Circulating tumor DNA and radiological tumor volume identify patients at risk for relapse with resected, early-stage non-small-cell lung cancer.

BACKGROUND: Predicting relapse and overall survival (OS) in early-stage non-small-cell lung cancer (NSCLC) patients remains challenging. Therefore, we hypothesized that detection of circulating tumor DNA (ctDNA) can identify patients with increased risk of relapse and that integrating radiological tumor volume measurement along with ctDNA detectability improves prediction of outcome. PATIENTS AND METHODS: We analyzed 366 serial plasma samples from 85 patients who underwent surgical resections and assessed ctDNA using a next-generation sequencing liquid biopsy assay, and measured tumor volume using a computed tomography-based three-dimensional annotation. RESULTS: Our results showed that patients with detectable ctDNA at baseline or after treatment and patients who did not clear ctDNA after treatment had a significantly worse clinical outcome. Integrating radiological analysis allowed the stratification in risk groups prognostic of clinical outcome as confirmed in an independent cohort of 32 patients. CONCLUSIONS: Our findings suggest ctDNA and radiological monitoring could be valuable tools for guiding follow-up care and treatment decisions for early-stage NSCLC patients.

Pneumonitis After Concurrent Chemoradiation and Immune Checkpoint Inhibition in Patients with Locally Advanced Non-small Cell Lung Cancer.

AIMS: Pneumonitis is a common and potentially deadly complication of combined chemoradiation and immune checkpoint inhibition (CRT-ICI) in patients with locally advanced non-small cell lung cancer (LA-NSCLC). In this study we sought to identify the risk factors for pneumonitis with CRT-ICI therapy in LA-NSCLC cases and determine its impact on survival. MATERIALS AND METHODS: We conducted a retrospective chart review of 140 patients with LA-NSCLC who underwent curative-intent CRT-ICI with durvalumab between 2018 and 2021. Pneumonitis was diagnosed by a multidisciplinary team of clinical experts. We used multivariable cause-specific hazard models to identify risk factors associated with grade ≥2 pneumonitis. We constructed multivariable Cox proportional hazard models to investigate the impact of pneumonitis on all-cause mortality. RESULTS: The median age of the cohort was 67 years; most patients were current or former smokers (86%). The cumulative incidence of grade ≥2 pneumonitis was 23%. Among survivors, 25/28 patients had persistent parenchymal scarring. In multivariable analyses, the mean lung dose (hazard ratio 1.14 per Gy, 95% confidence interval 1.03-1.25) and interstitial lung disease (hazard ratio 3.8, 95% confidence interval 1.3-11.0) increased the risk for pneumonitis. In adjusted models, grade ≥2 pneumonitis (hazard ratio 2.5, 95% confidence interval 1.0-6.2, P = 0.049) and high-grade (≥3) pneumonitis (hazard ratio 8.3, 95% confidence interval 3.0-23.0, P < 0.001) were associated with higher all-cause mortality. CONCLUSIONS: Risk factors for pneumonitis in LA-NSCLC patients undergoing CRT-ICI include the mean radiation dose to the lung and pre-treatment interstitial lung disease. Although most cases are not fatal, pneumonitis in this setting is associated with markedly increased mortality.

Ramucirumab plus erlotinib versus placebo plus erlotinib in previously untreated EGFR-mutated metastatic non-small-cell lung cancer (RELAY): exploratory analysis of next-generation sequencing results.

BACKGROUND: Ramucirumab plus erlotinib (RAM + ERL) demonstrated superior progression-free survival (PFS) over placebo + ERL (PBO + ERL) in the phase III RELAY study of patients with epidermal growth factor receptor (EGFR)-mutated metastatic non-small-cell lung cancer (EGFR+ mNSCLC; NCT02411448). Next-generation sequencing (NGS) was used to identify clinically relevant alterations in circulating tumor DNA (ctDNA) and explore their impact on treatment outcomes. PATIENTS AND METHODS: Eligible patients with EGFR+ mNSCLC were randomized 1 : 1 to ERL (150 mg/day) plus RAM (10 mg/kg)/PBO every 2 weeks. Liquid biopsies were to be prospectively collected at baseline, cycle 4 (C4), and postdiscontinuation follow-up. EGFR and co-occurring/treatment-emergent (TE) genomic alterations in ctDNA were analyzed using Guardant360 NGS platform. RESULTS: In those with valid baseline samples, detectable activating EGFR alterations in ctDNA (aEGFR+) were associated with shorter PFS [aEGFR+: 12.7 months (n = 255) versus aEGFR-: 22.0 months (n = 131); hazard ratio (HR) = 1.87, 95% confidence interval (CI) 1.42-2.51]. Irrespective of detectable/undetectable baseline aEGFR, RAM + ERL was associated with longer PFS versus PBO + ERL [aEGFR+: median PFS (mPFS) = 15.2 versus 11.1 months, HR = 0.63, 95% CI 0.46-0.85; aEGFR-: mPFS = 22.1 versus 19.2 months, HR = 0.80, 95% CI 0.49-1.30]. Baseline alterations co-occurring with aEGFR were identified in 69 genes, most commonly TP53 (43%), EGFR (other than aEGFR; 25%), and PIK3CA (10%). PFS was longer in RAM + ERL, irrespective of baseline co-occurring alterations. Clearance of baseline aEGFR by C4 was associated with longer PFS (mPFS = 14.1 versus 7.0 months, HR = 0.481, 95% CI 0.33-0.71). RAM + ERL improved PFS outcomes, irrespective of aEGFR mutation clearance. TE gene alterations were most commonly in EGFR [T790M (29%), other (19%)] and TP53 (16%). CONCLUSIONS: Baseline aEGFR alterations in ctDNA were associated with shorter mPFS. RAM + ERL was associated with improved PFS outcomes, irrespective of detectable/undetectable aEGFR, co-occurring baseline alterations, or aEGFR+ clearance by C4. aEGFR+ clearance by C4 was associated with improved PFS outcomes. Monitoring co-occurring alterations and aEGFR+ clearance may provide insights into mechanisms of EGFR tyrosine kinase inhibitor resistance and the patients who may benefit from intensified treatment schedules.

Molecular markers of metastatic disease in KRAS-mutant lung adenocarcinoma.

BACKGROUND: Prior studies characterized the association of molecular alterations with treatment-specific outcomes in KRAS-mutant (KRASMUT) lung adenocarcinoma (LUAD). Less is known about the prognostic role of molecular alterations and their associations with metastatic disease. PATIENTS AND METHODS: We analyzed clinicogenomic data from 1817 patients with KRASMUT LUAD sequenced at the Dana-Farber Cancer Institute (DFCI) and Memorial Sloan Kettering Cancer Center (MSKCC). Patients with metastatic (M1) and nonmetastatic (M0) disease were compared. Transcriptomic data from The Cancer Genome Atlas (TCGA) were investigated to characterize the biology of differential associations with clinical outcomes. Organ-specific metastasis was associated with overall survival (OS). RESULTS: KEAP1 (DFCI: OR = 2.3, q = 0.04; MSKCC: OR = 2.2, q = 0.00027) and SMARCA4 mutations (DFCI: OR = 2.5, q = 0.06; MSKCC: OR = 2.6, q = 0.0021) were enriched in M1 versus M0 tumors. On integrative modeling, NRF2 activation was the genomic feature most associated with OS. KEAP1 mutations were enriched in M1 versus M0 tumors independent of STK11 status (KEAP1MUT/STK11WT: DFCI OR = 3.0, P = 0.0064; MSKCC OR = 2.0, P = 0.041; KEAP1MUT/STK11MUT: DFCI OR = 2.3, P = 0.0063; MSKCC OR = 2.5, P = 3.6 × 10-05); STK11 mutations without KEAP1 loss were not associated with stage (KEAP1WT/STK11MUT: DFCI OR = 0.97, P = 1.0; MSKCC OR = 1.2, P = 0.33) or outcome. KEAP1/KRAS-mutated tumors with and without STK11 mutations exhibited high functional STK11 loss. The negative effects of KEAP1 were compounded in the presence of bone (HR = 2.3, P = 4.4 × 10-14) and negated in the presence of lymph node metastasis (HR = 1.0, P = 0.91). CONCLUSIONS: Mutations in KEAP1 and SMARCA4, but not STK11, were associated with metastatic disease and poor OS. Functional STK11 loss, however, may contribute to poor outcomes in KEAP1MUT tumors. Integrating molecular data with clinical and metastatic-site annotations can more accurately risk stratify patients.

Dissecting the clinicopathologic, genomic, and immunophenotypic correlates of KRASG12D-mutated non-small-cell lung cancer.

BACKGROUND: Allele-specific KRAS inhibitors are an emerging class of cancer therapies. KRAS-mutant (KRASMUT) non-small-cell lung cancers (NSCLCs) exhibit heterogeneous outcomes, driven by differences in underlying biology shaped by co-mutations. In contrast to KRASG12C NSCLC, KRASG12D NSCLC is associated with low/never-smoking status and is largely uncharacterized. PATIENTS AND METHODS: Clinicopathologic and genomic information were collected from patients with NSCLCs harboring a KRAS mutation at the Dana-Farber Cancer Institute (DFCI), Memorial Sloan Kettering Cancer Center, MD Anderson Cancer Center, and Imperial College of London. Multiplexed immunofluorescence for CK7, programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), Foxp3, and CD8 was carried out on a subset of samples with available tissue at the DFCI. Clinical outcomes to PD-(L)1 inhibition ± chemotherapy were analyzed according to KRAS mutation subtype. RESULTS: Of 2327 patients with KRAS-mutated (KRASMUT) NSCLC, 15% (n = 354) harbored KRASG12D. Compared to KRASnon-G12D NSCLC, KRASG12D NSCLC had a lower pack-year (py) smoking history (median 22.5 py versus 30.0 py, P < 0.0001) and was enriched in never smokers (22% versus 5%, P < 0.0001). KRASG12D had lower PD-L1 tumor proportion score (TPS) (median 1% versus 5%, P < 0.01) and lower tumor mutation burden (TMB) compared to KRASnon-G12D (median 8.4 versus 9.9 mt/Mb, P < 0.0001). Of the samples which underwent multiplexed immunofluorescence, KRASG12D had lower intratumoral and total CD8+PD1+ T cells (P < 0.05). Among 850 patients with advanced KRASMUT NSCLC who received PD-(L)1-based therapies, KRASG12D was associated with a worse objective response rate (ORR) (15.8% versus 28.4%, P = 0.03), progression-free survival (PFS) [hazard ratio (HR) 1.51, 95% confidence interval (CI) 1.45-2.00, P = 0.003], and overall survival (OS; HR 1.45, 1.05-1.99, P = 0.02) to PD-(L)1 inhibition alone but not to chemo-immunotherapy combinations [ORR 30.6% versus 35.7%, P = 0.51; PFS HR 1.28 (95%CI 0.92-1.77), P = 0.13; OS HR 1.36 (95%CI 0.95-1.96), P = 0.09] compared to KRASnon-G12D. CONCLUSIONS: KRASG12D lung cancers harbor distinct clinical, genomic, and immunologic features compared to other KRAS-mutated lung cancers and worse outcomes to PD-(L)1 blockade. Drug development for KRASG12D lung cancers will have to take these differences into account.

Distinct tumor-infiltrating lymphocyte landscapes are associated with clinical outcomes in localized non-small-cell lung cancer.

BACKGROUND: Despite the importance of tumor-infiltrating T lymphocytes (TILs) in cancer biology, the relationship between TIL phenotypes and their prognostic relevance for localized non-small-cell lung cancer (NSCLC) has not been well established. PATIENTS AND METHODS: Fresh tumor and normal adjacent tissue was prospectively collected from 150 patients with localized NSCLC. Tissue was comprehensively characterized by high-dimensional flow cytometry of TILs integrated with immunogenomic data from multiplex immunofluorescence, T-cell receptor sequencing, exome sequencing, RNA sequencing, targeted proteomics, and clinicopathologic features. RESULTS: While neither the magnitude of TIL infiltration nor specific TIL subsets were significantly prognostic alone, the integration of high-dimensional flow cytometry data identified two major immunotypes (IM1 and IM2) that were predictive of recurrence-free survival independent of clinical characteristics. IM2 was associated with poor prognosis and characterized by the presence of proliferating TILs expressing cluster of differentiation 103, programmed cell death protein 1, T-cell immunoglobulin and mucin-domain containing protein 3, and inducible T-cell costimulator. Conversely, IM1 was associated with good prognosis and differentiated by an abundance of CD8+ T cells expressing cytolytic enzymes, CD4+ T cells lacking the expression of inhibitory receptors, and increased levels of B-cell infiltrates and tertiary lymphoid structures. While increased B-cell infiltration was associated with good prognosis, the best prognosis was observed in patients with tumors exhibiting high levels of both B cells and T cells. These findings were validated in patient tumors from The Cancer Genome Atlas. CONCLUSIONS: Our study suggests that although the number of infiltrating T cells is not associated with patient survival, the nature of the infiltrating T cells, resolved in distinct TIL immunotypes, is prognostically relevant in NSCLC and may inform therapeutic approaches to clinical care.

Safety and activity of vandetanib in combination with everolimus in patients with advanced solid tumors: a phase I study.

BACKGROUND: Preclinical studies suggest that combining vandetanib (VAN), a multi-tyrosine kinase inhibitor of rearranged during transfection (RET) proto-oncogene, vascular endothelial growth factor receptor (VEGFR), and epidermal growth factor receptor (EGFR), with everolimus (EV), a mammalian target of rapamycin (mTOR) inhibitor, may improve antitumor activity. We determined the safety, maximum tolerated dose (MTD), recommended phase II dose (RP2D), and dose-limiting toxicities (DLTs) of VAN + EV in patients with advanced solid cancers and the effect of combination therapy on cancer cell proliferation and intracellular pathways. PATIENTS AND METHODS: Patients with refractory solid tumors were enrolled in a phase I dose-escalation trial testing VAN (100-300 mg orally daily) + EV (2.5-10 mg orally daily). Objective responses were evaluated using RECIST v1.1. RET mutant cancer cell lines were used in cell-based studies. RESULTS: Among 80 patients enrolled, 72 (90%) patients were evaluable: 7 achieved partial response (PR) (10%) and 37 had stable disease (SD) (51%; duration range: 1-27 cycles). Clinical benefit (SD or PR ≥ 6 months) was observed in 26 evaluable patients [36%, 95% confidence intervals (CI) (25% to 49%)]. In 80 patients, median overall survival (OS) was 10.5 months [95% CI (8.5-16.1)] and median progression-free survival (PFS) 4.1 months [95% CI (3.4-7.3)]. Six patients (7.5%) experienced DLTs and 20 (25%) required dose modifications. VAN + EV was safe, with fatigue, rash, diarrhea, and mucositis being the most common toxicities. In cell-based studies, combination therapy was superior to monotherapy at inhibiting cancer cell proliferation and intracellular signaling. CONCLUSIONS: The MTDs and RP2Ds of VAN + EV are 300 mg and 10 mg, respectively. VAN + EV combination is safe and active in refractory solid tumors. Further investigation is warranted in RET pathway aberrant tumors.

The value of interventional radiology in clinical trial teams: experience from the BATTLE lung cancer trials.

AIM: To report on the multidisciplinary approach, focusing specifically on the role of the interventional radiologist (IR), used to support the Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) and BATTLE-2 trials. MATERIALS AND METHODS: Patients who underwent percutaneous image-guided biopsy for the BATTLE and BATTLE-2 trials were reviewed. A radiology-based, three-point, lesion-scoring system was developed and used by two IRs. Lesions were given a score of 3 (most likely to yield sufficient material for biomarker analysis) if they met the following criteria: size >2 cm, solid mass, demonstrated imaging evidence of viability, and were technically easy to sample. Lesions not meeting all four criteria were scored 2 with the missing criteria noted as negative factors. Lesions considered to have risks that outweighed potential benefits receive a score of 1 and were not biopsied. Univariate and multivariate analyses were performed to evaluate the score's ability to predict successful yield for biomarker adequacy. RESULTS: A total of 555 biopsies were performed. The overall yield for analysis of the required biomarkers was 86.1% (478/555), and 84% (268/319) and 88.9% (210/236) for BATTLE and BATTLE-2, respectively (p=0.09). Lesions receiving a score of 3 were adequate for biomarker analysis in 89% of cases. Lesions receiving a score of 2 with more than two negative factors were adequate for molecular analysis in 69.2% (IR1, p=0.03) and 74% (IR2, p=0.04) of cases. The two IRs scored 78.4% of the lesions the same indicating moderate agreement (kappa=0.55; 95% confidence interval [CI]: 0.48, 0.61). CONCLUSIONS: IRs add value to clinical trial teams by optimising lesions selected for biopsy and biomarker analysis.

Clinical activity of programmed cell death 1 (PD-1) blockade in never, light, and heavy smokers with non-small-cell lung cancer and PD-L1 expression ≥50.

BACKGROUND: Immune checkpoint inhibitors (ICIs) are standard therapies for patients with advanced non-small-cell lung cancer (NSCLC) and a programmed death-ligand 1 (PD-L1) tumor proportion score (TPS) ≥50%. Tumor mutation burden (TMB) also predicts response to ICIs but is often not available in real time for decision making in the first-line setting. Smoking exposure can be a proxy for TMB in NSCLC. The impact of smoking status on efficacy of PD-1 blockade in NSCLC patients with PD-L1 TPS ≥50% has not been well defined. PATIENTS AND METHODS: To investigate the relationship between smoking and activity of ICIs in NSCLC, we retrospectively studied 315 patients with NSCLC and PD-L1 TPS ≥50% at five USA academic medical centers. Objective response rates (ORRs), progression-free survival (PFS), and duration of response (DOR) were compared between never (<100 lifetime cigarettes), light (≤10 pack-years), and heavy (>10 pack-years) smokers. A subset of patients underwent next-generation sequencing to estimate TMB. RESULTS: We identified 36 (11%) never, 42 (13%) light, and 237 (75%) heavy smokers with NSCLC and PD-L1 TPS ≥50% treated with ICIs. Objective responses were observed in 27%, 40%, and 40% of never, light, and heavy smokers, respectively (P = 0.180 never versus heavy; P = 1.000 light versus heavy). Median PFS and median DOR were numerically shorter in never and light smokers compared with heavy smokers (PFS 3.0 versus 4.0 versus 5.4 months; median DOR 6.9 versus 10.8 versus 17.8 months), but were not statistically different [PFS: hazard ratio (HR) 1.37, P = 0.135 and HR 1.24, P = 0.272; DOR: HR 1.92, P = 0.217 and HR 1.79, P = 0.141]. CONCLUSIONS: PD-(L)1 inhibitors are associated with antitumor activity in NSCLC with PD-L1 TPS ≥50% regardless of smoking status. Nevertheless, there is a signal of potentially decreased durability among never and light smokers that should be further evaluated. Distinct immunobiologic features may affect initial response versus durability of antitumor immunity to programmed cell death 1 (PD-1) blockade.

Outcomes to first-line pembrolizumab in patients with non-small-cell lung cancer and very high PD-L1 expression.

BACKGROUND: In non-small-cell lung cancers with programmed death-ligand 1 (PD-L1) expression on ≥50% of tumor cells, first-line treatment with the PD-1 inhibitor pembrolizumab improves survival compared with platinum-doublet chemotherapy. Whether higher PD-L1 levels within the expression range of 50%-100% predict for even greater benefit to pembrolizumab is currently unknown. PATIENTS AND METHODS: In this multicenter retrospective analysis, we analyzed the impact of PD-L1 expression levels on the overall response rate (ORR), median progression-free survival (mPFS), and median overall survival (mOS) in patients who received commercial pembrolizumab as first-line treatment of non-small-cell lung cancer (NSCLC) with a PD-L1 expression of ≥50% and negative for genomic alterations in the EGFR and ALK genes . RESULTS: Among 187 patients included in this analysis, the ORR was 44.4% [95% confidence interval (CI) 37.1% to 51.8%], the mPFS was 6.5 months (95% CI 4.5-8.5), and the mOS was not reached. The median PD-L1 expression level among patients who experienced a response to pembrolizumab was significantly higher than among patients with stable or progressive disease (90% versus 75%, P < 0.001). Compared with patients with PD-L1 expression of 50%-89% (N = 107), patients with an expression level of 90%-100% (N = 80) had a significantly higher ORR (60.0% versus 32.7%, P < 0.001), a significantly longer mPFS [14.5 versus 4.1 months, hazard ratio (HR) 0.50 (95% CI 0.33-0.74), P < 0.01], and a significantly longer mOS [not reached versus 15.9 months, HR 0.39 (95% CI 0.21-0.70), P = 0.002]. CONCLUSION: Among patients with NSCLC and PD-L1 expression of ≥50% treated with first-line pembrolizumab, clinical outcomes are significantly improved in NSCLCs with a PD-L1 expression of ≥90%. These findings have implications for treatment selection as well as for clinical trial interpretation and design.

A phase I dose-escalation and dose-expansion study of brontictuzumab in subjects with selected solid tumors.

Background: Brontictuzumab is a monoclonal antibody that targets Notch1 and inhibits pathway activation. The purpose of this first-in-human study was to determine the maximum tolerated dose (MTD), safety, pharmacokinetics, immunogenicity and preliminary efficacy of brontictuzumab in patients with solid tumors. Patients and methods: Subjects with selected refractory solid tumors were eligible. Brontictuzumab was administered intravenously at various dose levels and schedule during dose escalation, and at 1.5 mg/kg every 3 weeks (Q3W) during expansion. Evidence of Notch1 pathway activation as determined by an immunohistochemistry assay was required for entry in the expansion cohort. Adverse events were graded according to the NCI-CTCAE v 4.03. Efficacy was assessed by RECIST 1.1. Results: Forty-eight subjects enrolled (33 in dose escalation and 15 in the expansion phase). The MTD was 1.5 mg/kg Q3W. Dose-limiting toxicities were grade 3 diarrhea in two subjects and grade 3 fatigue in one subject. The most common drug-related adverse events of any grade were diarrhea (71%), fatigue (44%), nausea (40%), vomiting (21%), and AST increase (21%). Brontictuzumab exhibited nonlinear pharmacokinetics with dose-dependent terminal half-life ranging 1-4 days. Clinical benefit was seen in 6 of 36 (17%) assessable subjects: 2 had unconfirmed partial response (PR) and 4 subjects had prolonged (≥ 6 months) disease stabilization (SD). Both PRs and three prolonged SD occurred in adenoid cystic carcinoma (ACC) subjects with evidence of Notch1 pathway activation. Pharmacodynamic effects of brontictuzumab were seen in patients' blood and tumor. Conclusion: Brontictuzumab was well tolerated at the MTD. The main toxicity was diarrhea, an on-target effect of Notch1 inhibition. An efficacy signal was noted in subjects with ACC and Notch1 pathway activation. ClinicalTrials.gov identifier: NCT01778439.

Integrating cytokines and angiogenic factors and tumour bulk with selected clinical criteria improves determination of prognosis in advanced renal cell carcinoma.

BACKGROUND: In two clinical trials of the vascular endothelial growth factor (VEGF) receptor inhibitor pazopanib in advanced renal cell carcinoma (mRCC), we found interleukin-6 as predictive of pazopanib benefit. We evaluated the prognostic significance of candidate cytokines and angiogenic factors (CAFs) identified in that work relative to accepted clinical parameters. METHODS: Seven preselected plasma CAFs (interleukin-6, interleukin-8, osteopontin, VEGF, hepatocyte growth factor, tissue inhibitor of metalloproteinases (TIMP-1), and E-selectin) were measured using multiplex ELISA in plasma collected pretreatment from 343 mRCC patients participating in the phase 3 registration trial of pazopanib vs placebo (NCT00334282). Tumour burden (per sum of longest diameters (SLD)) and 10 other clinical factors were also analysed for association with overall survival (OS; based on initial treatment assignment). RESULTS: Osteopontin, interleukin-6, and TIMP-1 were independently associated with OS in multivariable analysis. A model combining the three CAFs and five clinical variables (including SLD) had higher prognostic accuracy than the International Metastatic Renal Cell Carcinoma Database Consortium criteria (concordance-index 0.75 vs 0.67, respectively), and distinguished two groups of patients within the original intermediate risk category. CONCLUSIONS: A prognostic model incorporating osteopontin, interleukin-6, TIMP-1, tumour burden, and selected clinical criteria increased prognostic accuracy for OS determination in mRCC patients.

MicroRNA-related genetic variants in iron regulatory genes, dietary iron intake, microRNAs and lung cancer risk.

Background: Genetic variations in MicroRNA (miRNA) binding sites may alter structural accessibility of miRNA binding sites to modulate risk of cancer. This large-scale integrative multistage study was aimed to evaluate the interplay of genetic variations in miRNA binding sites of iron regulatory pathway, dietary iron intake and lung cancer (LC) risk. Patients and methods: The interplay of genetic variant, dietary iron intake and LC risk was assessed in large-scale case-control study. Functional characterization of the validated SNP and analysis of target miRNAs were performed. Results: We found that the miRNA binding site SNP rs1062980 in 3' UTR of Iron-Responsive Element Binding protein 2 gene (IREB2) was associated with a 14% reduced LC risk (P value = 4.9×10 - 9). Comparing to AA genotype, GG genotype was associated with a 27% reduced LC risk. This association was evident in males and ever-smokers but not in females and never-smokers. Higher level of dietary iron intake was significantly associated with 39% reduced LC risk (P value = 2.0×10 - 8). This association was only present in individuals with AG + AA genotypes with a 46% reduced risk (P value = 1.0×10 - 10), but not in GG genotype. The eQTL-analysis showed that rs1062980 significantly alters IREB2 expression level. Rs1062980 is predicted to alter a miR-29 binding site on IREB2 and indeed the expression of miR-29 is inversely correlated with IREB2 expression. Further, we found that higher circulating miR-29a level was significantly associated with 78% increased LC risk. Conclusion: The miRNA binding site SNP rs1062980 in iron regulatory pathway, which may alter the expression of IREB2 potentially through modulating the binding of miR-29a, together with dietary iron intake may modify risk of LC both individually and jointly. These discoveries reveal novel pathway for understanding lung cancer tumorigenesis and risk stratification.

Phase II trial of everolimus and erlotinib in patients with platinum-resistant recurrent and/or metastatic head and neck squamous cell carcinoma.

BACKGROUND: Enhanced phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is one of the key adaptive changes accounting for epidermal growth factor receptor (EGFR) inhibitor-resistant growth in head and neck squamous cell carcinoma (HNSCC). We designed a phase II clinical trial of EGFR tyrosine kinase inhibitor (TKI), erlotinib, in association with the mTOR inhibitor, everolimus, based on the hypothesis that the downstream effects of Akt through inhibition of mTOR may enhance the effectiveness of the EGFR-TKI in patients with recurrent/metastatic HNSCC. PATIENTS AND METHODS: Patients with histologically or cytologically confirmed platinum-resistant HNSCC received everolimus 5 mg and erlotinib 150 mg daily orally until disease progression, intolerable toxicity, investigator or patient decision. Cytokines and angiogenic factors profile, limited mutation analysis and p16 immunohistochemistry status were included in the biomarker analysis. RESULTS: Of the 35 assessable patients, 3 (8%) achieved partial response at 4 weeks, 1 confirmed at 12 weeks; overall response rate at 12 weeks was 2.8%. Twenty-seven (77%) patients achieved disease stabilization at 4 weeks, 11 (31%) confirmed at 12 weeks. Twelve-week progression-free survival (PFS) was 49%, median PFS 11.9 weeks and median overall survival (OS) 10.25 months. High neutrophil gelatinase lipocalin (P = 0.01) and vascular endothelial growth factor (VEGF) (P = 0.04) plasma levels were significantly associated with worse OS. CONCLUSIONS: The combination of erlotinib and everolimus did not show significant benefit in unselected patients with platinum-resistant metastatic HNSCC despite a manageable toxicity profile. Markers of tumor invasion and hypoxia identify a group of patients with particularly poor prognosis. CLINICAL TRIAL NUMBER: NCT00942734.

Circulating cytokines and monocyte subpopulations as biomarkers of outcome and biological activity in sunitinib-treated patients with advanced neuroendocrine tumours.

BACKGROUND: Sunitinib is approved worldwide for treatment of advanced pancreatic neuroendocrine tumours (pNET), but no validated markers exist to predict response. This analysis explored biomarkers associated with sunitinib activity and clinical benefit in patients with pNET and carcinoid tumours in a phase II study. METHODS: Plasma was assessed for vascular endothelial growth factor (VEGF)-A, soluble VEGF receptor (sVEGFR)-2, sVEGFR-3, interleukin (IL)-8 (n=105), and stromal cell-derived factor (SDF)-1α (n=28). Pre-treatment levels were compared between tumour types and correlated with response, progression-free (PFS), and overall survival (OS). Changes in circulating myelomonocytic and endothelial cells were also analysed. RESULTS: Stromal cell-derived factor-1α and sVEGFR-2 levels were higher in pNET than in carcinoid (P=0.003 and 0.041, respectively). High (above-median) baseline SDF-1α was associated with worse PFS, OS, and response in pNET, and high sVEGFR-2 with longer OS (P⩽0.05). For carcinoid, high IL-8, sVEGFR-3, and SDF-1α were associated with shorter PFS and OS, and high IL-8 and SDF-1α with worse response (P⩽0.05). Among circulating cell types, monocytes showed the largest on-treatment decrease, particularly CD14+ monocytes co-expressing VEGFR-1 or CXCR4. CONCLUSIONS: Interleukin-8, sVEGFR-3, and SDF-1α were identified as predictors of sunitinib clinical outcome. Putative pro-tumorigenic CXCR4+ and VEGFR-1+ monocytes represent novel candidate markers and biologically relevant targets explaining the activity of sunitinib.

EGFR biomarkers predict benefit from vandetanib in combination with docetaxel in a randomized phase III study of second-line treatment of patients with advanced non-small cell lung cancer.

BACKGROUND: ZODIAC was a randomized phase III study of second-line treatment in patients with advanced non-small cell lung cancer (NSCLC) that evaluated the addition of vandetanib to docetaxel. The study showed a statistically significant improvement in progression-free survival and objective response rate, but not in overall survival for unselected patients. This study evaluated epidermal growth factor receptor (EGFR) gene mutation, copy number gain, and protein expression, and KRAS gene mutation, in pretreatment tumor samples as potential biomarkers predicting benefit from vandetanib as second-line treatment of NSCLC. PATIENTS AND METHODS: After progression following first-line chemotherapy, 1391 patients with locally advanced or metastatic (stage IIIB/IV) NSCLC were randomized 1 : 1 to receive vandetanib (100 mg/day) plus docetaxel (75 mg/m(2) every 21 days) or placebo plus docetaxel in the ZODIAC study. Archival tumor samples (n = 570) were collected from consenting patients (n = 958) for predefined, prospective biomarker analyses. RESULTS: Of evaluable samples, 14% were EGFR mutation positive, 35% were EGFR FISH positive, 88% were EGFR protein expression positive, and 13% were KRAS mutation positive. Compared with the overall study population, in which progression-free survival (PFS) [hazard ratio (HR) = 0.79] but not OS (HR = 0.91) were significantly improved with vandetanib, there was greater relative clinical benefit for patients with EGFR mutation-positive tumors [PFS HR 0.51, confidence interval (CI) 0.25-1.06 and OS HR 0.46, CI 0.14-1.57] and EGFR FISH-positive tumors (PFS HR 0.61, CI 0.39-0.94 and OS HR 0.48, CI 0.28-0.84). Similarly, patients with EGFR mutation or FISH-positive tumor samples who received vandetanib had an increased chance of objective tumor response (odds ratios 3.34, CI 0.8-13.89, and 3.90, CI 1.02-14.82, respectively). There did not appear to be benefit for vandetanib in patients with KRAS mutation-positive tumors. CONCLUSIONS: High EGFR gene copy number or activating EGFR mutations may identify patient subgroups who receive increased clinical benefit from vandetanib in combination with docetaxel in second-line NSCLC. CLINICALTRIALSGOV: NCT00312377.

A cytokine and angiogenic factor (CAF) analysis in plasma for selection of sorafenib therapy in patients with metastatic renal cell carcinoma.

BACKGROUND: We investigated cytokines and angiogenic factors (CAFs) in patients with metastatic renal cell carcinoma (mRCC) treated in a randomized phase II clinical trial of sorafenib versus sorafenib+ interferon-α (IFN-α) that yielded no differences in progression-free survival (PFS). We aimed to link the CAF profile to PFS and select candidate predictive and prognostic markers for further study. METHODS: The concentrations of 52 plasma CAFs were measured pretreatment (n = 69), day 28, and day 56 using multiplex bead arrays and enzyme-linked immunosorbent assay. We investigated the association between baseline levels of CAFs with PFS and posttreatment changes. RESULTS: Unsupervised CAF clustering analysis revealed two distinct mRCC patient groups with elevated proangiogenic or proinflammatory mediators. A six-marker baseline CAF signature [osteopontin, vascular endothelial growth factor (VEGF), carbonic anhydrase 9, collagen IV, VEGF receptor-2, and tumor necrosis factor-related apoptosis-inducing ligand] correlated with PFS benefit (hazard ratio 0.20 versus 2.25, signature negative versus positive, respectively; P = 0.0002). While changes in angiogenic factors were frequently attenuated by the sorafenib+ IFN combination, most key immunomodulatory mediators increased. CONCLUSIONS: Using CAF profiling, we identified two mRCC patient groups, a candidate plasma signature for predicting PFS benefit, and distinct marker changes occurring with each treatment. This platform may provide valuable insights into renal cell carcinoma biology and the molecular consequences of targeted therapies.

Antitumour efficacy of MEK inhibitors in human lung cancer cells and their derivatives with acquired resistance to different tyrosine kinase inhibitors.

BACKGROUND: To study the molecular mechanisms regulating cancer cell resistance to four different tyrosine kinase inhibitors (TKIs): erlotinib, gefitinib, vandetanib and sorafenib. METHODS: An in vitro model of acquired resistance to these TKIs was developed by continuously treating the human lung adenocarcinoma cell line CALU-3 with escalating doses of each drug. Transcriptional profiling was performed with Agilent whole genome microarrays. Western blot analysis, enzyme-linked immunosorbent (ELISA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation, migration, invasion and anchorage-independent colony growth assays were conducted in vitro and experiments with established xenografts in athymic nude mice were performed in vivo in parental (P) and TKI-resistant (R) CALU-3 cell lines. RESULTS: As compared with P-CALU-3 cells, in TKI-R CALU-3 cell lines a significant increase in the expression of activated, phosphorylated MET, IGF-1R, AKT, MEK, MAPK and of survivin was observed. Downregulation of E-cadherin and amphiregulin mRNAs and upregulation of vimentin, VE-cadherin, HIF-1α and vascular endothelial growth factor receptor-1 mRNAs were observed in all four TKI-R CALU-3 cell lines. All four TKI-R CALU-3 cells showed increased invasion, migration and anchorage-independent growth. Together, these data suggest epithelial to mesenchymal transition (EMT) in TKI-R CALU-3 cells. Treatment with several agents that target AKT, MET or IGF-1R did not affect TKI-R CALU-3 cell proliferation. In contrast, treatment with MSC19363669B and selumetinib, two selective MEK inhibitors, caused inhibition of cell proliferation, invasion, migration, anchorage-independent growth in vitro and of tumour growth in vivo of all four TKI-R CALU-3 cell lines. CONCLUSION: These data suggest that resistance to four different TKIs is characterised by EMT, which is MEK-inhibitor sensitive in human CALU-3 lung adenocarcinoma.

Increased mobilisation of circulating endothelial progenitors in von Hippel-Lindau disease and renal cell carcinoma.

BACKGROUND: Circulating endothelial cells (CECs) are a candidate biomarker for monitoring angiogenesis in cancer. Circulating endothelial cell subsets are mobilised by angiogenic mediators. Because of the highly angiogenic phenotype of renal cell carcinoma (RCC), we sought to assess the potential of CECs as a marker of RCC in patients with von Hippel-Lindau (VHL) disease and those with sporadic RCC. METHODS: We performed multicolour flow cytometry to enumerate CECs in patients with RCC, patients with VHL disease with and without RCC, and normal subjects. Two subsets of CECs were evaluated: mature CECs (mCECs) and circulating endothelial progenitors (CEPs). RESULTS: In patients with VHL disease and RCC and those with sporadic RCC (N=10), CEPs and the CEP:mCEC ratio were higher than in normal subjects (N=17) (median CEPs: 0.97 vs 0.19 cells µl(-1), respectively, P<0.01; median CEP:mCEC: 0.92 vs 0.58, respectively, P=0.04). However, in patients with VHL without RCC, CECs were not increased. In paired pre- and post-nephrectomy RCC patient samples (N=20), CEPs decreased after surgery (median difference 0.02 cells µl(-1), -0.06 to 1.2; P=0.05). CONCLUSION: Circulating endothelial progenitors were elevated in RCC, but not in patients with VHL without RCC. Circulating endothelial progenitor enumeration merits further investigation as a monitoring strategy for patients with VHL.

Multiple receptor tyrosine kinases regulate HIF-1alpha and HIF-2alpha in normoxia and hypoxia in neuroblastoma: implications for antiangiogenic mechanisms of multikinase inhibitors.

Novel treatment approaches are needed for children with advanced neuroblastoma. Studies with neuroblastoma cells have indicated the presence of a hypoxia-driven vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)-1 autocrine loop modulating hypoxia-inducible factor-1alpha (HIF-1alpha). Whether other receptor tyrosine kinases (RTKs) are capable of modulating HIF-1alpha levels and whether RTKs can regulate HIF-2alpha as well is largely unknown. We evaluated neuroblastoma cell lines for expression of various RTKs. Although cell lines were heterogeneous in the expression of VEGFR-1, -3, c-Kit and RET, most cells expressed PDGFR-alpha and -beta. Ligand-induced activation of multiple RTKs upregulated HIF-1alpha levels, whereas activation of VEGFR-1 alone upregulated HIF-2alpha. Multitargeted tyrosine kinase inhibitor sunitinib reduced hypoxia-induced rises in HIF-1alpha and HIF-2alpha through mechanisms involving effects on both mRNA levels and protein stability. In addition, sunitinib and sorafenib had direct effects on tumor cell viability in vitro. In a neuroblastoma xenograft model, tumor growth inhibition by sunitinib was associated with inhibition of angiogenesis and reduced HIF-1alpha levels. These findings show that multiple RTKs may regulate the HIF axis in normoxia and hypoxia and suggest that multikinase inhibitors may exert antiangiogenic effects not only by direct effects on endothelial cells, but also by blocking compensatory hypoxia- and ligand-induced changes in HIF-1alpha and HIF-2alpha.