Virginie Poulot

Long-Term Survival for Patients With Non–Small-Cell Lung Cancer With Intratumoral Lymphoid Structures

PURPOSE It has been established that the immune system plays an important role in tumor rejection. There is also compelling evidence that immune responses can develop independently of secondary lymphoid organs in tertiary lymphoid structures. We studied the presence and the correlation of tertiary lymphoid structures with clinical outcome in non-small-cell lung cancer (NSCLC), as the prognostic value of these structures in patients with cancer had not yet been established. PATIENTS AND METHODS This retrospective study was performed by immunohistochemistry on paraffin-embedded tissue specimens from 74 patients with early-stage NSCLC. RESULTS Tertiary lymphoid structures were detected in some tumors but not in nontumoral lungs. Thus we called these structures tumor-induced bronchus-associated lymphoid tissue (Ti-BALT). As in lymph nodes, Ti-BALTs were composed of mature dendritic cell (DC)/T-cell clusters adjacent to B-cell follicles and had features of an ongoing immune response. Because the quantitative counting of Ti-BALT was difficult to achieve, we used mature DCs that homed exclusively in Ti-BALT as a specific marker of these structures. Univariate analysis showed that the density of mature DCs was highly associated with a favorable clinical outcome (overall, disease-specific, and disease-free survival), suggesting that Ti-BALT may participate in antitumoral immunity. The density of tumor-infiltrating lymphocytes, in particular, CD4(+) and T-bet(+) Th1 T cells, was profoundly decreased in tumors weakly infiltrated by mature DCs. CONCLUSION The density of mature DCs was found to be a better predictor of clinical outcome than the other parameters tested. The number of tumor-infiltrating mature DCs may identify patients with early-stage NSCLC who have a high risk of relapse.

Non-mucinous and mucinous subtypes of adenocarcinoma with bronchioloalveolar carcinoma features differ by biomarker expression and in the response to gefitinib

There is no optimal established therapy for treating advanced or recurrent adenocarcinoma with bronchioloalveolar carcinoma features (ADC-BAC), and it remains unclear whether chemotherapy achieves therapeutic results comparable to those seen in the more common non-small lung carcinoma subtypes. In order to improve the decisions made during the treatment of advanced ADC-BAC, we attempted to better characterize the mucinous and non-mucinous ADC-BAC subtypes. Fifty pathological samples were obtained from 62 patients included in a multicenter prospective phase II trial (IFCT0401) conducted to evaluate gefitinib as a first-line therapy for non-resectable ADC-BAC. These samples were centrally reviewed and re-classified as non-mucinous (n=25) or mucinous (n=25) subtypes. We demonstrated that demographic data, clinical characteristics and stage at presentation (extrathoracic versus lung metastasis, as well as TNM staging) did not distinguish between the two subtypes. In contrast, three biological markers (PAS staining, TTF-1 expression and EGFR genomic gain combined with mutation analysis) enabled us to independently segregate all but 2 of the 50 patients into the mucinous and non-mucinous ADC-BAC subtypes. Finally, only mucinous tumors appeared to be resistant to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). Additional prospective studies are required to better approach therapeutic strategy in mucinous tumors, which are a distinct entity from non-mucinous tumors.

Impact of Systematic EGFR and KRAS Mutation Evaluation on Progression-Free Survival and Overall Survival in Patients with Advanced Non–Small-Cell Lung Cancer Treated by Erlotinib in a French Prospective Cohort (ERMETIC Project—Part 2)

Background: Epidermal growth factor and v-Ki-ras2 Kirsten ras sarcoma (KRAS) mutation status, although associated with EGFR- tyrosine kinase inhibitor (TKI) efficacy, has not been used in clinical practice until recently. The prospective Evaluation of the EGFR Mutation status for the administration of EGFR-TKIs in non small cell lung Carcinoma (ERMETIC) study aimed to implement these biomarkers in France. Methods: Between March 2007 and April 2008, EGFR and KRAS were studied by sequencing DNA tumor specimens from 522 consecutive advanced non–small-cell lung cancer patients treated with EGFR-TKI, mostly in second- or third-line settings. Cox models were used to investigate the impact of patient characteristics and mutations on progression-free survival (PFS) and overall survival (OS). Added value from mutation status was evaluated using likelihood ratio (LR) tests. Classification and regression tree analysis aimed to identify homogeneous groups in terms of survival. Results: Among the 522 patients, 87% were white, 32% were women, and 18% were never-smokers, with 65% presenting with adenocarcinoma. Biological data were available for 307 patients, showing 44 EGFR mutations (14%) and 42 KRAS (14%) mutations. Median PFS was 2.4 months (interquartile range, 1.4–4.6) and median OS 5.6 months (interquartile range, 2.2–14.0). Factors independently associated with PFS were performance status 1 or 2 to 3 (hazards ratio [HR] = 1.5, 95% confidence interval [CI] 1.1–1.9; and HR = 2.3, CI 1.7–3.1, respectively; p < 0.001); former or current smoker status (HR = 1.8, CI 1.4–2.4 and 2.0,CI 1.4–2.8, respectively; p < 0.001); nonadenocarcinoma histology (squamous cell: HR = 0.9 CI 0.7–1.2]; others: HR = 1.6, 1.3–2.1; p < 0.001); at least two metastatic sites (HR = 1.3, CI 1.1–1.6 and 1.6, CI 1.3–2.1, respectively; p < 0.001); prior taxane-based chemotherapy (HR = 1.3, CI 1.0–1.3, p = 0.01); non-white (HR = 0.7, CI 0.5–0.9, p = 0.009). Similar results were found for OS. In addition, EGFR and KRAS mutations were significantly associated with PFS (HR = 0.5, CI 0.3–0.7 and HR = 1.2, CI 0.8–1.8, respectively, versus no mutation; LR p = 0.001). In the OS model, adjusted HR was 0.7 (0.4–1.0) for EGFR mutation and 1.7 (1.1–2.4) for KRAS (LR p = 0.004). Classification and regression tree analysis revealed EGFR mutation to be the primary factor for identifying homogeneous patient subgroups in terms of PFS. Conclusions: EGFR and KRAS status independently impacts outcomes in advanced non–small-cell lung cancer patients treated with EGFR-TKI. However, EGFR status impacts both PFS and OS whereas KRAS only impacts OS. These findings support the nationwide use of EGFR status for patient selection before EGFR-TKI therapy. The role of KRAS mutations remains to be elucidated.

Neutrophils Promote Aerogenous Spread of Lung Adenocarcinoma with Bronchioloalveolar Carcinoma Features

Purpose: Adenocarcinoma with bronchioloalveolar carcinoma (BAC) features is a subtype of non–small cell lung cancers characterized by an intense inflammatory reaction composed of macrophages and neutrophils and by a distinct natural history with intrapulmonary spread leading to death due to respiratory failure. We hypothesized that neutrophils could promote aerogenous spread of lung adenocarcinoma with BAC features. Experimental Design: We examined the effect of neutrophils on A549 cell line detachment in vitro and we quantified desquamation of tumor cells on tumor tissue (n = 25) and on matched bronchioloalveolar lavage (n = 17) in vivo in a series of patients with adenocarcinoma with BAC features. Results: Neutrophils induced A549 detachment mediated by signals through cell-to-cell contact. Detached A549 cells were still viable and able to proliferate in vitro. Neutralization studies identified several membrane-bound molecules involved in detachment (i.e., intercellular adhesion molecule-1/lymphocyte function-associated antigen-1, tumor necrosis factor α/tumor necrosis factor α receptor inhibitor, interleukin-1α /interleukin-1α receptor, and neutrophil elastase). In tumor tissue, shedding was detected in all samples, with a median shedding score of 42% (range, 4-95%). Micropapillary clusters were detected in 23 of the 25 tumor tissue samples, with a median micropapillary score of 1.40 (range, 0-2.1), and tumor cells were detected in 7 of 17 lavages. The micropapillary score was associated with a high neutrophil count in bronchioloalveolar lavage (P = 0.051). The shedding cell percentage was a significant factor in shorter survival (P = 0.034, univariate Cox analysis). Conclusions: Tumor shedding is induced by neutrophils. It is a significant factor of shorter survival and may be an important event in adenocarcinoma progression.

Blood vessel invasion is a major feature and a factor of poor prognosis in sarcomatoid carcinoma of the lung

OBJECTIVES Pulmonary sarcomatoid carcinomas (SC) are highly disseminated types of non-small-cell lung carcinoma. Their prognosis is poor. New therapeutic targets are needed to improve disease management. MATERIALS AND METHODS From 1995 to 2013, clinical and survival data from all consecutive patients with surgically treated SC were collected. Pathological and biomarker analyses were performed: TTF1, P63, c-MET and ALK expression (immunohistochemistry), PAS staining, ALK rearrangement (FISH), and EGFR, KRAS, HER2, BRAF, PIK3CA, and MET genes mutations (PCR). RESULTS Seventy-seven patients were included. Median age was 61 years (53-69). Histological subtypes were pleomorphic carcinoma (78%), carcinosarcoma (12%), and giant-cell and/or spindle-cell carcinoma (10%). Blood vessel invasion (BVI) was present in 90% of cases. Morphology and immunohistochemistry were indicative of an adenocarcinoma, squamous, and adenosquamous origin in 41.5%, 17% and 11.5%, respectively, 30% remained not-otherwise-specified. KRAS, PIK3CA, EGFR, and MET mutations were found in 31%, 8%, 3%, and 3%, respectively. No tumors had HER2 or BRAF mutations, or ALK rearrangement, whereas 34% had a c-MET positive score. Five-year overall survival (OS) was 29% for the whole population. At multivariate analysis, tumor size <50mm (HR=1.96 [1.04-3.73], p=0.011), no lymph-node metastasis (HR=3.25 [1.68-6.31], p<0.0001), no parietal pleural invasion (HR=1.16 [1.06-1.28], p=0.002), no BVI (HR=1.22 [1.06-1.40], p=0.005), and no squamous component (HR=3.17 [1.48-6.79], p=0.01) were associated with longer OS. Biomarkers did not influence OS. CONCLUSION Dedifferentiation in NSCLC could lead to SC and an epithelial subtype component could influence outcome. BVI was present in almost all SCs and was an independent factor of poor prognosis.

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

EGFR and KRAS mutation status in non-small-cell lung cancer occurring in HIV-infected patients

Non-small-cell lung cancer (NSCLC) is the most common non-acquired immune deficiency syndrome-related malignancy responsible for death. Mutational status is crucial for choosing treatment of advanced NSCLC, yet no data is available on the frequency of epidermal growth factor receptor (EGFR) and Kirsten ras (KRAS) mutations and their impact on NSCLC in human immunodeficiency virus (HIV)-infected patients (HIV-NSCLC). All consecutive HIV-NSCLC patients diagnosed between June 1996 and August 2013 at two Paris university hospitals were reviewed, with tumor samples analyzed for EGFR and KRAS mutational status. Overall, 63 tumor samples were analyzed out of 73 HIV-NSCLC cases, with 63% of advanced NSCLC. There were 60 non-squamous and nine squamous cell carcinomas, with EGFR and KRAS mutations identified in two (3.3%) and seven (11.5%) tumors, respectively. The proportion of KRAS mutations was 29% if solely the more sensitive molecular techniques were considered. The two patients with advanced adenocarcinoma harboring EGFR mutations exhibited lasting partial response to EGFR-tyrosine kinase inhibitors. Overall survival for patients with advanced NSCLC were >30 months for those with EGFR mutations, <3 months for KRAS mutations (n=2), and the median was 9 months [4.1-14.3] for wild-type (n=34). In multivariate analysis, KRAS mutation and CD4<200 cells/μL were associated with poor prognosis (hazard ratio (HR): 24 [4.1-140.2], p=0.0004; HR: 3.1 [1.3-7.5], p=0.01, respectively). EGFR mutation must be investigated in HIV-NSCLC cases due to its predictive and prognostic impact, whereas KRAS mutation is of poor prognostic value. Clinicians should search for drugs dedicated to this target population.

Pro-tumoural CXCL10/CXCR3-A autocrine loop in invasive mucinous lung adenocarcinoma

Invasive mucinous adenocarcinoma (IMA) is a mucinous variant of lepidic predominant lung adenocarcinoma (LPA) and associated with a worse prognosis. We postulated that cytokine expression would enable us to differentiate IMA from LPA in terms of prognosis and acquisition of pro-tumoural capacities. A 30-cytokine panel was assessed in bronchoalveolar lavage fluids (BALF) from IMA (n=38), LPA (n=25) and control samples (n=7). We investigated the expression of differentially expressed cytokines and splice variants of their receptors in surgical samples. The presence of EGFR and KRAS mutations were determined. We also examined the expression of cytokines and splice variants of their receptors in different cell lines, exploring their functional impact on signalling pathways, proliferation and migration. Only C-X-C motif chemokine 10 (CXCL10) was differentially expressed, namely overexpressed in IMA BALF compared with LPA. CXCL10 overexpression in BALF was linked to a worse prognosis. In surgical samples, CXCL10 and its receptor C-X-C motif chemokine receptor 3 (CXCR3) were overexpressed in IMA compared to LPA. A pro-tumoural CXCR3-A splice variant was overexpressed in IMA, suggesting a CXCL10/CXCR3-A autocrine loop in IMA. CXCL10 and CXCR3 expression were not correlated with EGFR or KRAS status. CXCL10 up-regulated CXCR3-A expression, Erk1/2 phosphorylation and enhanced migration in the mucinous H2228 cell line. CXCL10/CXCR3-A may play a pro-tumoural role in IMA via an autocrine mechanism. Invasive mucinous lung adenocarcinoma derived pro-tumoural advantages by means of a CXCL10/CXCR3-A autocrine loop http://ow.ly/2Gjj308QxwK

NRG1 fusion in a French cohort of invasive mucinous lung adenocarcinoma

Invasive mucinous lung adenocarcinoma (IMA) is a rare subtype of lung adenocarcinoma with no effective treatment option in advanced disease. KRAS mutations occur in 28–87% of the cases. NRG1 fusions were recently discovered in KRAS‐negative IMA cases and otherwise negative for known driver oncogenes and could represent an attractive therapeutic target. Published data suggest that NRG1 fusions occur essentially in nonsmoking Asian women. From an IMA cohort of 25 French patients of known ethnicity, driver oncogenes EGFR, KRAS, BRAF, ERBB2 mutations, and ALK and ROS1 rearrangements presence were analyzed. In the IMA samples remaining negative for these driver oncogenes, an NRG1 rearrangement detection was performed by FISH. A driver oncogene was identified in 14/25 IMA, namely 12 KRAS mutations (48%), one ROS1 rearrangement (4%), and one ALK rearrangement (4%). The detection of NRG1 rearrangement by FISH was conducted in the 11 pan‐negative IMA. One sample was NRG1FISH‐positive and 100% of the tumor nuclei analyzed were positive. This NRG1‐positive patient was a 61‐year‐old nonsmoking woman of Vietnamese ethnicity and was the sole patient of Asian ethnicity of the cohort. She died 6 months after the diagnosis with a pulmonary multifocal disease. NRG1FISH detection should be considered in patients with IMA pan‐negative for known driver oncogenes. These results might suggest that NRG1 fusion is more frequent in IMA from Asian patient. Larger studies are needed.