonged overall survival (OS) in metastatic HER2neu+ gastric carcinoma (GC). Lapatinib inhibits both EGFR and HER2neu. We investigated the efficacy and safety of lapatinib with epirubicin (E) + CF/X in GC according to HER2neu and EGFR status. METHODS: Tumors from chemotherapy-naïve patients were screened centrally by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Patients with EGFR and/or HER2neu expression or amplification were allocated to three strata based on EGFR/HER2neu status and were randomized to lapatinib (arm A) or placebo (arm B), with 6 cycles of ECF or ECX (investigator-selected). The primary endpoint was progression-free survival (PFS) in stratum 3. RESULTS: 29 of 72 screened patients were randomized to strata 1 (HER2neu+: by FISH and IHC, n = 6), 2 (HER2neu-: by FISH/+ by IHC, n = 5) and 3 (HER2neu-/EGFR+, n = 18), of which 28 patients were eligible (14 per arm). Enrollment was curtailed after announcement of the negative LOGiC trial results. Median PFS was 8.0 versus 5.9 months (HR = 0.86, 95% CI 0.37-1.99) in the per protocol population, and 8.0 versus 6.3 months (HR = 0.85, 95% CI 0.30-2.46) for stratum 3, in the lapatinib versus placebo arm respectively. Median OS was 13.8 versus 10.1 months, respectively (HR = 0.90, 95% CI 0.35-2.27). There were no safety concerns. CONCLUSIONS: Central EGFR and HER2neu stratification by IHC and FISH can be used for further pan-HER strategies. Lapatinib with ECF/X was well tolerated, but did not show clear activity in patients with metastatic GC.

Cytokeratin-18 fragments predict treatment response and overall survival in gastric cancer in a randomized controlled trial.

BACKGROUND: Gastric cancer is common malignancy and exhibits a poor prognosis. At the time of diagnosis, the majority of patients present with metastatic disease which precludes curative treatment. Non-invasive biomarkers which discriminate early from advanced stages or predict the response to treatment are urgently required. This study explored the cytokeratin-18 fragment M30 and full-length cytokeratin-18 M65 in predicting treatment response and survival in a randomized, placebo-controlled trial of advanced gastric cancer. METHODS: Patients enrolled in the SUN-CASE study received sunitinib or placebo as an adjunct to standard therapy with leucovorin (Ca-folinate), 5-fluorouracil, and irinotecan in second or third line. Treatment response rates, progression-free survival and overall survival were assessed during a follow-up period of 12 months. Cytokeratin-18 fragments were analyzed in 52 patients at baseline and day 14 of therapy. RESULTS: Levels of M30 correlated with the presence of metastasis and lymph node involvement and decreased significantly during chemotherapy. Importantly, baseline levels of M30 were significantly higher in patients who failed therapy. In addition, patients who did not respond to treatment were also identifiable at day 14 based on elevated M30 levels. By stepwise regression analysis, M30 at day 14 was identified as independent predictor of treatment response. Likewise, serum levels of full-length cytokeratin-18 M65 at baseline also correlated with treatment failure and progression-free survival. The addition of sunitinib did not exert any effects on serum levels of M30 or M65. CONCLUSION: The cytokeratin-18 fragment M30 at day 14 identifies patients that fail to second- or third-line therapy for advanced gastric cancer. Validation of this non-invasive biomarker in gastric cancer is warranted.

Perioperative chemotherapy with or without epidermal growth factor receptor blockade in unselected patients with locally advanced oesophagogastric adenocarcinoma: Randomized phase II study with advanced biomarker program of the German Cancer Society (AIO/CAO STO-0801).

BACKGROUND: Perioperative chemotherapy significantly improves survival in patients with locally advanced oesophagogastric cancer (EGC). However, as approximately 60% of patients will die from their disease, new therapeutic agents such as molecular-targeted drugs are needed. PATIENTS AND METHODS: To evaluate the role of panitumumab with perioperative chemotherapy, previously untreated patients with locally advanced EGC received, in an open-label randomised phase II study (NEOPECX), standard epirubicin, cisplatin, capecitabine (ECX) chemotherapy with or without panitumumab. The primary end-point was the histological response rate after neoadjuvant therapy. The expression status and gene copy number of EGFR, HER2, and MET were determined by immunohistochemistry and fluorescence in situ hybridization (FISH). Plasma samples were collected before the first cycle of neoadjuvant chemotherapy. RESULTS: Overall, 160 patients (80 versus 80) were eligible. The majority (82% versus 80%) showed lymph node involvement. Rate of R0-resection, percentage of patients with downstaging to ypT0-2 at pathohistological evaluation, and rate of major histological response was equal in both arms. Toxicity was increased by panitumumab with regard to thromboembolic events and skin toxicity. Patients with tumour EGFR, HER2 or MET expression had shorter progression-free and overall survival. FISH positivity for these markers was associated with shorter survival independent of therapy. High levels of soluble EGFR in particular predicted poor survival in the panitumumab arm. CONCLUSION: The addition of panitumumab to ECX did not improve downstaging of locally advanced EGC. Low plasma levels of pathway-associated proteins such as sEGFR may identify a group of patients that benefit from EGFR-directed therapy. CLINICALTRIALS.GOV: NCT01234324.

Phase I Study of Definitive Radio-chemotherapy with Cisplatin, 5-Fluorouracil and Cetuximab for Unresectable Locally Advanced Esophageal Cancer.

BACKGROUND/AIM: Prognoses of patients receiving radio-chemotherapy with 5-fluorouracil (5-FU) and cisplatin for unresectable esophageal cancer may be improved with the addition of cetuximab. This phase I study aimed to define the maximum tolerated dose of 5-FU when combined with cisplatin, cetuximab and radiotherapy. PATIENTS AND METHODS: Treatment included 59.4 Gy of radiotherapy concurrently with two courses of cisplatin (20 mg/m2, d1-4) and 5-FU (dose level 0: 500 mg/m2, dose level 1: 750 mg/m2, d1-4; dose level 2: 1,000 mg/m2, d1-4), followed by two courses of chemotherapy. Cetuximab was given for 14 weeks (400 mg/m2 loading dose followed by 250 mg/m2 weekly). RESULTS: At dose level 1 (n=3) and 2 (n=3), no patient experienced a dose-limiting toxicity. Minor treatment modifications were due to organization or request by physicians/patients. At dose level 2, only five grade 3 adverse events occurred. CONCLUSION: Dose level 2 appears safe and is used in a subsequent randomized phase II study.

Sunitinib added to FOLFIRI versus FOLFIRI in patients with chemorefractory advanced adenocarcinoma of the stomach or lower esophagus: a randomized, placebo-controlled phase II AIO trial with serum biomarker program.

BACKGROUND: As a multi-targeted anti-angiogenic receptor tyrosine kinase (RTK) inhibitor sunitinib (SUN) has been established for renal cancer and gastrointestinal stromal tumors. In advanced refractory esophagogastric cancer patients, monotherapy with SUN was associated with good tolerability but limited tumor response. METHODS: This double-blind, placebo-controlled, multicenter, phase II clinical trial was conducted to evaluate the efficacy, safety and tolerability of SUN as an adjunct to second and third-line FOLFIRI (NCT01020630). Patients were randomized to receive 6-week cycles including FOLFIRI plus sodium folinate (Na-FOLFIRI) once every two weeks and SUN or placebo (PL) continuously for four weeks followed by a 2-week rest period. The primary study endpoint was progression-free survival (PFS). Preplanned serum analyses of VEGF-A, VEGF-D, VEGFR2 and SDF-1α were performed retrospectively. RESULTS: Overall, 91 patients were randomized, 45 in each group (one patient withdrew). The main grade ≥3 AEs were neutropenia and leucopenia, observed in 56 %/20 % and 27 %/16 % for FOLFIRI + SUN/FOLFIRI + PL, respectively. Median PFS was similar, 3.5 vs. 3.3 months (hazard ratio (HR) 1.11, 95 % CI 0.70-1.74, P = 0.66) for FOLFIRI + SUN vs. FOLFIRI + PL, respectively. For FOLFIRI + SUN, a trend towards longer median overall survival (OS) compared with placebo was observed (10.4 vs. 8.9 months, HR 0.82, 95 % CI 0.50-1.34, one-sided P = 0.21). In subgroup serum analyses, significant changes in VEGF-A (P = 0.017), VEGFR2 (P = 0.012) and VEGF-D (P < 0.001) serum levels were observed. CONCLUSIONS: Although sunitinib combined with FOLFIRI did not improve PFS and response in chemotherapy-resistant gastric cancer, a trend towards better OS was observed. Further biomarker-driven studies with other anti-angiogenic RTK inhibitors are warranted. TRIAL REGISTRATION: This study was registered prospectively in the NCT Clinical Trials Registry (ClinicalTrials.gov) under NCT01020630 on November 23, 2009 after approval by the leading ethics committee of the Medical Association of Rhineland-Palatinate, Mainz, in coordination with the participating ethics committees (see Additional file 2) on September 16, 2009.

Inclusion of targeted therapies in the standard of care for metastatic colorectal cancer patients in a German cancer center: the more the better?!

PURPOSE: Significant prolongation of overall survival (OS) has been reached in metastatic colorectal cancer (mCRC) treatment within the last 5-10 years. Our study was conducted in order to evaluate and compare OS of different standard of care treatment options in a university-based outpatient clinic. METHODS: One hundred and three mCRC patients were identified by retrospective analysis and treated according to available guidelines. OS was analyzed according to the different combinations of first- and second-line treatments. RESULTS: mCRC patients revealed an mOS of 34.4 months. Patients receiving anti-vascular endothelial growth factor (VEGF) blockade in at least one treatment line showed a significantly longer survival time (p = 0.0056) versus patients without any bevacizumab. No OS differences were detected comparing the different first- and second-line chemotherapy (CTX) strategies in the unselected population. However, wild-type (wt) Kras patients treated with anti-epidermal growth factor receptor (EGFR) therapy plus CTX in first-line therapy showed significantly longer OS compared to those receiving only additional VEGF inhibition or no targeted therapy (p = 0.0056; mOS 46.8 vs. 20.4 months, respectively). wt Kras patients profited in trend (p = 0.076) from CTX combinations of first-line anti-EGFR followed by second-line anti-VEGF compared to first-line anti-VEGF followed by second-line anti-EGFR (mOS 46.8 vs. 19.2 months, respectively). CONCLUSIONS: Our results indicate successful allocation of the current mCRC treatment according to the Kras status. Differences in OS of wt Kras patients indicated the further need for randomized trials to define the potential benefit of sequential therapy with EGFR inhibition in first-line therapy followed by VEGFR inhibition vice versa.

Proteins of the VEGFR and EGFR pathway as predictive markers for adjuvant treatment in patients with stage II/III colorectal cancer: results of the FOGT-4 trial.

BACKGROUND: Unlike metastatic colorectal cancer (CRC) there are to date few reports concerning the predictive value of molecular biomarkers on the clinical outcome in stage II/III CRC patients receiving adjuvant chemotherapy. Aim of this study was to assess the predictive value of proteins related with the EGFR- and VEGFR- signalling cascades in these patients. METHODS: The patients' data examined in this study were from the collective of the 5-FU/FA versus 5-FU/FA/irinotecan phase III FOGT-4 trial. Tumor tissues were stained by immunohistochemistry for VEGF-C, VEGF-D, VEGFR-3, Hif-1 α, PTEN, AREG and EREG expression and evaluated by two independent, blinded investigators. RESULTS: Patients with negative AREG and EREG expression on their tumor had a significant longer DFS in comparison to AREG/EREG positive ones (p< 0.05). The benefit on DFS in AREG-/EREG- patients was even stronger in the group that received 5-FU/FA/irinotecan as adjuvant treatment (p=0.002). Patients with strong expression of PTEN profited more in terms of OS under adjuvant treatment containing irinotecan (p< 0.05). Regarding markers of the VEGFR- pathway we found no correlation of VEGF-C- and VEGFR-3 expression with clinical outcome. Patients with negative VEGF-D expression had a trend to live longer when treated with 5-FU/FA (p=0.106). Patients who were negative for Hif-1 α, were disease-free in more than 50% at the end of the study and showed significant longer DFS-rates than those positive for Hif-1 α (p=0.007). This benefit was even stronger at the group treated with 5-FU/FA/irinotecan (p=0.026). Finally, AREG-/EREG-/PTEN+ patients showed a trend to live longer under combined treatment combination. CONCLUSIONS: The addition of irinotecan to adjuvant treatment with 5-FU/FA does not provide OS or DFS benefit in patients with stage II/III CRC. Nevertheless, AREG/EREG negative, PTEN positive and Hif-1 α negative patients might profit significantly in terms of DFS from a treatment containing fluoropyrimidines and irinotecan. Our results suggest a predictive value of these biomarkers concerning adjuvant chemotherapy with 5-FU/FA +/- irinotecan in stage II/III colorectal cancer.

VEGFR-3 and CXCR4 as predictive markers for treatment with fluorouracil, leucovorin plus either oxaliplatin or cisplatin in patients with advanced esophagogastric cancer: a comparative study of the Arbeitsgemeinschaft Internistische Onkologie (AIO).

BACKGROUND: Combination of fluoropyrimidines and a platinum derivative are currently standards for systemic chemotherapy in advanced adenocarcinoma of the stomach and gastroesophageal junction (GEJ). Nevertheless, individual likelihood for response to these therapeutic regimes remains uncertain. Even more, no predictive markers are available to determine which patients may benefit more from oxaliplatin versus cisplatin or vice versa. The new invasion and stem cell markers VEGFR-3 and CXCR4 have been linked prognostically with more aggressive esophagogastric cancer types. Thus, we aimed to assess correlations of VEGFR-3 and CXCR4 expression levels with clinical outcome in a randomized phase III study of patients with oxaliplatin/leucovorin/5-FU (FLO) versus cisplatin/leucovorin/5-FU (FLP). METHODS: The patients data examined in this study (n = 72) were from the collective of the FLO vs. FLP phase III AIO trial. Tumour tissues were stained via immunohistochemistry for VEGFR-3 and CXCR4 expression and results were evaluated by two independent, blinded investigators.Outcome parameter: Survival analysis was calculated for patients receiving FLO vs. FLP in relation to VEGFR-3 and CXCR4 expression. RESULTS: 54% and 36% of the examined tumour tissues showed strong positive expression of VEGFR-3 and CXCR4 respectively. No superiority of each regime was detected in terms of overall survival (OS) in the whole population. Patients with strong expression of CXCR4 on their tumour tissues profited more in terms of OS under the treatment of FLP (mOS: 28 vs 15 months, p = 0.05 respectively). Patients with negative VEGFR-3 and CXCR4 expression had a trend to live longer when FLO regime was applied (mOS: 22 vs. 9 months, p = 0.099 and 20 vs. 10 months, p = 0.073 respectively). In an exploratory analysis of patients older than 60 years at diagnosis, we observed a significant benefit in overall survival for VEGFR-3 and CXCR4-positive patients when treated with FLP (p = 0.002, p = 0.021 respectively). CONCLUSIONS: CXCR4 positive patients profited in terms of OS from FLP, whereas FLO proved to be more effective in CXCR4 and VEGFR-3 negative patients. Our results suggest, despite the limited size of the study, a predictive value of these biomarkers concerning chemotherapy with FLP or FLO in advanced esophagogastric cancer.

Moguntinones--new selective inhibitors for the treatment of human colorectal cancer.

3-Indolyl and 3-azaindolyl-4-aryl maleimide derivatives, called moguntinones (MOG), have been selected for their ability to inhibit protein kinases associated with angiogenesis and induce apoptosis. Here, we characterize their mode of action and their potential clinical value in human colorectal cancer in vitro and in vivo. MOG-19 and MOG-13 were characterized in vitro using kinase, viability, and apoptosis assays in different human colon cancer (HT-29, HCT-116, Caco-2, and SW480) and normal colon cell lines (CCD-18Co, FHC, and HCoEpiC) alone or in combination with topoisomerase I inhibitors. Intracellular signaling pathways were analyzed by Western blotting. To determine their potential to inhibit tumor growth in vivo, the human HT-29 tumor xenograft model was used. Moguntinones prominently inhibit several protein kinases associated with tumor growth and metastasis. Specific signaling pathways such as GSK3ß and mTOR downstream targets were inhibited with IC(50) values in the nanomolar range. GSK3ß signaling inhibition was independent of KRAS, BRAF, and PI3KCA mutation status. While moguntinones alone induced apoptosis only in concentrations >10 µmol/L, MOG-19 in combination with topoisomerase I inhibitors induced apoptosis synergistically at lower concentrations. Consistent with in vitro data, MOG-19 significantly reduced tumor volume and weight in combination with a topoisomerase I inhibitor in vivo. Our in vitro and in vivo data present significant proapoptotic, antiangiogenic, and antiproliferative effects of MOG-19 in different human colon cancer cells. Combination with clinically relevant topoisomerase I inhibitors in vitro and xenograft mouse model demonstrate a high potency of moguntinones to complement and improve standard chemotherapy options in human colorectal cancer.

Single-agent therapy with sorafenib or 5-FU is equally effective in human colorectal cancer xenograft--no benefit of combination therapy.

BACKGROUND: We initiated this preclinical study in order to analyze the impact of sorafenib single treatment versus combination treatment in human colorectal cancer. METHODS: The effect of increasing sorafenib doses on proliferation, apoptosis, migration, and activation of signal cascades was analyzed in vitro. The effect of sorafenib single treatment versus 5-fluorouracil (5-FU) single treatment and combination therapy on in vivo proliferation and target cytokine receptor/ligand expression was analyzed in a human colon cancer xenograft mouse model using HT29 tumor cells. RESULTS: In vitro, SW480 and HT29 cell lines were sensitive to sorafenib, as compared to Caco2 and SW620 cell lines, independent of the mutation status of K-ras, Raf, PTEN, or PI3K. The effect on migration was marginal, but distinct differences in caspases activation were seen. Combination strategies were beneficial in some settings (sorafenib + 5-FU; irinotecan) and disadvantageous in others (sorafenib + oxaliplatin), depending on the chemotherapeutic drug and cell line chosen. Sensitive cell lines revealed a downregulation of AKT and had a weak expression level of GADD45ß. In resistant cell lines, pp53 and GADD45ß levels decreased upon sorafenib exposure. In vivo, the combination treatment of sorafenib and 5-FU was equally effective as the respective monotherapy concerning tumor proliferation. Interestingly, treatment with either sorafenib or 5-FU resulted in a significant decrease of VEGFR1 and PDGFRß expression intensity. CONCLUSIONS: In colorectal cancer, a sensitivity towards sorafenib exists, which seems similarly effective as a 5-FU monotherapy. A combination therapy, in contrast, does not show any additional effect.