Rosa Conforti

Opposing Effects of Toll-like Receptor (TLR3) Signaling in Tumors Can Be Therapeutically Uncoupled to Optimize the Anticancer Efficacy of TLR3 Ligands

Many cancer cells express Toll-like receptors (TLR) that offer possible therapeutic targets. Polyadenylic-polyuridylic acid [poly(A:U)] is an agonist of the Toll-like receptor TLR3 that displays anticancer properties. In this study, we illustrate how the immunostimulatory and immunosuppressive effects of this agent can be uncoupled to therapeutic advantage. We took advantage of two TLR3-expressing tumor models that produced large amounts of CCL5 (a CCR5 ligand) and CXCL10 (a CXCR3 ligand) in response to type I IFN and poly(A:U), both in vitro and in vivo. Conventional chemotherapy or in vivo injection of poly(A:U), alone or in combination, failed to reduce tumor growth unless an immunochemotherapeutic regimen of vaccination against tumor antigens was included. CCL5 blockade improved the efficacy of immunochemotherapy, whereas CXCR3 blockade abolished its beneficial effects. These findings show how poly(A:U) can elicit production of a range of chemokines by tumor cells that reinforce immunostimulatory or immunosuppressive effects. Optimizing the anticancer effects of TLR3 agonists may require manipulating these chemokines or their receptors.

TLR3 as a Biomarker for the Therapeutic Efficacy of Double-stranded RNA in Breast Cancer

The discovery of a targeted therapeutic compound along with its companion predictive biomarker is a major goal of clinical development for a personalized anticancer therapy to date. Here we present evidence of the predictive value of TLR3 expression by tumor cells for the efficacy of Poly (A:U) dsRNA in 194 breast cancer patients enrolled in a randomized clinical trial. Adjuvant treatment with double-stranded RNA (dsRNA) was associated with a significant decrease in the risk of metastatic relapse in TLR3 positive but not in TLR3-negative breast cancers. Moreover, we show the functional relevance of TLR3 expression by human tumor cells for the antitumor effects mediated by dsRNA in several preclinical mouse models carried out in immunocompromised animals. These 2 independent lines of evidence relied upon the generation of a novel tool, an anti-TLR3 antibody (40F9.6) validated for routine detection of TLR3 expression on paraffin-embedded tissues. Altogether, these data suggest that dsRNA mediates its therapeutic effect through TLR3 expressed on tumor cells, and could therefore represent an effective targeted treatment in patients with TLR3-positive cancers.

The Janus face of dendritic cells in cancer

On the basis of experimental models and some human data, we can assume that tumor outgrowth results from the balance between immunosurveillance (the extrinsic tumor suppressor mechanisms) and immunosubversion dictated by transformed cells and/or the corrupted surrounding microenvironment. Cancer immunosurveillance relies mainly upon conventional lymphocytes exerting either lytic or secretory functions, whereas immunosubversion results from the activity of regulatory T or suppressor myeloid cells and soluble mediators. Although specific tools to target or ablate dendritic cells (DCs) became only recently available, accumulating evidence points to the critical role of the specialized DC system in dictating most of the conventional and regulatory functions of tumor-specific T lymphocytes. Although DC can be harnessed to silence tumor development, tumors in turn can exploit DC to evade immunity. Indeed, DCs harbor defects in their differentiation and stimulatory functions in cancer-bearing hosts and can actively promote T-cell tolerance to self-tumor antigens. In this review, we will focus on the dual role of DC during tumor progression and discuss pharmacoimmunological strategies to harness DC against cancer.

CXCR4 Expression in Early Breast Cancer and Risk of Distant Recurrence

BACKGROUND Chemokine receptor 4 (CXCR4) has been demonstrated to have a critical role in the early metastatic process. The aim of this study was to evaluate the prognostic value of CXCR4 expression in primary breast tumors and describe correlations with the occurrence of metastasis in organs expressing the CXCR4 ligand stromal cell-derived factor 1 (i.e., liver, lung, brain, and bone). PATIENTS AND METHODS CXCR4 expression in primary breast tumors was evaluated by immunohistochemistry in 823 patients included in two prospective clinical trials. CXCR4 expression was considered positive when >1% of tumor cells were stained. The prognostic value of CXCR4 expression was assessed by a Cox regression model adjusted for clinical characteristics. We assessed the association of CXCR4 expression with the rate of distant metastasis to specific organ sites. RESULTS CXCR4 was expressed in 92 of 794 primary tumors (12%). CXCR4 expression was not associated with clinical characteristics. CXCR4 was not prognostic for overall survival and showed a nonsignificant trend toward a higher risk for distant metastasis. CXCR4(+) tumors showed a significantly higher risk for bone metastasis. The 10-year incidences of bone metastases were 23% (13.6%-32.6%) and 12% (9.7%-15%) in CXCR4(+) and CXCR4(-) tumors, respectively. CONCLUSION This study suggests that expression of CXCR4 in primary breast tumors is associated with a higher likelihood of developing bone metastases. This finding could open new avenues for the development of novel adjuvant strategies, including bone-targeting agents.

Pulmonary toxicity related to systemic treatment of nonsmall cell lung cancer.

Physicians who are responsible for the delivery of systemic treatment in lung cancer should be aware of the potential risk of drug‐induced pulmonary toxicity (DIPT), because such toxicity may develop in the context of a multifactorial clinical condition. First, most patients with lung cancer may suffer from other non‐neoplastic, smoking‐related lung diseases, such as emphysema and chronic obstructive lung disease, which may generate pathologic changes in lung parenchyma. In addition, lung cancer itself may worsen the respiratory function, inducing atelectasis and lymphangitic carcinomatosis. The superimposed iatrogenic damage may lead to respiratory failure and, sometimes, death. The risk of DIPT from chemotherapeutic agents has been widely examined in the past; and, currently, the potential for lung toxicity has been extended by the introduction of molecular targeted therapies. Because there are no univocal criteria with which to recognize DIPT, the diagnosis often is made by exclusion; consequently, it is hard to establish an early diagnosis. The objective of this review was to describe the major DIPTs associated with antineoplastic agents against nonsmall cell lung cancer to help physicians with this difficult diagnostic challenge. Cancer 2011.

Association between the nuclear to cytoplasmic ratio of p27 and the efficacy of adjuvant polychemotherapy in early breast cancer

BACKGROUND The purpose of this study was to evaluate the prognostic and predictive value of p27 expression in patients with early breast cancer. PATIENTS AND METHODS Quantitative immunofluorescence assays for p27 were done on a tissue microarray that included 823 samples from patients randomized between anthracycline-based chemotherapy and no chemotherapy. Quantification of p27 was done using the AQUA® system (HistoRx, Inc., Branford, CT). Both p27 nuclear expression and the nuclear to cytoplasmic ratio were assessed. RESULTS Nuclear p27 expression was not predictive for the efficacy of anthracycline-based chemotherapy [adjusted P=0.18 for disease-free survival (DFS)] nor prognostic [95% confidence interval (CI) 0.99-1.01, P=0.49]. However, p27 nuclear/cytoplasmic ratio was predictive for the efficacy of adjuvant chemotherapy (adjusted P=0.016 DFS). The adjusted hazard ratio (HR) for relapse associated with adjuvant chemotherapy was 0.56 (95% CI 0.37-0.84, P=0.005) and 1.06 (95% CI 0.76-1.47, P=0.74) for patients with high and low nuclear/cytoplasmic ratio, respectively. p27 N/C ratio was prognostic in patients treated with chemotherapy (HR for relapse or death for a 1 unit increase in p27 N/C ratio was 0.30, 95% CI 0.12-0.77) but not in the untreated arm (HR for relapse or death was 1.27, 95% CI 0.58-2.8). CONCLUSIONS This study did not confirm the role of p27 nuclear expression as a prognostic parameter. However, the p27 nuclear/cytoplasmic ratio was predictive in patients treated with anthracycline-based chemotherapy.

An inhibitor of cyclin-dependent kinases suppresses TLR signaling and increases the susceptibility of cancer patients to herpes viridae

Cyclin-dependent kinase (CDK) inhibitors have been considered as excellent drug candidates for cancer therapy owing to their potential capacity to restore cell cycle control. The first generation of CDK inhibitors showed modest clinical advantages that could be attributed to off-target effects preventing them from reaching therapeutic concentrations. A phase I dose-escalation study using the second generation multi-CDK inhibitor PHA-793887 was conducted on a total of 19 patients with advanced refractory malignancies in two sites in Europe: the University of Leeds and St. James’s Institute of Oncology, Leeds, UK, and the Institut Gustave Roussy, Villeujf, France (IGR). Fifteen patients were treated at IGR. Six among these patients manifested the reactivation of herpes virus replication. In vitro experiments revealed that PHA-793887 severely impaired signaling by toll-like receptors (such as TLR3, TLR4 and TLR9) in dendritic cells (DC), thus suppressing the production of multiple cytokines (type 1 interferon, interleukin-6,-10, -12, and tumor necrosis factor- α) by mature DC, as well as the DC-stimulated production of interferon-γ by natural killer cells. Pharmacological inhibition of glycogen synthase-3β (GSK-3β), one of the off-targets of PHA-793887, did not cause such immunological defects. Altogether, these data underscore a hitherto unsuspected immunosuppressive effect of PHA-793887.

Abstract 371: Expression and circulating level of VEGFR-3 in renal cell cancer: Implication for antiangiogenic treatments

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Regional lymph node metastasis is a common event in solid tumors and is considered a marker for dissemination, increased stage, and worse prognosis. In renal cell cancer (RCC) lymphatic tumor spread exists, but, despite impressive therapeutic advances in metastatic RCC, including targeting of VEGF induced angiogenesis, data on the role of vegfr-3 thats appears to be the signaling pathway for tumor-induced lymphangiogenesis in RCC are rare. In this study, we analyzed the expression and the presence of the soluble VEGFR-3 form in 30 RCC patients and results were correlated with clinicopathological parameters. Patients and Methods: Tumor and sera from 30 RCC patients (20 clear cell (ccRCC) and 10 papillary (pRCC) were included in this study. The expressions of VEGFR-3 on tumor were evaluated by immunohistochemestry. Using ELISA assays, sVEGFR–3 were measured in sera of RCC patients in comparison to 20 healthy controls. Results: A high expression of VEGFR-3 was observed in more than 55% of the patients as compared to the negative control. Regarding circulating VEGF R-3 the level were variable in all samples, but, the median sVEGF- R3 level was significantly higher (p=0.005) in RCC patients as compared to healthy donors. An inverse correlation between VEGFR-3 expression & its soluble form was noted (r=-0.33 p=0.040).. However, when patients were divided into subgroups, the expression as well as the circulating form of VEGFR-3 were significantly higher in pRCC than ccRCC (p= 0.035 and 0.022 respectively). No significant correlation with lymph node status was observed. Furthermore, there was no correlation with any clinicopathological variable in the two subgroups. Conclusions: we showed that in renal cell cancer, VEGFR-3 is expressed and present as soluble form, a different expression pattern in ccRCC and pRCC exist. Therefore, any clinical treatment strategy designed to target lymphangiogenesis in RCC should differentiate between ccRCC and pRCC Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 371.

Abstract 3443: Lymphangiogenic parameters in metastatic malignant melanoma: association with clinicopathological parameters and prognosis

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background and aims: Regional lymph node metastasis is one of the important indicators of malignant melanoma. Lymphatic or vascular systems are considered to provide a way whereby tumor cells can migrate to the lymph nodes either by direct invasion of surrounding tissue or spreading. Despite their clinical relevance, the mechanisms that guide the way of spreading are not well known. Recent studies have shown that Flt-4, a VEGF receptor, is activated by its ligand, VEGF-C. The resultant signaling pathway promotes angiogenesis and/or lymphangiogenesis, but the prognostic significance of these axes in malignant melanoma remains controversial. The aim of this study is to clarify therole of these factors and to evaluate the relationships between the VEGF-C/Flt-4 axis and lymphangiogenesis, lymph node metastasis, and prognosis in patients with metastatic malignant melanoma patients (MMM). The expressions of VEGF-C and VEGFR −3 were detected on 15 metastatic melanoma tumor by immunohistochemistry. Circulating form of VEGFC and Flt-4 were measured using ELISA assays in the sera of 90 patients with MMM in comparison to 50 healthy controls. Disease free Survival (DFS) and overall survival (OS) were analyzed using the Kaplan-Meier method. Results: High expressions of VEGF-C and VEGFR-3 proteins were concomitantly detected in the cytoplasm of melanoma tumors. VEGF-C was associated with VEGFR-3 expression and was significantly correlated with lymph node metastasis. Pretreatment median levels of sVEGF-C and sFlt-4 were significantly higher in MMM patients as compared to healthy ones (p=0.005, p<0.00001). None of the studied markers correlated with gender, age or LDH levels. An inverse correlation between Flt-4 expression & its soluble form was noted (r=-0.33 p=0.040). A positive correlation between sVEGF-C & sFlat-4 was observed (r’=0.430, p=0.0014). Furthermore, high expression & levels of sVEGF-C, sFlat-4 correlated to high tumor burden (p=0.02, p=0.045). Only sVEGFC/sFlat-4 ratio showed a relationship with lymph node metastasis. Pretreatment sFlat-4 level was significantly different (p=0.0125) between R (n=40) & NR patients (n=50) but not sVEGF-C level. After treatment, no change in the median sFlat-4 level was observed. By contrast, sVEGF-C increased specifically in NR patients with high tumor burden. Only elevated level of sFlat-4 concentration exert a significantly unfavorable impact on disease free survival (X2= 3.855, p=0.045). Conclusions: This study suggests that both VEGF-C and VEGFR-3 expression as well as their soluble forms are involved in peritumoral lymphangiogenesis and lymphatic metastasis. Furthermore, the VEGF-C/Flt-4 axis enhances cancer cell invasiveness, contributes to the promotion of cancer cell metastasis and may be clinically useful indicators for prognostic evaluation in patients with metastatic malignant melanoma. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3443.

Predictive value of p27 for the benefit of adjuvant anthracycline-based chemotherapy in early breast cancer.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #6063 Background: p27 is a cyclin-dependent kinase (cdk) inhibitor that plays a role in cell cycle regulation. The expression of this protein, assessed by immunohistochemistry (IHC), has prognostic value for overall survival in breast cancer patients (Porter P.L., J Natl Cancer Inst., 2007). Based on this, we hypothesized that p27 could be a predictive factor for the efficacy of adjuvant anthracycline (A)-based chemotherapy in early breast cancer. Methods: Tumor samples from 823 patients, included at Institut Gustave Roussy (IGR) in two randomized trials comparing an A-based chemotherapy with no adjuvant treatment, were used to construct a tissue microarray. We previously reported predictive values of ER, Her2 expression and molecular subclassification using the same tissue array (Conforti R., Ann Oncol., 2007). Nuclear and cytoplasmic expression of p27 was assessed by the AQUA system. A p27 ratio of nuclear and cytoplasmic expression (p27 N/C ratio) was calculated for each tumor sample. The prognostic and predictive value of the continuous value of p27 N/C ratio was assessed by a Cox regression model adjusted for treatment, age, tumor grade, stage and ER expression (IHC) and stratified by trial. Results: p27 was assessable in 715 primary tumors. The p27 N/C ratio was significantly different according to the molecular subclasses (p=0.001) and was higher in Her2- versus Her2+ and in EGFR+ versus EGFR- tumors (p=0.0075 and p<0.0001 respectively). There was an almost significant interaction between p27 N/C ratio and disease-free survival in the adjusted Cox model (p=0.052). The adjusted hazard ratios (HR) of relapse or death of a unit increase in p27 N/C ratio was HR = 1.27 (95% CI = [0.58-2.81], p = 0.55) in the control arm and HR = 0.30 (95% CI = [0.12-0.77], p= 0.01) in the A-arm. For overall survival, the adjusted interaction p-value was equal to 0.08. The adjusted HR of death of a unit increase in p27 N/C ratio was HR=1.67 (95% CI = [0.61-1.73], p=0.32) in the control arm and HR=0.32 (95% CI = [0.10-0.99], p=0.05) in the A-arm. Conclusion: p27 N/C ratio, determined by the AQUA system, was borderline predictive for the efficacy of adjuvant A-based chemotherapy. However, this study is hampered by a relatively low number of events, a low dose of anthracycline, and multi-hypotheses testing (overall 20 biomarkers tested in different studies). Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6063.