EURECCA colorectal: multidisciplinary mission statement on better care for patients with colon and rectal cancer in Europe

Background: Care for patients with colon and rectal cancer has improved in the last twenty years however still considerable variation exists in cancer management and outcome between European countries. Therefore, EURECCA, which is the acronym of European Registration of cancer care, is aiming at defining core treatment strategies and developing a European audit structure in order to improve the quality of care for all patients with colon and rectal cancer. In December 2012 the first multidisciplinary consensus conference about colon and rectum was held looking for multidisciplinary consensus. The expert panel consisted of representatives of European scientific organisations involved in cancer care of patients with colon and rectal cancer and representatives of national colorectal registries. Methods: The expert panel had delegates of the European Society of Surgical Oncology (ESSO), European Society for Radiotherapy & Oncology (ESTRO), European Society of Pathology (ESP), European Society for Medical Oncology (ESMO), European Society of Radiology (ESR), European Society of Coloproctology (ESCP), European CanCer Organisation (ECCO), European Oncology Nursing Society (EONS) and the European Colorectal Cancer Patient Organisation (EuropaColon), as well as delegates from national registries or audits. Experts commented and voted on the two web-based online voting rounds before the meeting (between 4th and 25th October and between the 20th November and 3rd December 2012) as well as one online round after the meeting (4th20th March 2013) and were invited to lecture on the subjects during the meeting (13th15th December 2012). The sentences in the consensus document were available during the meeting and a televoting round during the conference by all participants was performed. All sentences that were voted on are available on the EURECCA website www.canceraudit.eu. The consensus document was divided in sections describing evidence based algorithms of diagnostics, pathology, surgery, medical oncology, radiotherapy, and follow-up where applicable for treatment of colon cancer, rectal cancer and stage IV separately. Consensus was achieved using the Delphi method. Results: The total number of the voted sentences was 465. All chapters were voted on by at least 75% of the experts. Of the 465 sentences, 84% achieved large consensus, 6% achieved moderate consensus, and 7% resulted in minimum consensus. Only 3% was disagreed by more than 50% of the members. Conclusions: It is feasible to achieve European Consensus on key diagnostic and treatment issues using the Delphi method. This consensus embodies the expertise of professionals from all disciplines involved in the care for patients with colon and rectal cancer. Diagnostic and treatment algorithms were developed to implement the current evidence and to define core treatment guidance for multidisciplinary team management of colon and rectal cancer throughout Europe.

Inhibition of tumor angiogenesis by non-steroidal anti-inflammatory drugs: emerging mechanisms and therapeutic perspectives.

Chronic intake of non steroidal anti-inflammatory drugs (NSAIDs) is associated with a reduced risk of developing gastrointestinal tumors, in particular colon cancer. Increasing evidence indicates that NSAID exert tumor-suppressive activity on pre-malignant lesions (polyps) in humans and on established experimental tumors in mice. Some of the tumor-suppressive effects of NSAIDs depend on the inhibition of cyclooxygenase-2 (COX-2), a key enzyme in the synthesis of prostaglandins and thromboxane, which is highly expressed in inflammation and cancer. Recent findings indicate that NSAIDs exert their anti-tumor effects by suppressing tumor angiogenesis. The availability of COX-2-specific NSAIDs opens the possibility of using this drug class as anti-angiogenic agents in combination with chemotheapy or radiotherapy for the treatment of human cancer. Here we will briefly review recent advances in the understanding of the mechanism by which NSAIDs suppress tumor angiogenesis and discuss their potential clinical application as anti-cancer agents.

Tumor targeting with newly designed biparatopic antibodies directed against two different epitopes of the carcinoembryonic antigen (CEA).

In an attempt to improve tumor targeting and tumor retention time of monoclonal antibodies (MAbs), we prepared biparatopic antibodies (BpAbs) having the capability of binding 2 different non-overlapping epitopes on the same target antigen molecule, namely, the carcinoembryonic antigen (CEA). Six BpAbs were constructed by coupling 2 different Fab' fragments from 4 different specific anti-CEA MAbs recognizing 4 CEA epitopes (Gold 1-4). Demonstration of the double paratopic binding of these antibodies for CEA was confirmed in vitro by inhibition radioimmunoassay and cross-inhibition analysis by surface plasmon resonance (SPR; BIACORE) technology. Using the latter technique, the affinity constants for CEA immobilized onto the sensor chip were found to range from 0.37 to 1.54 x 10(9) M(-1) for the 4 parental F(ab')2 fragments and from 1.88 to 10.14 x 10(9) M(-1) for the BpAbs, demonstrating the advantage of biparatopic binding over conventional F(ab')2 binding. The Ka improvement was particularly high for BpAb F6/35A7 and BpAb F6/B17 with a 9.5- and 8.1-fold increase, respectively, as compared with the parental F(ab')2. In vivo, the 6 BpAbs were compared with their 2 respective parental F(ab')2 by injection of 131I-BpAb/125I-F(ab')2 parental fragments into nude mice xenografted with the human colon carcinoma T380. Dissection 72 hr post-injection demonstrated that BpAb B17/CE25 and BpAb F6/B17 gave higher tumor uptake than that of their parental F(ab')2. This finding is particularly interesting for BpAb F6/B17, which compared favorably with the F6 F(ab')2, one of the best parental F(ab')2 fragments used in our study.

CD1d-antibody fusion proteins target iNKT cells to the tumor and trigger long-term therapeutic responses.

Despite the well-established antitumor activity of CD1d-restricted invariant natural killer T lymphocytes (iNKT), their use for cancer therapy has remained challenging. This appears to be due to their strong but short-lived activation followed by long-term anergy after a single administration of the CD1d agonist ligand alpha-galactosylceramide (αGC). As a promising alternative, we obtained sustained mouse iNKT cell responses associated with prolonged antitumor effects through repeated administrations of tumor-targeted recombinant sCD1d-antitumor scFv fusion proteins loaded with αGC. Here, we demonstrate that CD1d fusion proteins bound to tumor cells via the antibody fragment specific for a tumor-associated antigen, efficiently activate human iNKT cell lines leading to potent tumor cell lysis. The importance of CD1d tumor targeting was confirmed in tumor-bearing mice in which only the specific tumor-targeted CD1d fusion protein resulted in tumor inhibition of well-established aggressive tumor grafts. The therapeutic efficacy correlated with the repeated activation of iNKT and natural killer cells marked by their release of TH1 cytokines, despite the up-regulation of the co-inhibitory receptor PD-1. Our results demonstrate the superiority of providing the superagonist αGC loaded on recombinant CD1d proteins and support the use of αGC/sCD1d-antitumor fusion proteins to secure a sustained human and mouse iNKT cell activation, while targeting their cytotoxic activity and cytokine release to the tumor site.

Endothelial cells as key determinants of the tumor microenvironment: interaction with tumor cells, extracellular matrix and immune killer cells.

Besides tumor cells, the tumor microenvironment harbors a variety of host-derived cells, such as endothelial cells, fibroblasts, innate and adaptive immune cells. It is a complex and highly dynamic environment, providing very important cues to tumor development and progression. Tumor-associated endothelial cells play a key role in this process. On the one hand, they form tumor-associated (angiogenic) vessels through sprouting from locally preexisting vessels or recruitment of bone marrow-derived endothelial progenitor cells, to provide nutritional support to the growing tumor. On the other hand, they are the interface between circulating blood cells, tumor cells and the extracellular matrix, thereby playing a central role in controlling leukocyte recruitment, tumor cell behavior and metastasis formation. Hypoxia is a critical parameter modulating the tumor microenvironment and endothelial/tumor cell interactions. Under hypoxic stress, tumor cells produce factors that promote tumor angiogenesis, tumor cell motility and metastasis. Among these factors, VEGF, a main angiogenesis modulator, can also play a critical role in the control of immune tolerance. This review discusses some aspects of the role of endothelial cells within tumor microenvironment and emphasizes their interaction with tumor cells, the extracellular matrix and with immune killer cells. We will also address the role played by circulating endothelial progenitor cells and illustrate their features and mechanism of recruitment to the tumor microenvironment and their role in tumor angiogenesis.

Development of a Liver-specific Tet-On Inducible System for AAV Vectors and Its Application in the Treatment of Liver Cancer.

Recombinant adeno-associated virus (rAAV) are effective gene delivery vehicles that can mediate long-lasting transgene expression. However, tight regulation and tissue-specific transgene expression is required for certain therapeutic applications. For regulatable expression from the liver we designed a hepatospecific bidirectional and autoregulatory tetracycline (Tet)-On system (Tet(bidir)Alb) flanked by AAV inverted terminal repeats (ITRs). We characterized the inducible hepatospecific system in comparison with an inducible ubiquitous expression system (Tet(bidir)CMV) using luciferase (luc). Although the ubiquitous system led to luc expression throughout the mouse, luc expression derived from the hepatospecific system was restricted to the liver. Interestingly, the induction rate of the Tet(bidir)Alb was significantly higher than that of Tet(bidir)CMV, whereas leakage of Tet(bidir)Alb was significantly lower. To evaluate the therapeutic potential of this vector, an AAV-Tet(bidir)-Alb-expressing interleukin-12 (IL-12) was tested in a murine model for hepatic colorectal metastasis. The vector induced dose-dependent levels of IL-12 and interferon-γ (IFN-γ), showing no significant toxicity. AAV-Tet(bidir)-Alb-IL-12 was highly efficient in preventing establishment of metastasis in the liver and induced an efficient T-cell memory response to tumor cells. Thus, we have demonstrated persistent, and inducible in vivo expression of a gene from a liver-specific Tet-On inducible construct delivered via an AAV vector and proved to be an efficient tool for treating liver cancer.

Proteasome-assisted identification of a SSX-2-derived epitope recognized by tumor-reactive CTL infiltrating metastatic melanoma.

The tumor Ag SSX-2 (HOM-MEL-40) was found by serological identification of Ags by recombinant expression cloning and was shown to be a cancer/testis Ag expressed in a wide variety of tumors. It may therefore represent a source of CD8(+) T cell epitopes useful for specific immunotherapy of cancer. To identify potential SSX-2-derived epitopes that can be recognized by CD8(+) T cells, we used an approach that combined: 1) the in vitro proteasomal digestion of precursor peptides overlapping the complete SSX-2 sequence; 2) the prediction of SSX-2-derived peptides with an appropriate HLA-A2 binding score; and 3) the analysis of a tumor-infiltrated lymph node cell population from an HLA-A2(+) melanoma patient with detectable anti-SSX-2 serum Abs. This strategy allowed us to identify peptide SSX-2(41-49) as an HLA-A2-restricted epitope. SSX2(41-49)-specific CD8(+) T cells were readily detectable in the tumor-infiltrated lymph node population by multimer staining, and CTL clones isolated by multimer-guided cell sorting were able to lyse HLA-A2(+) tumor cells expressing SSX-2.

Carcinoembryonic antigen expression, antibody localisation and immunophotodetection of human colon cancer liver metastases in nude mice: a model for radioimmunotherapy.

Colorectal cancer frequently disseminates through the portal vein into the liver. In this study, outbred Swiss nude mice were adapted to facilitate the induction of liver metastases by a pre-grafting treatment with 6 Gy total body irradiation and i.v. injection of anti-asialo GM1 antibody. One day later, cultured LS 174T human colon cancer cells were injected into the surgically exposed spleen, which was resected 3 min later. In 48 of 65 mice, a few to several hundred liver metastases were macroscopically observed at dissection 3 to 4 weeks after transplantation. Ten of 10 mice, followed-up for survival, died with multiple large confluent liver metastases. By reducing the radiation dose to 4 or 0 Gy, or omitting the anti-asialo GM1 antibody injection, only 60%, 37% or 50% of mice, respectively, had visible metastases 3 weeks after transplantation. Carcinoembryonic antigen (CEA) measured in tumour extracts was in the mean 25.6 micrograms/g in liver metastases compared with 9.2 micrograms/g in s.c. tumours. Uptake of radiolabelled anti-CEA monoclonal antibody (MAb) in the metastases 12, 24 and 48 hr after injection gave a mean value of 39% of the injected dose per gram of tissue (ID/g). In comparison, MAb uptake in s.c. and intrasplenic tumours or lung metastases gave a mean percentage ID/g of 20, 18 and 15, respectively. Laser-induced fluorescence after injection of indocyanin-MAb conjugate allowed direct visual detection of small liver metastases, including some that were not visible under normal light. Preliminary results showed that mice, pre-treated with 4 Gy irradiation and the anti-asialo GM1 injection, were tolerant to radioimmunotherapy with a total dose of 500 muCi 131I labeled anti-CEA intact MAbs given in 3 injections.

The Oncogene Metadherin Modulates the Apoptotic Pathway Based on the Tumor Necrosis Factor Superfamily Member TRAIL (Tumor Necrosis Factor-related Apoptosis-inducing Ligand) in Breast Cancer.

Metadherin (MTDH), the newly discovered gene, is overexpressed in more than 40% of breast cancers. Recent studies have revealed that MTDH favors an oncogenic course and chemoresistance. With a number of breast cancer cell lines and breast tumor samples, we found that the relative expression of MTDH correlated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity in breast cancer. In this study, we found that knockdown of endogenous MTDH cells sensitized the MDA-MB-231 cells to TRAIL-induced apoptosis both in vitro and in vivo. Conversely, stable overexpression of MTDH in MCF-7 cells enhanced cell survival with TRAIL treatment. Mechanically, MTDH down-regulated caspase-8, decreased caspase-8 recruitment into the TRAIL death-inducing signaling complex, decreased caspase-3 and poly(ADP-ribose) polymerase-2 processing, increased Bcl-2 expression, and stimulated TRAIL-induced Akt phosphorylation, without altering death receptor status. In MDA-MB-231 breast cancer cells, sensitization to TRAIL upon MTDH down-regulation was inhibited by the caspase inhibitor Z-VAD-fmk (benzyloxycarbonyl-VAD-fluoromethyl ketone), suggesting that MTDH depletion stimulates activation of caspases. In MCF-7 breast cancer cells, resistance to TRAIL upon MTDH overexpression was abrogated by depletion of Bcl-2, suggesting that MTDH-induced Bcl-2 expression contributes to TRAIL resistance. We further confirmed that MTDH may control Bcl-2 expression partly by suppressing miR-16. Collectively, our results point to a protective function of MTDH against TRAIL-induced death, whereby it inhibits the intrinsic apoptosis pathway through miR-16-mediated Bcl-2 up-regulation and the extrinsic apoptosis pathway through caspase-8 down-regulation.

Circulating Tumor-reactive CD8(+) T cells in melanoma patients contain a CD45RA(+)CCR7(-) effector subset exerting ex vivo tumor-specific cytolytic activity.

To defend the host from malignancies, the immune system can spontaneously raise CD8(+) T-cell responses against tumor antigens. Investigating the functional state of tumor-reactive cytolytic T cells in cancer patients is a key step for understanding the role of these cells in tumor immunosurveillance and for evaluating the potential of immunotherapeutic approaches of vaccination against cancer. In this study we identified a subset of circulating tumor-reactive CD8(+) T lymphocytes, which specifically secreted IFN-gamma after exposition to autologous tumor cell lines in stage IV metastatic melanoma patients. Additional phenotypic characterization using multicolor flow cytometry revealed that a significant fraction of these cells were CD45RA(+)CCR7(-), a phenotype that has been proposed recently to characterize cytolytic effectors potentially able to home into inflamed tissues. In the case of an HLA-A2-expressing patient, the antigen specificity of this population was identified by using HLA-A2/peptide multimers incorporating a tyrosinase-derived peptide. Consistently with their phenotypic characteristics, A2/tyrosinase peptide multimer(+) CD8(+) T cells, isolated by cell sorting, were directly lytic ex vivo and able to specifically recognize tyrosinase-expressing tumor cells. Overall, these results provide the first evidence that a proportion of melanoma patients have circulating tumor-reactive T cells, which are lytic effectors cells.

Effect of RasGAP N2 fragment-derived peptide on tumor growth in mice.

Peptides that interfere with the natural resistance of cancer cells to genotoxin-induced apoptosis may improve the efficacy of anticancer regimens. We have previously reported that a cell-permeable RasGAP-derived peptide (TAT-RasGAP(317-326)) specifically sensitizes tumor cells to genotoxin-induced apoptosis in vitro. Here, we examined the in vivo stability of a protease-resistant D-form of the peptide, RI.TAT-RasGAP(317-326), and its effect on tumor growth in nude mice bearing subcutaneous human colon cancer HCT116 xenograft tumors. After intraperitoneal injection, RI.TAT-RasGAP(317-326) persisted in the blood of nude mice for more than 1 hour and was detectable in various tissues and subcutaneous tumors. Tumor-bearing mice treated daily for 7 days with RI.TAT-RasGAP(317-326) (1.65 mg/kg body weight) and cisplatin (0.5 mg/kg body weight) or doxorubicin (0.25 mg/kg body weight) displayed reduced tumor growth compared with those treated with either genotoxin alone (n = 5-7 mice per group; P = .004 and P = .005, respectively; repeated measures analysis of variance [ANOVA, two-sided]). This ability of the RI.TAT-RasGAP(317-326) peptide to enhance the tumor growth inhibitory effect of cisplatin was still observed at peptide doses that were at least 150-fold lower than the dose lethal to 50% of mice. These findings provide the proof of principle that RI.TAT-RasGAP(317-326) may be useful for improving the efficacy of chemotherapy in patients.

Retroviral infection of neonatal Peyer's patch lymphocytes: the mouse mammary tumor virus model.

Mouse mammary tumor virus is known to infect newborn mice via mother's milk. A proposed key step for viral spread to the mammary gland is by the infection of lymphocytes. We show here that although in suckling mice retroviral proteins are found in all epithelial cells of the gut, viral DNA is exclusively detectable in the Peyer's patches. As early as 5 d after birth the infection leads to a superantigen response in the Peyer's patches but not in other lymphoid organs draining the intestine. Viral DNA can be detected before the superantigen response and becomes first evident in the Peyer's patches followed by mesenteric lymph nodes and finally all lymphoid organs.

Allorestricted T lymphocytes with a high avidity T-cell receptor towards NY-ESO-1 have potent anti-tumor activity.

The cancer-testis antigen NY-ESO-1 has been targeted as a tumor-associated antigen by immunotherapeutical strategies, such as cancer vaccines. The prerequisite for a T-cell-based therapy is the induction of T cells capable of recognizing the NY-ESO-1-expressing tumor cells. In this study, we generated human T lymphocytes directed against the immunodominant NY-ESO-1(157-165) epitope known to be naturally presented with HLA-A*0201. We succeeded to isolate autorestricted and allorestricted T lymphocytes with low, intermediate or high avidity TCRs against the NY-ESO-1 peptide. The avidity of the established CTL populations correlated with their capacity of lysing HLA-A2-positive, NY-ESO-1-expressing tumor cell lines derived from different origins, e.g. melanoma and myeloma. The allorestricted NY-ESO-1-specific T lymphocytes displayed TCRs with the highest avidity and best anti-tumor recognition activity. TCRs derived from allorestricted, NY-ESO-1-specific T cells may be useful reagents for redirecting primary T cells by TCR gene transfer and, therefore, may facilitate the development of adoptive transfer regimens based on TCR-transduced T cells for the treatment of NY-ESO-1-expressing hematological malignancies and solid tumors.

Early prediction of response to sunitinib after imatinib failure by 18F-fluorodeoxyglucose positron emission tomography in patients with gastrointestinal stromal tumor.

PURPOSE: Positron emission tomography with (18)F-fluorodeoxyglucose (FDG-PET) was used to evaluate treatment response in patients with gastrointestinal stromal tumors (GIST) after administration of sunitinib, a multitargeted tyrosine kinase inhibitor, after imatinib failure. PATIENTS AND METHODS: Tumor metabolism was assessed with FDG-PET before and after the first 4 weeks of sunitinib therapy in 23 patients who received one to 12 cycles of sunitinib therapy (4 weeks of 50 mg/d, 2 weeks off). Treatment response was expressed as the percent change in maximal standardized uptake values (SUV). The primary end point of time to tumor progression was compared with early PET results on the basis of traditional Response Evaluation Criteria in Solid Tumors (RECIST) criteria. RESULTS: Progression-free survival (PFS) was correlated with early FDG-PET metabolic response (P < .0001). Using -25% and +25% thresholds for SUV variations from baseline, early FDG-PET response was stratified in metabolic partial response, metabolically stable disease, or metabolically progressive disease; median PFS rates were 29, 16, and 4 weeks, respectively. Similarly, when a single FDG-PET positive/negative was considered after 4 weeks of sunitinib, the median PFS was 29 weeks for SUVs less than 8 g/mL versus 4 weeks for SUVs of 8 g/mL or greater (P < .0001). None of the patients with metabolically progressive disease subsequently responded according to RECIST criteria. Multivariate analysis showed shorter PFS in patients who had higher residual SUVs (P < .0001), primary resistance to imatinib (P = .024), or nongastric GIST (P = .002), regardless of the mutational status of the KIT and PDGFRA genes. CONCLUSION: Week 4 FDG-PET is useful for early assessment of treatment response and for the prediction of clinical outcome. Thus, it offers opportunities to individualize and optimize patient therapy.