Circulating tumor DNA in advanced non-small-cell lung cancer patients with HIV is associated with shorter overall survival: Results from a Phase II trial (IFCT-1001 CHIVA).

INTRODUCTION: HIV is an exclusion criterion for most lung cancer (LC) trials, however LC is the most common non-AIDS-defined malignancy in people living with HIV (PLHIV), poorer prognosis than the general population. Circulating tumor DNA (ctDNA) was a prognostic marker in LC patients from the general population. This study assessed ctDNA's prognostic value in PLHIV from a dedicated phase II trial. METHODS: Overall, 61 PLHIV with advanced non-squamous non-small-cell lung cancer (NSCLC) participated in the IFCT Phase II trial evaluating first-line four-cycle carboplatin (Ca) AUC5 pemetrexed (P) 500 mg/m2 induction therapy every 3 weeks, followed by P maintenance therapy. Blood samples collected before treatment were analyzed to detect ctDNA using ultra-deep targeted next-generation-sequencing (NGS). RESULTS: Appropriate samples were available from 55 PLVIH and analyzed for ctDNA detection. Including 42 males (76.4 %), 52.9 years median age, 51 smokers (92.7 %), five with non-squamous NSCLC Stage III (9%), 50 Stage IV (91 %), and performance status (PS) 0-2. ctDNA was detected in 35 patients (64 %), 22 with high and 13 with low ctDNA levels. Overall, 77 % were positive for TP53, 29 % for KRAS, and 11 % for STK11 mutations, more than one alteration was detected in 43 % of samples. Multivariate analysis showed that positive ctDNA was significantly associated with shorter PFS (HR, 4.31, 95 %CI: 2.06-8.99, p < 0.0001), and shorter OS (HR, 3.52, 95 %CI: 1.72-7.19, p < 0.001). Moreover, OS was significantly longer for patients with low ctDNA levels at diagnosis as compared to high (p = 0.01). CONCLUSION: We show that ctDNA detection using ultra-deep NGS is an independent prognostic factor in PLHIV with advanced NSCLC.

Efficacy of Immune Checkpoint Inhibitors in Lung Sarcomatoid Carcinoma.

INTRODUCTION: Immune checkpoint inhibitors (ICIs) have improved cancer prognosis but have not been evaluated specifically in sarcomatoid carcinoma (SC), a rare lung cancer subtype with poor prognosis. As such, our study sought to retrospectively assess the efficacy of ICI in SC. METHODS: All consecutive patients with centrally confirmed SC treated using ICI as a second-line treatment or beyond between 2011 and 2017 were enrolled. Programmed death-ligand 1 (PD-L1) tumor expression was assessed using immunohistochemistry (SP263 clone) and the tumor mutational burden (TMB) with the Foundation One panel. TMB was considered high if it was greater than or equal to 10 mutations per megabase. RESULTS: Overall, 37 patients with SC were evaluated, predominantly men (73%) with a median age of 63.2 years (36.8-79.7) and who were current or former smokers (94.6%). Immunotherapy (nivolumab, 86.5% of cases) was given as a second-line treatment in 54% of the patients and as third-line treatment or beyond in 46% of the patients. The objective response rate was 40.5% and disease control rate was 64.8%, regardless of PD-L1 status. Median overall survival was 12.7 months (range: 0.3-45.7). One-third of patients exhibited early progression. The median PD-L1 expression was 70% (0-100). There was a trend toward higher PD-L1 expression in responsive diseases, with an objective response rate of 58.8% in patients with PD-L1+ and 0% in the one patient with PD-L1- (p = 0.44). The median TMB was 18 (4-39) mutations per megabase, with 87.5% of the cases displaying a high TMB. There was a trend toward higher TMB in responders versus stable or progressive diseases (p = 0.2). CONCLUSIONS: Patients with SC exhibited high response rates and prolonged overall survival under ICI treatment. These data support the prospective investigation of ICI in patients with SC who are under first-line treatment.

Relevance of Detection of Mechanisms of Resistance to ALK Inhibitors in ALK-Rearranged NSCLC in Routine Practice.

BACKGROUND: Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) have shown efficacy in the treatment of ALK-rearranged non-small-cell lung cancer (NSCLC), but the disease eventually progresses in all patients. In many cases, resistance to ALK TKIs arises through ALK mutations. Although clinical and biological data suggest variations in TKI efficacy according to the mechanism of resistance, ALK mutations are still rarely investigated in routine practice. MATERIALS AND METHODS: We performed a retrospective multicentric study with an aim to determine the frequency and clinical relevance of ALK alterations detected using targeted next-generation sequencing in patients with advanced ALK-rearranged NSCLC after progression during an ALK TKI treatment. Data on clinical, pathological, and molecular characteristics and patient outcomes were collected. RESULTS: We identified 23 patients with advanced ALK-rearranged NSCLC who, between January 2012 and May 2017, had undergone at least 1 repeat biopsy at progression during an ALK TKI treatment. A resistance mechanism was identified in 9 of the 23 patients (39%). The anomalies involved included 9 ALK mutations in 8 patients and one ALK amplification. The ALK mutation rate was 15% after failure of a first ALK TKI and 33% after failure of 2 ALK TKI treatments. Five of 7 patients who received a different ALK TKI after detection of an ALK mutation achieved an objective response. All of the patients who received a TKI presumed to act on the detected ALK mutant achieved disease control. CONCLUSION: Targeted next-generation sequencing is suitable for detecting ALK resistance mutations in ALK-rearranged NSCLC patients in routine practice. It might help select the best treatment at the time of disease progression during treatment with an ALK TKI.

Calpain 1 in bronchoalveolar lavage fluid is associated with poor prognosis in lepidic predominant pulmonary adenocarcinoma.

Calpain 1 is a proinflammatory calcium-activated cysteine protease, which can be partly externalized. Extracellular calpains limit inflammatory processes and promote tissue repair, through cell proliferation and migration. Toll like receptor (TLR) 2 has been identified as a target of extracellular calpains in lymphocytes. The aim was to investigate the externalization of calpain 1 and the release of soluble TLR2 during tumor progression of pulmonary lepidic predominant adenocarcinoma (LPA). Extracellular calpain 1, soluble fragment of TLR2 and cytokines were analyzed by ELISA in bronchoalveolar lavage fluid (BALF) supernatants from patients with LPA (n=68). Source of calpain was analyzed by immunohistochemistry and soluble TLR2 by flow cytometry on polymorphonuclear neutrophils (PMN) and human lung cancer cell lines. Extracellular calpain 1, secreted by tumor cells, was associated to tumor progression, neutrophilic inflammation, with a poor prognostic factor on survival (P=0.003). TLR2 was expressed on PMN and tumor cells and decreased after calpain exposure. Soluble fragment of TLR2 in BALF supernatants was correlated to the extracellular calpain 1 concentration (r=0.624; P<0.001), and its high level was associated with tumor progression and a pro-inflammatory environment. Extracellular calpain 1 secreted by tumor cells, could participate in inflammatory microenvironment and tumor progression through TLR2 in LPA.

c-MET Overexpression as a Poor Predictor of MET Amplifications or Exon 14 Mutations in Lung Sarcomatoid Carcinomas.

INTRODUCTION: MNNG HOS transforming gene (MET) abnormalities such as amplification and exon 14 mutations may be responsive to targeted therapies. They are prevalent in lung sarcomatoid carcinomas (LSCs) and must be diagnosed as efficiently as possible. Hypothetically, c-MET overexpression by immunohistochemistry (IHC) may prove effective as a screening test for MET abnormalities. METHODS: Tissue samples were obtained from consecutive patients with a resected LSC in four oncologic centers. IHC was performed using the SP44 antibody (Ventana, Tucson, Arizona) and evaluated using the MetMab score and H-score. Fluorescence in situ hybridization was applied with the dual color probe set from Zytovision (Clinisciences, Nanterre, France). True MET amplification was diagnosed when MET gene copy number was 5 or greater and the ratio between MET gene copy number and chromosome 7 number was greater than 2. All MET exon 14 alterations including those affecting splice sites occurring within splice donor and acceptor sites were detected in the routine molecular testing on genetic platforms. RESULTS: A total of 81 LSCs were included. Fourteen (17%) exhibited positive IHC using the MetMab score and 15 (18.5%) using the H-score. MET amplification was detected in six tumors (8.5%) and MET exon 14 mutation in five (6%). A weak positive correlation between IHC and fluorescence in situ hybridization was found (r = 0.27, p = 0.0001). IHC sensitivity for MET amplification was 50%, with a specificity of 83%, positive predictive value of 21.4%, and negative predictive value of 94.7%. IHC sensitivity for MET exon 14 mutations was 20%, with a specificity of 83%, positive predictive value of 7%, and negative predictive value of 94%. CONCLUSION: IHC is not a relevant screening tool for MET abnormalities in LSC.

Nonsmall cell lung cancer from HIV-infected patients expressed programmed cell death-ligand 1 with marked inflammatory infiltrates.

OBJECTIVE: Immunotherapies targeting the programmed cell death-1 (PD-1)/PD-ligand 1 (PD-L1) checkpoint improved prognosis in lung cancer. PD-1/PD-L1 status, however, has not been investigated in human immunodeficiency virus (HIV)-positive patients. This study assessed PD-L1 status and tumor immune-cell infiltration in nonsmall cell lung cancer (NSCLC) in HIV patients. METHODS: Consecutive HIV patients treated between 1996 and 2014 were enrolled. PD-L1 tumor expression was assessed using immunohistochemistry with two antibodies (clones 5H1 and E1L3N), and tumor immune-cell infiltration with CD3, CD4, CD8, CD20, CD163, and MPO. PD-L1 expression and immune infiltration results were compared with those of 54 NSCLCs from unknown HIV status patients. RESULTS: Thirty-four HIV-positive patients were evaluated: predominantly men (88.2%) (median age: 51.1 years) presenting stage IV (38.2%) adenocarcinomas (76.5%). The median blood CD4 count was 480 cells/µL (86-1120) and 64% exhibited undetectable viral load. The PD-L1 score (percentage of positive cells × intensity) was higher in HIV-positive than HIV-undetermined patients with the E1L3N clone [median (range) 0 (0-150) versus 0 (0-26.7), P = 0.047], yet not with the 5H1 clone [0 (0-120) versus 0 (0-26.7) P = 0.07, respectively]. PD-L1 expression frequency did not differ between both cohorts (18.7 versus 9.3% using E1L3N and 10 versus 5.6% using 5H1 clone, respectively). There were significantly greater cytotoxic T-cell (P < 0.001), B-lymphocyte (P = 0.005), and activated macrophage (P < 0.001) infiltrations in the HIV-positive patients, but no differences for CD4 T cells. CONCLUSION: Tumors in HIV-positive patients seem to express higher PD-L1 levels with increased immune infiltration, supporting their inclusion in clinical trials assessing immune checkpoint inhibitors.

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.

Lepidic predominant adenocarcinoma and invasive mucinous adenocarcinoma of the lung exhibit specific mucin expression in relation with oncogenic drivers.

OBJECTIVES: To evaluate MUC1, MUC2, MUC5B, MUC5AC, and MUC6 expression in invasive lepidic predominant adenocarcinoma (LPA) and invasive mucinous adenocarcinoma (IMA) of the lung, and the impact of oncogenic drivers. MATERIALS AND METHODS: MUC1, MUC2, MUC5B, MUC5AC, MUC6, TTF1 and Hnf4α immunohistochemistry was performed on surgical samples from 52 patients with IMA (n=25) or LPA (n=27). We searched for EGFR, KRAS, BRAF, and HER2 mutations and ALK, ROS1, and NRG1 rearrangements. RESULTS: MUC1, MUC2, MUC5B, MUC5AC, and MUC6 expression was detected in tumor cells in 77%, 2%, 63%, 36%, and 21% of cases, respectively. MUC1 was significantly more overexpressed in LPA. MUC5B, MUC5AC, and MUC6 were typically detected in goblet cells and overexpressed in IMA. Hnf4α-positive IMA (n=11) were TTF1-negative and typically did not expressed MUC1 and expressed MUC5AC and MUC6. Hnf4α-negative IMA (n=14) showed a reverse profile of mucins expression, with MUC1 expression and a lack of MUC5AC and MUC6 expression. EGFR-positive status was significantly associated with LPA, MUC1 expression, and no MUC5B, MUC5AC, or MUC6 expression. KRAS-positive status was significantly associated with IMA and MUC5B and MUC5AC expression. CONCLUSIONS: LPA and IMA exhibit specific mucin expression profiles, with MUC1 being associated with LPA, while MUC5B, MUC5AC, and MUC6 were associated with IMA. Hnf4α expression and EGFR and KRAS mutations may play a role in mucin expression profiles of these lung adenocarcinoma subtypes.

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.

Sarcomatoid lung carcinomas show high levels of programmed death ligand-1 (PD-L1) and strong immune-cell infiltration by TCD3 cells and macrophages.

OBJECTIVES: Pulmonary sarcomatoid carcinomas (SC) are rare tumors, associated with worse prognosis and resistant to platinum-based regimens. Therapies targeting the PD-1/PD-L1 pathway are an emerging treatment for lung cancer. By characterizing intra-tumoral immune infiltration and evaluating PD-L1 expression, it could be possible to predict the efficacy of these new treatments. MATERIALS AND METHODS: From 1997 to 2013, data from all patients with SC who underwent lung resection was collected. Tumor-immune infiltration and PD-L1 expression were studied by immunochemistry tests, analyzing CD3 (clone SP7), CD4 (clone 1F6), CD8 (clone C8/144b), CD20 (clone L26), CD163 (clone 10D6), MPO (clone 59A5), and PD-L1 (clone 5H1). Results were compared to those of 54 NSCLC. RESULTS: In total, 75 SC were included. Forty (53%) SC expressed PD-L1 vs 11 NSCLC (20%) (p<0.0001). CD3+ tumor-infiltrating lymphocytes and CD163+ tumor-associated macrophages were more important in SC than in NSCLC (median 23% [17-30] of tumoral surface vs 17% [7-27], p=0.011 and 23% [17-30] vs 20% [13-23], p=0.002, respectively). In SC, the presence of Kirsten Ras (KRAS) mutations, blood vessel invasion, and TTF1+ positivity were associated with PDL1 expression. On multivariate analysis, only CD163+ macrophages and blood-vessel invasion were associated with tumoral PD-L1 expression. High levels of tumor-infiltrating lymphocytes (CD3+ or CD4+ and not CD8+) constituted a factor of good prognosis on survival. Interestingly, PD-L1 expression distinguishes subpopulations within tumor-infiltrating lymphocytes (CD3+ or CD4+) with different prognosis CONCLUSIONS: PD-L1 expression was higher in SC than in NSCLC as well as immune-cell infiltration by TCD3 cells and macrophages. This suggests that targeting the PD-1/PD-L1 pathway could represent a new potential therapy.

Sonic Hedgehog Pathway Activation Is Associated With Resistance to Platinum-Based Chemotherapy in Advanced Non-Small-Cell Lung Carcinoma.

INTRODUCTION: Chemoresistance is a major challenge in the treatment of advanced non-small-cell lung cancer (NSCLC). Because the Sonic hedgehog (Shh) pathway is reactivated in NSCLC, we investigated an association between chemoresistance and Shh activation. PATIENTS AND METHODS: From a cohort of 178 patients with advanced NSCLC treated with platinum-based chemotherapy as first-line treatment, we selected all surgical tumor samples at diagnosis (n = 36). Shh activation was evaluated through Gli1 and Gli2 expression using immunohistochemistry (quantitative score). In vitro treatment studies with cisplatin or vismodegib (Shh pathway inhibitor), or both, were performed on NSCLC cell lines (H322 and A549) and primary cultures from patients with sarcomatoid carcinoma (n = 4). RESULTS: Of the 36 patients, 12 had NSCLC refractory to chemotherapy (R-patients, 33.3%) and 24 had controlled disease (C-patients). Gli1 expression did not differ between the R- and C-patients (P = .35). Gli2 expression was more often positive in the R-patients (41.7% vs. 8.3%; P = .02). Progression-free survival (PFS) and overall survival (OS) in patients with a Gli2-positive score was 2.1 and 8.0 months, respectively, compared with 6.7 and 18.0 months for patients with a Gli2-negative score (P = .03 and P = .002, respectively). On multivariate analysis, the Gli2 score correlated independently with PFS (hazard ratio [HR], 2.64; 95% confidence interval [CI], 1.05-6.63; P = .04) and OS (HR, 4.36; 95% CI, 1.67-11.36; P = .003). The sarcomatoid carcinoma cell lines were more resistant to cisplatin than were the H838 and A549 cell lines. The cisplatin-vismodegib combination displayed a synergistic cytotoxic effect in the most chemoresistant cells in vitro. CONCLUSION: The Shh pathway is associated with resistance to platinum-based chemotherapy in NSCLC.

VEGF neutralizing aerosol therapy in primary pulmonary adenocarcinoma with K-ras activating-mutations.

K-ras mutations promote angiogenesis in lung cancer and contribute to the drug resistance of cancer cells. It is not clear whether K-ras mutated adenocarcinomas are sensitive to anti-angiogenic therapy with monoclonal antibodies (mAbs) that target vascular endothelial growth factor (VEGF). Anti-angiogenic mAbs are usually delivered systemically, but only a small proportion reaches the lung after intravenous injection. We investigated the relevance of a non-invasive pulmonary route for the delivery of anti-VEGF mAbs in the mouse K-ras(LA1) model. We found that pulmonary delivery of these mAbs significantly reduced the number of tumor lesions and inhibited malignant progression. The antitumor effect involves the VEGFR2-dependent inhibition of blood vessel growth, which impairs tumor proliferation. Pharmacokinetic analysis of aerosolized anti-VEGF showed its low rate of passage into the bloodstream, suggesting that this delivery route is associated with reduced systemic side effects. Our findings highlight the value of the aerosol route for administration of anti-angiogenic mAbs in pulmonary adenocarcinoma with K-ras activating-mutations.

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.

Factors associated with early progression of non-small-cell lung cancer treated by epidermal growth factor receptor tyrosine-kinase inhibitors.

Epidermal growth factor receptor tyrosine-kinase inhibitors (EGFR-TKI) are a therapeutic option as second-line therapy in non-small-cell lung carcinoma (NSCLC), regardless of the EGFR gene status. Identifying patients with early progression during EGFR-TKI treatment will help clinicians to choose the best regimen, TKI or chemotherapy. From a prospective database, all patients treated with gefitinib or erlotinib between 2001 and 2010 were retrospectively reviewed. Patients were classified into two groups according to their tumor response by RECIST after 45 days of treatment, progressive disease (PD) or controlled disease (CD). Two hundred and sixty-eight patients were treated with EGFR-TKI, among whom 239 were classified as PD (n = 75) and CD (n = 164). Median overall survival was 77 days (95% CI 61-109) for PD and 385 days (95% CI 267-481) for CD. Patients with PD were of younger age (P = 0.004) and more frequently current smokers (P = 0.001) had more frequently a performance status ≥2 (P = 0.012), a weight loss ≥10% (P = 0.025), a shorter time since diagnosis (P < 0.0001), a pathological classification as non-otherwise-specified NSCLC (P = 0.01), and the presence of abdominal metastases (P = 0.008). In multivariate analysis, abdominal metastases were the only factor associated with early progression (odds ratio (OR) 2.17, 95% CI [1.12-4.19]; P = 0.021). Wild-type EGFR versus mutated EGFR was associated with early progression. The presence of abdominal metastasis was independently associated with early progression in metastatic NSCLC receiving EGFR-TKI.

The impact of intracytoplasmic mucin in lung adenocarcinoma with pneumonic radiological presentation.

OBJECTIVES: This study investigated the clinical and prognostic impact of intracytoplasmic mucin in lung adenocarcinoma with "pneumonic" radiological presentation, formerly known as bronchioloalveolar carcinoma (BAC). PATIENTS AND METHODS: Between 1986 and 2011, clinical and pathological data from 120 consecutive patients with lung adenocarcinoma with "pneumonic" radiological presentation were reviewed. Intracytoplasmic mucin was assessed using a diastase-resistant periodic acid-Schiff staining. The presence of EGFR or K-Ras mutations and ALK rearrangement were determined in surgical samples. RESULTS: The two predominant histological patterns were invasive mucinous adenocarcinoma (40%) and lepidic predominant adenocarcinoma (32%). Intracytoplasmic mucin was detected in 71 patients (59.2%) who were more likely to be non-smokers (p=0.04) and have bronchorrhea (p=0.006), crepitant rales (p=0.02), or neutrophil alveolitis (p=0.0004). In mucin-producing tumors, EGFR mutation was not detected, K-Ras mutations and ALK rearrangement were present in 32% and 3% of cases, respectively. In non-mucin-producing tumors, EGFR and K-Ras mutations were detected in 17% and 10% of cases, respectively, no ALK rearrangement was detected. In univariate analysis, performance status>0, crepitant rales, bronchorrhea, neutrophil alveolitis, bilateral extension, intracytoplasmic mucin and no surgery were associated with worse survival. In multivariate analyses, intracytoplasmic mucin, neutrophil alveolitis, and no surgery were independent factors for worse survival. CONCLUSION: Intracytoplasmic mucin is associated with specific clinical characteristics and is an independent factor for worse survival in lung adenocarcinoma formerly known as BAC.

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-ras(LA1) ), with and without TLR9 inactivation (K-ras(LA1) TLR9(-/-) and K-ras(LA1) TLR9(+/+) , respectively). TLR9 was functionally expressed only in mononuclear cells of K-ras(LA1) TLR9(+/+) mice. These mice had significantly worse survival and a higher tumor burden than K-ras(LA1) TLR9(-/-) 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-ras(LA1) TLR9(+/+) mice were characterized by an angiogenic phenotype with higher concentrations of vascular endothelial growth factor (VEGF) and higher microvessel density than from K-ras(LA1) TLR9(-/-) mice. LKR13 cells, an ADC cell line derived from K-ras(LA1) 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.

Specific targeting of caspase-9/PP2A interaction as potential new anti-cancer therapy.

PURPOSE: PP2A is a serine/threonine phosphatase critical to physiological processes, including apoptosis. Cell penetrating peptides are molecules that can translocate into cells without causing membrane damage. Our goal was to develop cell-penetrating fusion peptides specifically designed to disrupt the caspase-9/PP2A interaction and evaluate their therapeutic potential in vitro and in vivo. EXPERIMENTAL DESIGN: We generated a peptide containing a penetrating sequence associated to the interaction motif between human caspase-9 and PP2A (DPT-C9h), in order to target their association. Using tumour cell lines, primary human cells and primary human breast cancer (BC) xenografts, we investigated the capacity of DPT-C9h to provoke apoptosis in vitro and inhibition of tumour growth (TGI) in vivo. DPT-C9h was intraperitoneally administered at doses from 1 to 25 mg/kg/day for 5 weeks. Relative Tumour Volume (RTV) was calculated. RESULTS: We demonstrated that DPT-C9h specifically target caspase-9/PP2A interaction in vitro and in vivo and induced caspase-9-dependent apoptosis in cancer cell lines. DPT-C9h also induced significant TGI in BC xenografts models. The mouse-specific peptide DPT-C9 also induced TGI in lung (K-Ras model) and breast cancer (PyMT) models. DPT-C9h has a specific effect on transformed B cells isolated from chronic lymphocytic leukemia patients without any effect on primary healthy cells. Finally, neither toxicity nor immunogenic responses were observed. CONCLUSION: Using the cell-penetrating peptides blocking caspase-9/PP2A interactions, we have demonstrated that DPT-C9h had a strong therapeutic effect in vitro and in vivo in mouse models of tumour progression.

Insulin-like growth factor-1 receptor inhibition overcomes gefitinib resistance in mucinous lung adenocarcinoma.

The appropriate selection of patients is a major challenge in the treatment of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). Prospective trials in adenocarcinoma demonstrated that the mucinous subtype presents a poorer outcome under EGFR-TKI treatment than the non-mucinous subtype. Our aim was to determine the molecular characteristics associated with resistance to EGFR-TKIs in mucinous and non-mucinous adenocarcinoma. Eighty adenocarcinoma samples, including 34 tumours from patients treated with gefitinib in a phase II clinical trial (IFCT0401), were classified as mucinous (n = 32) or non-mucinous (n = 48) adenocarcinoma. We demonstrated that four biological markers were differentially expressed between the two subtypes: mucinous tumours that overexpressed IGF1R (p < 0.0001) and amphiregulin (p = 0.004) with a tendency for more frequent KRAS mutations, in contrast to non-mucinous tumours that overexpressed EGFR (p < 0.0001) and TTF-1 (p < 0.0001) with more frequent EGFR mutations (p = 0.037). Higher IGF1R (p = 0.02) and lower TTF-1 (p = 0.02) expression was associated with disease progression under gefitinib treatment. We observed in vitro cross-talk between EGFR and IGF1R signalling pathways in gefitinib-resistant H358 mucinous cells. Anti-amphiregulin siRNAs and anti-IGF1R treatments sensitized the H358 cells to gefitinib-induced apoptosis with additive effects, suggesting that these treatments could overcome the resistance of mucinous tumours to EGFR-TKIs, including those with KRAS mutation. Our results highlighted that mucinous and non-mucinous adenocarcinoma subtypes are different entities with different therapeutic responses to EGFR-TKIs. These data will foster the development of therapeutic strategies for treating adenocarcinoma with mucinous component.

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.

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 alpha/tumor necrosis factor alpha receptor inhibitor, interleukin-1alpha /interleukin-1alpha 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.