Laure Belmont

Expression of TLR9 in tumor-infiltrating mononuclear cells enhances angiogenesis and is associated with a worse survival in lung cancer

Toll‐like receptors (TLRs) play a crucial role in the innate and adaptive immune responses against microbial infection, tissue injury and cancer. Ligands of TLR9 have been developed as therapy in non‐small‐cell lung carcinoma (NSCLC). However, phase III clinical trials in metastatic NSCLC were negative. Our objective was to determine whether TLR9 affects tumor growth. We generated a mouse model of lung adenocarcinoma (ADC) mutated for K‐ras (K‐rasLA1), with and without TLR9 inactivation (K‐rasLA1TLR9−/− and K‐rasLA1TLR9+/+, respectively). TLR9 was functionally expressed only in mononuclear cells of K‐rasLA1TLR9+/+ mice. These mice had significantly worse survival and a higher tumor burden than K‐rasLA1TLR9−/− mice. Lung tumors were analyzed for 24 cytokines/growth factors using Bio‐Plex multiplex bead‐based assays. Factor VIII was assessed by immunochemistry. Tumors from K‐rasLA1TLR9+/+ mice were characterized by an angiogenic phenotype with higher concentrations of vascular endothelial growth factor (VEGF) and higher microvessel density than from K‐rasLA1TLR9−/− mice. LKR13 cells, an ADC cell line derived from K‐rasLA1 mice, were subcutaneously injected into TLR9−/− and TLR9+/+ mice. Syngeneic tumors regressed in TLR9−/− but not in TLR9+/+ mice. Peripheral blood mononuclear cells from TLR9−/− mice released less VEGF than those from TLR9+/+ mice. In 61 patients with early‐stage NSCLC, TLR9 was expressed in mononuclear cells that infiltrated tumors, as assessed by immunochemistry, and contributed to worse survival. Our results suggest that TLR9 expression in mononuclear cells was associated with an angiogenic phenotype and promoted lung cancer progression. These findings may aid clinical development of TLR9 ligands to treat cancers.

Clinical and molecular features in patients with advanced non-small-cell lung carcinoma refractory to first-line platinum-based chemotherapy

Most of the cases of non-small-cell lung cancer (NSCLC) are diagnosed at an advanced stage and are treated with platinum-doublet chemotherapy. However, some patients are refractory to this treatment. The aim of this study was to identify the clinical and molecular characteristics of patients with refractory disease. All consecutive patients between 2003 and 2006, who received a platinum-doublet chemotherapy as first-line treatment for stage IIIb-IV NSCLC, were included. Refractory patients were defined as early progressive disease (PD) at the first evaluation of chemotherapy according to WHO criteria. The clinical, histo-pathological, and molecular characteristics (EGFR: exon 19, 20, 21 and KRAS: exon 2 by PCR sequencing; ALK by immunohistochemistry) and survival of refractory patients with initial PD (r-patients) and controlled disease (c-patients) were compared by univariate analyses. Factors that differed between the two groups (p-value <0.25 in univariate analyses) were entered into multivariate analysis. In this study, 178 patients were included. The first tumor assessment was carried out after a median of three cycles (range 1-4). Forty-six (25.8%) patients were refractory. Clinical presentation was similar between r- and c-patients. The sarcomatoid histological subtype was more common in r-patients than c-patients (10.9% vs. 1.5%, respectively; p=0.057). The proportion of EGFR (5.2% vs. 9.6%, p=0.224) and KRAS mutations (11.1% vs. 5.7%, p=0.357), and the expression of ALK (6.3% vs. 2.5%, p=0.327) did not differ significantly between the two groups. In multivariate analysis, sarcomatoid histological subtype was the only factor associated with early PD (OR=7.50; 95%CI: 1.02-55.45; p=0.048). r-Patients had significantly shorter survival than c-patients (median 5 months (IQR 3.2-9.9) vs. 15.4 months (IQR 9.9-22.5), respectively; p<0.0001). In conclusion, patients with early PD under platinum-doublet chemotherapy had shorter survival than c-patients. Sarcomatoid histological subtype was the only independent factor associated with early PD.

Secondary Resistance to Erlotinib: Acquired T790M Mutation and Small-Cell Lung Cancer Transformation in the Same Patient

CASE REPORT A 44-year-old woman a former smoker, presented with a 2-month history of progressive dyspnea. Computed tomography (CT)-scan showed diffuse alveolar opacities and micronodules in both lungs. Bronchoscopy was macroscopically normal, and bronchial biospsies were negative. Bronchioloalveolar lavage displayed revealed many TTF1 positive adenocarcinomatous cells harboring an exon 19 deletion (E746-A750) of endothelial growth factor receptor (EGFR) gene by sequencing. No extrathoracic metastases were detected on brain CT-scan or positron emission tomography scan. A first-line chemotherapy with carboplatin (area under the curve = 6), paclitaxel (175 mg/ m) and bevacizumab (15 mg/kg) every 3 weeks was started in December 2009. After stabilization at three cyles, the patient presented a pulmonary progression at six cycles. Erlotinib (150 mg/day) was started in June 2010 (Fig. 1A). After a partial

Routine administration of a single dose of cisplatin ≥ 75 mg/m2 after short hydration in an outpatient lung-cancer clinic

BACKGROUND Cisplatin is a pivotal drug in combined chemotherapy for non-small cell and small-cell lung cancers (NSCLC or SCLC), but its renal toxicity limits its use. Current guidelines recommend 24 h hydration: thus hospitalization is required. The aim of this retrospective study was to confirm the safety of short hydration before giving an intermediate-to-high dose of cisplatin in an outpatient clinic. PATIENTS AND METHODS Patients eligible had NSCLC or SCLC and were being treated with a chemotherapy regimen that included cisplatin ≥ 75 mg/m(2). They were given the same short hydration protocol for 1 day. Nephrotoxicity was defined as ≥ grade 1 according to NCIC common toxicity criteria. Predictive factors for nephrotoxicity were analyzed. RESULTS Three hundred and fifty-seven consecutive patients (median age 58 years, range: 25-81) were reviewed. Twenty-one patients (6%) had ≥ grade 1 nephrotoxicity and all except one had grade 1 toxicity according to NCIC criteria for common toxicity (SC < 1,5 N). Predictive factors independently associated with nephrotoxicity included associated co-morbid conditions (hypertension, diabetes, heart disease) (OR = 4.97 CI 95% [1.8-13.7] P = 0.002), initial serum creatinine ≥ 100 μmol/L (OR = 8.3 CI 95% [2.55-27.4] P = 0.0005), and dose cycle of cisplatin ≥ 100 mg/m(2) (OR = 10.8 CI 95% [3.6-32.5] P < 0.0001). CONCLUSION Rapid outpatient administration of a single dose of cisplatin at ≥ 75 mg/m(2) is feasible without a high risk of nephrotoxicity.

Abstract 1343: Toll-like Receptor 9 (TLR9) stimulation results in a change in tumor macrophage infiltration associated with an anti tumor effect in a mouse model of lung adenocarcinoma induced by oncogenic K-ras

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Microenvironment and immune system contribute to tumor escape in cancer. Toll-like Receptor 9 (TLR9) stimulation with a CpG ODN (PF-3512676) was expected to restore antitumor response in a mouse model of lung adenocarcinoma (ADC) induced by oncogenic K-ras (K-rasLA1 mice). In these mice, TLR9 was not expressed by normal lung, nor tumor cells but only by the inflammatory tumor component as shown by TLR9 mRNA quantification by real time quantitative fluorescent PCR and immunohistochemistry. Sixteen week-old K-rasLA1 mice received PF-3512676 (n=13) or vehicle (n=13) through subcutaneous administration for 3 weeks. Mice were killed after completion of treatment. During autopsy procedure, lesions were counted on lung surfaces by investigators blinded to treatment group. Compared to the vehicle group, there was a decrease of tumor cell proliferation (PCNA staining score: 28.5% ± 1 vs 22.4% ± 0.8; p<0.0001, respectively) and an increase of tumor cell apoptosis (CC3 western blot and staining score: 0.058% ± 0.01 vs 0.105% ± 0.01; p=0.0154, respectively) in the PF group. No effect was observed on the autopsy count (26.5 ± 3.7 tumors vs 24 ± 2.3; p=0.85) nor on the histological count (27 ± 3.4 tumors vs 20.7 ± 2.2; p=0.23). Macrophage infiltration studied by F4/80 staining was different between the two groups with a higher number of macrophages inside the tumors in PF group while a higher number in peritumor area in the vehicle group (intra/peritumoral macrophage ratio: 3.35 ± 0.5, PF group vs 1.68 ± 0.1 vehicle, p=0.003). LKR-13, a lung ADC cell line derived from K-rasLA1 mice, does not express TLR9 and was resistant to treatment with PF in vitro. However, LKR-13 cells grown as syngeneic tumors recruited macrophages, and those tumors regressed in response to treatment with PF. These findings provide evidence that TLR9 stimulation might reduced the expansion of lung ADC induced by oncogenic K-ras and suggest that phenotypic modification of macrophages may contribute to ADC regression. 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 1343.