Nathan A. Pennell

Gene expression-based survival prediction in lung adenocarcinoma: A multi-site, blinded validation study

Although prognostic gene expression signatures for survival in early-stage lung cancer have been proposed, for clinical application, it is critical to establish their performance across different subject populations and in different laboratories. Here we report a large, training–testing, multi-site, blinded validation study to characterize the performance of several prognostic models based on gene expression for 442 lung adenocarcinomas. The hypotheses proposed examined whether microarray measurements of gene expression either alone or combined with basic clinical covariates (stage, age, sex) could be used to predict overall survival in lung cancer subjects. Several models examined produced risk scores that substantially correlated with actual subject outcome. Most methods performed better with clinical data, supporting the combined use of clinical and molecular information when building prognostic models for early-stage lung cancer. This study also provides the largest available set of microarray data with extensive pathological and clinical annotation for lung adenocarcinomas.

Prognostic and Predictive Gene Signature for Adjuvant Chemotherapy in Resected Non–Small-Cell Lung Cancer

PURPOSE The JBR.10 trial demonstrated benefit from adjuvant cisplatin/vinorelbine (ACT) in early-stage non-small-cell lung cancer (NSCLC). We hypothesized that expression profiling may identify stage-independent subgroups who might benefit from ACT. PATIENTS AND METHODS Gene expression profiling was conducted on mRNA from 133 frozen JBR.10 tumor samples (62 observation [OBS], 71 ACT). The minimum gene set that was selected for the greatest separation of good and poor prognosis patient subgroups in OBS patients was identified. The prognostic value of this gene signature was tested in four independent published microarray data sets and by quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR). RESULTS A 15-gene signature separated OBS patients into high-risk and low-risk subgroups with significantly different survival (hazard ratio [HR], 15.02; 95% CI, 5.12 to 44.04; P < .001; stage I HR, 13.31; P < .001; stage II HR, 13.47; P < .001). The prognostic effect was verified in the same 62 OBS patients where gene expression was assessed by qPCR. Furthermore, it was validated consistently in four separate microarray data sets (total 356 stage IB to II patients without adjuvant treatment) and additional JBR.10 OBS patients by qPCR (n = 19). The signature was also predictive of improved survival after ACT in JBR.10 high-risk patients (HR, 0.33; 95% CI, 0.17 to 0.63; P = .0005), but not in low-risk patients (HR, 3.67; 95% CI, 1.22 to 11.06; P = .0133; interaction P < .001). Significant interaction between risk groups and ACT was verified by qPCR. CONCLUSION This 15-gene expression signature is an independent prognostic marker in early-stage, completely resected NSCLC, and to our knowledge, is the first signature that has demonstrated the potential to select patients with stage IB to II NSCLC most likely to benefit from adjuvant chemotherapy with cisplatin/vinorelbine.

ALK Status Testing in Non–Small Cell Lung Carcinoma: Correlation Between Ultrasensitive IHC and FISH

ALK gene rearrangements in advanced non-small cell lung carcinomas (NSCLC) are an indication for targeted therapy with crizotinib. Fluorescence in situ hybridization (FISH) using a recently approved companion in vitro diagnostic class FISH system commonly assesses ALK status. More accessible IHC is challenged by low expression of ALK-fusion transcripts in NSCLC. We compared ultrasensitive automated IHC with FISH for detecting ALK status on 318 FFPE and 40 matched ThinPrep specimens from 296 patients with advanced NSCLC. IHC was concordant with FFPE-FISH on 229 of 231 dual-informative samples (31 positive and 198 negative) and with ThinPrep-FISH on 34 of 34 samples (5 positive and 29 negative). Two cases with negative IHC and borderline-positive FFPE-FISH (15% and 18%, respectively) were reclassified as concordant based on negative matched ThinPrep-FISH and clinical data consistent with ALK-negative status. Overall, after including ThinPrep-FISH and amending the false-positive FFPE-FISH results, IHC demonstrated 100% sensitivity and specificity (95% CI, 0.86 to 1.00 and 0.97 to 1.00, respectively) for ALK detection on 249 dual-informative NSCLC samples. IHC was informative on significantly more samples than FFPE-FISH, revealing additional ALK-positive cases. The high concordance with FISH warrants IHCs routine use as the initial component of an algorithmic approach to clinical ALK testing in NSCLC, followed by reflex FISH confirmation of IHC-positive cases.

Combined Inhibition of the VEGFR and EGFR Signaling Pathways in the Treatment of NSCLC

Multitargeted agents represent the next generation of targeted therapies in solid tumors. The benefits of individually targeting the vascular endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR) signaling pathways have been clinically validated in recent years in a number of solid tumor types including non-small cell lung cancer (NSCLC). Given the heterogeneity of this tumor type and potential crosstalk between these key signaling pathways (which are known to play a critical role in tumor growth, metastasis, and angiogenesis), dual inhibition of the VEGFR and EGFR signaling pathways has the potential to offer additional clinical benefits in NSCLC. A number of approaches to inhibiting both VEGFR and EGFR signaling are currently under investigation, including monotherapy with a multitargeted tyrosine kinase inhibitor (e.g., vandetanib, AEE788, XL647, BMS-690514) or a combination of single-targeted therapies (e.g., bevacizumab, cetuximab, erlotinib, gefitinib). Preclinical and early clinical data (phase I and II trials) support combined inhibition of the VEGFR and EGFR pathways in NSCLC. Overall, combined inhibition strategies are well tolerated and have shown promise in early clinical studies. Ongoing phase II and phase III trials will determine the clinical potential of a number of dual inhibition strategies in the treatment of advanced NSCLC.

Improved tumor vascularization after anti-VEGF therapy with carboplatin and nab-paclitaxel associates with survival in lung cancer

Significance A better mechanistic understanding of the survival benefits and identification of biomarkers of response would greatly enhance the optimal utilization of antiangiogenic agents such as bevacizumab in combination with chemotherapy in the treatment of cancer. This study indicates that the benefits of bevacizumab with chemotherapy in non–small cell lung cancer (NSCLC) patients may depend on tumor vascular function during treatment. These correlative studies also provide new insights into the selection of NSCLC patients most likely to benefit from the addition of bevacizumab treatment to chemotherapy. The imaging and circulating biomarker candidates should be further evaluated in larger studies. Addition of anti-VEGF antibody therapy to standard chemotherapies has improved survival and is an accepted standard of care for advanced non–small cell lung cancer (NSCLC). However, the mechanisms by which anti-VEGF therapy increases survival remain unclear. We evaluated dynamic CT-based vascular parameters and plasma cytokines after bevacizumab alone and after bevacizumab plus chemotherapy with carboplatin and nab-paclitaxel in advanced NSCLC patients to explore potential biomarkers of treatment response and resistance to this regimen. Thirty-six patients were enrolled in this study. The primary end point was 6-mo progression-free survival rate, which was 74% (95% CI: 57, 97). This regimen has a promising overall response rate of 36% and median time to progression of 8.5 (6.0, 38.7) mo and overall survival of 12.2 (9.6, 44.1) mo. We found that anti-VEGF therapy led to a sustained increase in plasma PlGF, a potential pharmacodynamic marker. We also found that higher levels of soluble VEGFR1 measured before starting bevacizumab with chemotherapy were associated with worse survival, supporting its potential role as biomarker of treatment resistance. Our imaging biomarker studies indicate that bevacizumab-based treatment—while reducing blood flow, volume, and permeability in the overall population—may be associated with improved survival in patients with improved tumor vasculature and blood perfusion after treatment. This hypothesis-generating study supports the notion that excessively decreasing vascular permeability and pruning/rarefaction after bevacizumab therapy may negatively impact the outcome of combination therapy in NSCLC patients. This hypothesis warrants further dose-titration studies of bevacizumab to examine the dose effect on tumor vasculature and treatment efficacy.

XL647—A Multitargeted Tyrosine Kinase Inhibitor: Results of a Phase II Study in Subjects with Non-small Cell Lung Cancer Who Have Progressed after Responding to Treatment with Either Gefitinib or Erlotinib

Introduction: Although patients with non-small cell lung cancer (NSCLC) whose tumors harbor epidermal growth factor receptor (EGFR) activating mutations commonly experience significant regressions when treated with erlotinib or gefitinib, they uniformly develop resistance to these agents. The secondary EGFR T790M mutation is found in 50% of patients with acquired resistance. Herein, we studied XL647, an oral small molecule inhibitor of multiple receptor tyrosine kinases, including EGFR, VEGFR2, HER2, and EphB4, in NSCLC patients known or suspected of having tumors harboring T790M. Methods: Eligible patients included those with relapsed or recurrent advanced NSCLC who progressed after ≥12 weeks of stable disease or response to erlotinib or gefitinib and/or those patients with a documented EGFR T790M. XL647 300 mg was administered once daily. The primary end point was objective response rate. Pretreatment plasma samples were collected for mutation testing of circulating tumor DNA. Results: Forty-one patients were enrolled; 33 were evaluable for efficacy. One partial response was observed (response rate 3% and 90% confidence interval, 0% to 14%). Of patients whose tumors harbored T790M, 67% (8/12) had progression of disease as best response compared with 14% (3/21) of those without this mutation. Plasma samples from 40 patients were available for mutation testing, 14 (35%) of which were found to have EGFR mutations. Conclusions: The 3% response rate observed did not meet the prespecified threshold to recommend further study of XL647 in patients who develop acquired resistance to erlotinib or gefitinib. Patients with T790M had a significantly worse progression-free survival.

Incidence of Pneumonitis With Use of Programmed Death 1 and Programmed Death-Ligand 1 Inhibitors in Non-Small Cell Lung Cancer: A Systematic Review and Meta-Analysis of Trials

Background Programmed death 1 (PD‐1) programmed death‐ligand 1 (PD‐L1) inhibitors show significant clinical activity in non‐small cell lung carcinoma (NSCLC). However, they are often associated with potentially fatal immune‐mediated pneumonitis. Preliminary reports of trials suggest a difference in the rate of pneumonitis with PD‐1 and PD‐L1 inhibitors. We sought to determine the overall incidence of pneumonitis and differences according to type of inhibitors and prior chemotherapy use. Methods MEDLINE, Embase, and Scopus databases were searched up to November 2016. Rates of pneumonitis of any grade and grade ≥ 3 from all clinical trials investigating nivolumab, pembrolizumab, atezolizumab, durvalumab, and avelumab as single agents in NSCLC were collected. The incidence of pneumonitis across trials was calculated using DerSimonian‐Laird random effects models. We compared incidences between PD‐1 and PD‐L1 inhibitors and between treatment naive and previously treated patients. Results Nineteen trials (12 with PD‐1 inhibitors [n = 3,232] and 7 with PD‐L1 inhibitors [n = 1,806]) were identified. PD‐1 inhibitors were found to have statistically significant higher incidence of any grade pneumonitis compared with PD‐L1 inhibitors (3.6%; 95% CI, 2.4%‐4.9% vs 1.3%; 95% CI, 0.8%‐1.9%, respectively; P = .001). PD‐1 inhibitors were also associated with higher incidence of grade 3 or 4 pneumonitis (1.1%; 95% CI, 0.6%‐1.7% vs 0.4%; 95% CI, 0%‐0.8%; P = .02). Treatment naive patients had higher incidence of grade 1 through 4 pneumonitis compared with previously treated patients (4.3%; 95% CI, 2.4%‐6.3% vs 2.8%; 95% CI, 1.7%‐ 4%; P = .03). Conclusions There was a higher incidence of pneumonitis with use of PD‐1 inhibitors compared with PD‐L1 inhibitors. Higher rate of pneumonitis was more common in treatment naive patients.

Incidence of pneumonitis with use of PD-1 and PD-L1 inhibitors in non-small cell lung cancer: A Systematic Review and Meta-analysis of trials

Background Programmed death 1 (PD‐1) programmed death‐ligand 1 (PD‐L1) inhibitors show significant clinical activity in non‐small cell lung carcinoma (NSCLC). However, they are often associated with potentially fatal immune‐mediated pneumonitis. Preliminary reports of trials suggest a difference in the rate of pneumonitis with PD‐1 and PD‐L1 inhibitors. We sought to determine the overall incidence of pneumonitis and differences according to type of inhibitors and prior chemotherapy use. Methods MEDLINE, Embase, and Scopus databases were searched up to November 2016. Rates of pneumonitis of any grade and grade ≥ 3 from all clinical trials investigating nivolumab, pembrolizumab, atezolizumab, durvalumab, and avelumab as single agents in NSCLC were collected. The incidence of pneumonitis across trials was calculated using DerSimonian‐Laird random effects models. We compared incidences between PD‐1 and PD‐L1 inhibitors and between treatment naive and previously treated patients. Results Nineteen trials (12 with PD‐1 inhibitors [n = 3,232] and 7 with PD‐L1 inhibitors [n = 1,806]) were identified. PD‐1 inhibitors were found to have statistically significant higher incidence of any grade pneumonitis compared with PD‐L1 inhibitors (3.6%; 95% CI, 2.4%‐4.9% vs 1.3%; 95% CI, 0.8%‐1.9%, respectively; P = .001). PD‐1 inhibitors were also associated with higher incidence of grade 3 or 4 pneumonitis (1.1%; 95% CI, 0.6%‐1.7% vs 0.4%; 95% CI, 0%‐0.8%; P = .02). Treatment naive patients had higher incidence of grade 1 through 4 pneumonitis compared with previously treated patients (4.3%; 95% CI, 2.4%‐6.3% vs 2.8%; 95% CI, 1.7%‐ 4%; P = .03). Conclusions There was a higher incidence of pneumonitis with use of PD‐1 inhibitors compared with PD‐L1 inhibitors. Higher rate of pneumonitis was more common in treatment naive patients.

Prospective Clinical Study of Precision Oncology in Solid Tumors

Systematic studies evaluating clinical benefit of tumor genomic profiling are lacking. We conducted a prospective study in 250 patients with select solid tumors at the Cleveland Clinic. Eligibility required histopathologic diagnosis, age of 18 years or older, Eastern Cooperative Oncology Group performance status 0-2, and written informed consent. Tumors were sequenced using FoundationOne (Cambridge, MA). Results were reviewed at the Cleveland Clinic Genomics Tumor Board. Outcomes included feasibility and clinical impact. Colorectal (25%), breast (18%), lung (13%), and pancreatobiliary (13%) cancers were the most common diagnoses. Median time from consent to result was 25 days (range = 3-140). Of 223 evaluable samples, 49% (n = 109) of patients were recommended a specific therapy, but only 11% (n = 24) received such therapy: 12 on clinical trials, nine off-label, three on-label. Lack of clinical trial access (n = 49) and clinical deterioration (n = 29) were the most common reasons for nonrecommendation/nonreceipt of genomics-driven therapy.

ALK Status Testing in Non–Small-Cell Lung Carcinoma by FISH on ThinPrep Slides with Cytology Material

Introduction: Oncogenic anaplastic lymphoma kinase (ALK) gene rearrangements in non–small-cell lung carcinomas (NSCLC) provide the basis for targeted therapy with crizotinib and other specific ALK inhibitors. Treatment eligibility is conventionally determined by the Food and Drug Administration–approved companion diagnostic fluorescence in situ hybridization (FISH) assay on paraffin-embedded tissue (PET). On limited samples such as fine needle aspiration–derived cytoblocks, FISH for ALK is often uninformative. FISH performed on liquid-based ThinPrep slides (ThinPrep-FISH) may represent a robust alternative. Methods: Two hundred thirty cytology samples from 217 patients with advanced NSCLC, including a consecutive series of 179 specimens, were used to generate matched ThinPrep slides and paraffin cytoblocks. The same ThinPrep slides used for cytologic diagnosis were assessed by standard ALK break-apart two-color probe FISH, after etching of tumor areas. Ultrasensitive ALK immunohistochemistry (IHC) on corresponding cytoblocks [D5F3 antibody, OptiView signal amplification] served as the reference data set. Results: ThinPrep-FISH ALK signals were robust in 228 of 230 cases and not compromised by nuclear truncation inherent in paraffin-embedded tissue–FISH; only two samples displayed no signals. Nine of 178 informative cases (5%) in the consecutive series and 18 of 228 informative cases (7.8%) overall were ALK rearranged by ThinPrep-FISH. In 154 informative matched ThinPrep-FISH and cytoblock-IHC samples, 152 were concordant (10, 6.5% ALK status positive; 142, 92.2% ALK status negative), and two (1.3%) were ThinPrep-FISH positive but IHC negative (sensitivity 100%, specificity 98.6%, overall agreement 98.7%). Conclusion: Detection of ALK gene rearrangements in liquid cytology ThinPrep slides derived from patients with NSCLC can be confidently used for clinical ALK molecular testing.