Purpose: We aimed to maximize the performance of detecting genetic alterations in lung cancer using high-throughput sequencing for patient-derived xenografts (PDXs). Experimental Design: We undertook an integrated RNA and whole-exome sequencing of 14 PDXs. We focused on the genetic and functional analysis of β2-microglobulin (B2M), a component of the HLA class-I complex. Results: We identified alterations in genes involved in various functions, such as B2M involved in immunosurveillance. We extended the mutational analysis of B2M to about 230 lung cancers. Five percent of the lung cancers carried somatic mutations, most of which impaired the correct formation of the HLA-I complex. We also report that genes such as CALR, PDIA3, and TAP1, which are involved in the maturation of the HLA-I complex, are altered in lung cancer. By gene expression microarrays, we observed that restitution of B2M in lung cancer cells upregulated targets of IFNα/IFNγ. Furthermore, one third of the lung cancers lacked the HLA-I complex, which was associated with lower cytotoxic CD8⁺ lymphocyte infiltration. The levels of B2M and HLA-I proteins correlated with those of PD-L1. Finally, a deficiency in HLA-I complex and CD8⁺ infiltration tended to correlate with reduced survival of patients with lung cancer treated with anti-PD-1/anti-PD-L1. Conclusions: Here, we report recurrent inactivation of B2M in lung cancer. These observations, coupled with the mutations found at CALR, PDIA3, and TAP1, and the downregulation of the HLA-I complex, indicate that an abnormal immunosurveillance axis contributes to lung cancer development. Finally, our observations suggest that an impaired HLA-I complex affects the response to anti-PD-1/anti-PD-L1 therapies. Clin Cancer Res; 1–11. ©2016 AACR.

Background: Circulating DNA is observed at higher concentrations in patients with lung cancer than in controls. Qualitative and quantitative analysis of circulating DNA is a promising noninvasive tool. Our aim was to prospectively study the association between the catalytic subunit of telomerase (human telomerase reverse transcriptase [hTERT]) in plasma and clinical variables and survival in a large-scale non-small cell lung cancer (NSCLC) study. Methods: Four hundred forty-six patients with stages IIIB and IV NSCLC with a median follow-up of 9.7 months (range, 0.5–45) were analyzed. Blood samples were collected before therapy start (cisplatin/docetaxel). Quantification of baseline circulating DNA was determined as the amount of free hTERT in plasma, by using real-time quantitative polymerase chain reaction. Results: Patients with hTERT ≤49.8 ng/ml (median value) had a median time to progression (TTP) of 6.3 months compared with 4.9 for hTERT more than 49.8 ng/ml (p = 0.001). Overall survival (OS) was significantly higher (10.9 versus 9.3 months) at lower hTERT levels (p = 0.012). When calculations were done using hTERT as continuous variable, we did not observe independent significant differences. Thus, there is an apparent discrepancy in p values when hTERT is considered as a continuous versus dichotomized variable. There was a tendency to differentiate median hTERT levels with respect to response rates (complete response + partial response: 33.1 versus stable disease + progressive disease: 50.7 ng/ml, p = 0.12), but other clinical variables such as age, gender, performance status, stage, histology, and number of metastatic locations were not associated with hTERT. In multivariate analysis, hTERT was an independent prognostic variable for both TTP (hazard ratio: 1.44, p < 0.001) and OS (hazard ratio: 1.33, p = 0.007). Conclusions: In advanced NSCLC, high pretreatment circulating hTERT level is an independent poor prognostic marker for TTP and OS. Circulating DNA is a noninvasive marker, which may help to improve the prognostic profile of these patients.

Although EGFR mutant–selective tyrosine kinase inhibitors (TKI) are clinically effective, acquired resistance can occur by reactivating ERK. We show using in vitro models of acquired EGFR TKI resistance with a mesenchymal phenotype that CXCR7, an atypical G protein-coupled receptor, activates the MAPK–ERK pathway via β-arrestin. Depletion of CXCR7 inhibited the MAPK pathway, significantly attenuated EGFR TKI resistance, and resulted in mesenchymal-to-epithelial transition. CXCR7 overexpression was essential in reactivation of ERK1/2 for the generation of EGFR TKI–resistant persister cells. Many patients with non–small cell lung cancer (NSCLC) harboring an EGFR kinase domain mutation, who progressed on EGFR inhibitors, demonstrated increased CXCR7 expression. These data suggest that CXCR7 inhibition could considerably delay and prevent the emergence of acquired EGFR TKI resistance in EGFR-mutant NSCLC. Significance: Increased expression of the chemokine receptor CXCR7 constitutes a mechanism of resistance to EGFR TKI in patients with non–small cell lung cancer through reactivation of ERK signaling.

Background: Occult lymph node (LN) metastases are clinically relevant and confer a worse prognosis in non-small-cell lung cancer (NSCLC) patients. Current staging methods are unable to identify patients with poor outcome. Their detection requires both a more sensitive and specific technique. We aimed to assess the role of messenger RNA expression in pathologically negative LNs (pN0) of stage I NSCLC patients as markers of occult micrometastases and to correlate the results with local or distant tumor recurrence and survival. Patients and methods: Potential molecular markers were evaluated in 344 LNs and 38 tumors by quantitative real-time RT-PCR. Only CEACAM5 and PLUNC showed high expression in lung tumor tissue and null expression in RNA from benign LNs. Results: Thirteen per cent of the LNs were positive for CEACAM5 and 16% for PLUNC. Eight of 38 NSCLC patients had positive expression in pN2 nodes by CEACAM5 and/or PLUNC and disease-free survival (P = 0.028) and overall survival time was significantly worse in these patients compared with those with negative expression (P = 0.0083). Conclusions: Quantitative real-time RT-PCR of CEACAM5 and PLUNC can estimate the presence of micrometastatic cells in LNs with greater precision than current staging method used for assessing tumor recurrence risk.

BACKGROUND.: The objective of this study was to investigate the diagnostic value of methylation profiles for discrimination between malignant and benign pleural effusions. A secondary objective was to examine the concordance of methylation in samples of serum and pleural fluid.METHODS.: The authors used methylation‐specific polymerase chain reaction (MSP) analysis to examine the promoter methylation status of 4 genes in patients with pleural effusion: death‐associated protein kinase (DAPK), Ras association domain family 1A (RASSF1A), retinoic acid receptor β (RARβ), and p16/INK4a. Pleural effusions were collected from 87 patients who had their diagnoses confirmed on cytologic and/or histologic examinations and clinical evolution. Pleural effusions were classified as malignant (n = 53 patients) or benign (n = 34 patients).RESULTS.: Methylation was detected in serum from 45.3% of patients with malignant pleural effusions and from 0% of patients with benign pleural effusions, and it was detected in pleural fluid samples from 58.5% of patients with malignant pleural effusions and from 0% of patients with benign pleural effusions (P = .001). The sensitivity of MSP was greater than that of cytologic examination alone (39.1%; P = .001). When MSP was used together with cytologic examination, sensitivity increased to 69.8% (P = .001).CONCLUSIONS.: Cell‐free methylated DNA in pleural fluid can be detected in patients with neoplastic malignancy in a single extraction by thoracocentesis. Adequate management of the extracted pleural fluid can provide a rapid and reliable diagnosis in patients with pleural effusions who have suspected malignancy. MSP, used together with cytologic examination, may obviate the need for other invasive diagnostic tests. Cancer 2006. © 2006 American Cancer Society.