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PDF file - 83K, Baseline characteristics of 52 patients with RCC lung metastasis. * determined by Heng et al.

PDF file - 90K, Baseline characteristics of 140 patients with CRC lung metastasis. The stage was determined by pathological examination at the time of diagnosis.

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Purpose: If immune cells are involved in tumor surveillance and have a prognostic impact in most primary tumors, little is known about their significance in metastases. Because patients' survival is heterogeneous, even at metastatic stages, we hypothesized that immune cells may be involved in the control of metastases. We therefore characterized the tumor immune microenvironment and its prognostic value in colorectal and renal cell carcinoma (RCC) metastases, and compared it to primary tumors.Experimental Design: We analyzed by immunohistochemistry (n = 192) and qPCR (n = 32) the immune environments of colorectal carcinoma and RCC lung metastases.Results: Metastases from colorectal carcinoma and RCC have different immune infiltrates. Higher densities of DC-LAMP+ mature dendritic cells (P < 0.0001) and lower densities of NKp46+ NK cells (P < 0.0001) were observed in colorectal carcinoma as compared to RCC metastases, whereas densities of T cells were similar. High densities of CD8+ and DC-LAMP+ cells correlated with longer overall survival (OS) in colorectal carcinoma (P = 0.008) and shorter OS in RCC (P < 0.0001). High NK-cell densities were associated with improved survival in RCC (P = 0.002) but not in colorectal carcinoma. Densities of immune cells correlated significantly from primary to relapsing metastases for the same patient. A TH1 orientation was found in colorectal carcinoma metastases, whereas a heterogeneous immune gene expression was found in RCC metastases.Conclusions: Our results show a major prognostic value of the immune pattern (CD8+/DC-LAMP+ cell densities) in colorectal carcinoma and RCC, reproducible from primary to metastatic tumors, although with opposite clinical impacts, and highlight the role of the tumor cell in shaping its immune environment. Clin Cancer Res; 19(15); 4079–91. ©2013 AACR.

Purpose: Adding oxaliplatin to adjuvant 5-fluorouracil (5-FU) chemotherapy improves 3-year disease-free survival (DFS) after resection of stage III colon cancer. Several studies suggest that patients with tumors exhibiting defective mismatch repair (MMR) do not benefit from adjuvant 5-FU chemotherapy, but there are few data on 5-FU–oxaliplatin (FOLFOX) adjuvant chemotherapy in this setting. The aim of this study was to evaluate the prognostic value of MMR status for DFS in patients with stage III colon cancer receiving adjuvant FOLFOX chemotherapy.Experimental Design: MMR status was determined by microsatellite instability testing or immunohistochemistry in 303 unselected patients with stage III colon cancer receiving adjuvant FOLFOX chemotherapy in 9 centers. Cox proportional hazards models were used to examine the association between MMR status and 3-year DFS.Results: The 3-year DFS rate was significantly higher in the 34 patients (11.2% of the study population) with defective MMR tumors (90.5%) than in patients with proficient MMR tumors (73.8%; log-rank test; HR = 2.16; 95% CI, 1.09–4.27; P = 0.027). In multivariate analysis, MMR status remained an independent significant prognostic factor for DFS (HR = 4.48; 95% CI, 1.34–14.99; P = 0.015).Conclusion: MMR status is an independent prognostic biomarker for DFS in patients with stage III colon cancer receiving adjuvant FOLFOX chemotherapy. Clin Cancer Res; 17(23); 7470–8. ©2011 AACR.

PDF file - 101K, Influence of pre-operative patient treatments on the distribution of CD8+, DC-LAMP+ and NKp46+ immune cells in CRC (a, b and c) and RCC lung metastases (d, e and f). 63/140 patients with CRC lung metastases have been treated with neo-adjuvant chemotherapy and 9/52 patients with RCC lung metastases have been treated with IL2/IFN. Whiskers length represents 10-90 percentile. ns, not significant (Mann-Whitney test).

PDF file - 88K, Gene expression in lung metastases from CRC and RCC. Expression of genes related to (a) immune cell populations, (b) Th1/Th2 orientations, (c) inflammation and angiogenesis, (d) immunosuppression, (e) cytotoxicity, (f) chemokines/chemokine receptors in lung metastases from CRC (grey bars) and RCC (white bars). Expression levels of genes were determined using threshold cycle (Ct) values normalized to actin B ACTB (ΔCt). Whiskers length represents 10-90 percentile. ns, not significant; *P

PDF file - 107K, Kaplan-Meier curves for the duration of OS according to the presence of CD8+ T cells in the center of the tumor (CT) (a), in the invasive margin (IM) (b) and in combined regions (CT+IM) (c) of CRC lung metastases. Statistical comparison was performed by the log-rank test.

PDF file - 56K, P values corresponding to different cutoff (minimum P value, first, second and third quartile) in CRC and RCC lung metastases. {section sign}P values were corrected by the formula proposed by Altman et al.

PDF file - 148K, Kaplan-Meier curves for the duration of OS according to the presence of CD8+ T cells in the center of the tumor (CT) (a), in the invasive margin (IM) (b) and in combined regions (CT+IM) (c) of RCC lung metastases. Representation of the Kaplan-Meier curves for the duration of OS according to the presence of NKp46+ cells in the CT (d), in the IM (e) and in combined region (CT+IM) (f) of RCC lung metastases. Statistical comparison was performed by the log-rank test.

Purpose: Pathologic TNM staging is currently the best prognostic factor for non‐small cell lung carcinoma (NSCLC). However, even in early-stage NSCLC, the recurrence rates after surgery range from 25% to 50%. The preoperative detection of circulating tumor cells (CTC) could be useful to tailor new therapeutic strategies in NSCLC. We assessed the presence of CTC in NSCLC patients undergoing surgery, using cytologic analyses, after their isolation by size of epithelial tumor cells (ISET method). The presence and the number of CTCs were considered and correlated with clinicopathologic parameters including patient follow-up.Experimental Design: Of the 247 blood samples tested, 208 samples were from patients with resectable NSCLC and 39 from healthy subjects. The mean follow-up was 24 months. An image of detected cells with presumably nonhematologic features [initially defined as “circulating nonhematologic cells” (CNHC)] was recorded. The presence of CNHC was assessed blindly and independently by 10 cytopathologists, using cytologic criteria of malignancy on stained filters. The count of detected CNHCs was made for each filter.Results: One hundred two of 208 (49%) patients showed CNHCs corresponding to CNHC with malignant cytopathologic features in 76 of 208 (36%) cases. CNHCs were not detected in the control group. A level of 50 or more CNHCs corresponding to the third quartile was associated with shorter overall and disease-free-survival, independently of disease staging, and with a high risk of recurrence and death in early-stage I + II-resectable NSCLC.Conclusion: A high percentage of NSCLC patients show preoperative detection of CNHC by the ISET method. The presence and level of 50 or more CNHCs are associated with worse survival of patients with resectable NSCLC. Clin Cancer Res; 17(3); 505–13. ©2010 AACR.

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Supplementary Figures S1-S2; Supplementary Table S1.

Purpose: Adding oxaliplatin to adjuvant 5-fluorouracil (5-FU) chemotherapy improves 3-year disease-free survival (DFS) after resection of stage III colon cancer. Several studies suggest that patients with tumors exhibiting defective mismatch repair (MMR) do not benefit from adjuvant 5-FU chemotherapy, but there are few data on 5-FU–oxaliplatin (FOLFOX) adjuvant chemotherapy in this setting. The aim of this study was to evaluate the prognostic value of MMR status for DFS in patients with stage III colon cancer receiving adjuvant FOLFOX chemotherapy. Experimental Design: MMR status was determined by microsatellite instability testing or immunohistochemistry in 303 unselected patients with stage III colon cancer receiving adjuvant FOLFOX chemotherapy in 9 centers. Cox proportional hazards models were used to examine the association between MMR status and 3-year DFS. Results: The 3-year DFS rate was significantly higher in the 34 patients (11.2% of the study population) with defective MMR tumors (90.5%) than in patients with proficient MMR tumors (73.8%; log-rank test; HR = 2.16; 95% CI, 1.09–4.27; P = 0.027). In multivariate analysis, MMR status remained an independent significant prognostic factor for DFS (HR = 4.48; 95% CI, 1.34–14.99; P = 0.015). Conclusion: MMR status is an independent prognostic biomarker for DFS in patients with stage III colon cancer receiving adjuvant FOLFOX chemotherapy. Clin Cancer Res; 17(23); 7470–8. ©2011 AACR.

Purpose: Epidermal growth factor receptor (EGFR) and VEGF(R) signaling show extensive cross-talk, providing a rationale for joint targeting of the two pathways. However, combinations of monoclonal antibodies (mAb) targeting EGFR and VEGF showed disappointing activity in patients with colorectal cancer (CRC). We speculated that inhibition of surface receptors and ligands might only partly prevent oncogenic signaling whereas small-molecule tyrosine kinase inhibitors (TKI) would also influence intracellular signaling. Experimental Design: Mice with CRC xenografts were treated with two TKIs, vargatef and afatinib, or with two mAbs, bevacizumab and cetuximab, and their influence on tumor growth, viability, in vivo DNA synthesis, and the presence of phosphorylated EGFR and VEGFR was determined. The activity of the TKIs was further characterized in CRC cells with different KRAS status. Results: Vargatef and afatinib together showed strong tumor growth inhibition toward HT-29 xenografts compared with either drug alone, which was associated with a 5-fold increase in apoptotic tumor cell death. In comparison, bevacizumab and cetuximab together were exclusively cytostatic with no more activity than either drug alone. Exposure to the two TKIs was accompanied by a marked decrease of tumor-associated intracellular phospho-VEGFR1 and phospho-EGFR, whereas similar exposure to the two mAbs had no detectable effect. A synergistic activity of vargatef plus afatinib was observed in all eight CRC cell lines examined, independent of KRAS status. Conclusions: Our results indicate that attenuation of intracellular EGFR and/or VEGF signaling is required for cytotoxic activity. These findings provide a rationale for trials of the TKIs, even in patients with mutant KRAS. Clin Cancer Res; 17(20); 6522–30. ©2011 AACR.

Primary tumor localization determines the metastatic immune profile Background Most cancer patients die from their metastatic disease and not from their primary tumor. However, little is known on the immune microenvironment of metastatic lesions and on its impact on clinical outcome. Indeed, the immune pattern of the tumor microenvironment has been recently identified as being a major prognostic factor in primary tumors. Thus, a high density of memory T cells with a Th1 and CD8 cytotoxic orientation is beneficial in many cancers. However, there are a few exceptions since CD8+ T cells have been reported to be associated with bad prognosis in renal cell carcinoma. The question of the respective roles of the tumor cell and organ microenvironment to explain these differences has not been addressed. Methods We studied the density and organization of tumor-infiltrating immune cells (CD3+, CD8+, Foxp3+, granzyme B+, NK cells and mature DCs) in retrospective cohorts of primary and lung metastases of colorectal cancer (CRC, n=124) and renal cell carcinoma (RCC, n=55) by immunohistochemistry. We investigated by qPCR the gene expression levels related to immune populations and their functions in the lung metastases from 19 CRC and 12 RCC. Biological data's were compared to clinical data's and patient's outcome. Results The cell composition (CD3+, CD4+ and Foxp3+ T cells, mature DCs and NK cells) and organization (tertiary lymphoid structures) of the immune infiltrate in metastases depend on the origin of the primary tumor. In both cases (colorectal cancer and renal cell carcinoma lung metastases), the density of CD8+ T cells and mature DCs appeared to be strongest prognosticator of patient's survival as reported for many primary tumors, but with opposite impact. High densities of mature DCs and CD8+ cells correlate with good overall survival in lung metastases of colorectal cancer and with poor survival in lung metastases of renal cell cancer. In the latter, a strong Th2 and inflammatory context was present associated with a strong Th1 polarization. Conclusions Altogether, our data demonstrate that the immune pattern depends on the tumor origin and has a major prognostic role in advanced cancer stages with an impact on patient therapeutic management. We demonstrated also the complexity of the immune response polarization and its impact upon tumor immune surveillance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-497. doi:1538-7445.AM2012-LB-497

Backgound: Combining different classes of targeted agents represents a major challenge in modern cancer therapy. Agents targeting EGFR- or VEGF-signaling are approved for treatment of metastatic colorectal cancer paving the way for novel therapeutic strategies combining the two classes of agents. Unexpectedly, large clinical studies with combinations of EGFR-targeted antibodies and bevacizumab showed no added benefit compared to bevacizumab alone. It is currently unclear whether these findings are linked to incompatibility between the antibodies or rather to incompatibility between the two classes of targeted agents. Experimental: Combinations of two small orally active molecules, the irreversible EGFR and HER2 inhibitor BIBW2992 (B2) and the triple angiokinase inhibitor BIBF1120 (B1) were characterized in human colorectal cancer cell lines in vitro and in mice using the HT-29 colorectal xenograft model that is BRAF V600E mutated. Growth inhibitory and cytotoxic effects were characterized by flow cytometry complemented by immunological analyses for p27Kip1, phospho-Erk and phospho-Akt in cellular models and by quantitative immunohistochemistry analyses of EdU incorporation and TUNEL staining in tumor xenografts. Results: Exposure of colorectal cancer cells to B1 or to B2 resulted in growth inhibition linked to prolonged p27Kip1-mediated G1 arrest, which in most cell lines was accompanied by delayed apoptosis. Combinations of B1 and B2 resulted in increased cell death in all cellular models studied. In xenografts, four weeks treatment with combinations of B1 and B2 were associated with a clear synergistic effect on tumor growth inhibition compared to either agent alone. In contrast, no additional activity was observed for combinations of cetuximab and bevacizumab in the same xenograft model. Quantitative immunohistochemistry analyses revealed that all 4 agents inhibited in vivo DNA synthesis, as shown by EdU incorporation, and that none of the combinations tested showed superiority. TUNEL staining was not increased following single agent treatment with B1, cetuximab and bevacizumab while B2 resulted in increased TUNEL staining. Combinations of B1 and B2 were characterized by a pronounced increase in TUNEL staining, while no increase was observed for combinations of cetuximab and bevacizumab. Conclusions: We here show that combinations of small molecules that simultaneously target the HER and the angiogenic pathways show synergistic cytotoxic and tumor-growth inhibitory activities in the HT-29 colorectal xenograft model which is refractory to the combinations of cetuximab and bevacizumab. Therefore, the lack of superior clinical activity for combinations of cetuximab/panitumumab and bevacizumab is not due to an inherent incompatibility between the 2 classes of targeted agents. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A240.