Pembrolizumab for the treatment of non-small-cell lung cancer.

BACKGROUND: We assessed the efficacy and safety of programmed cell death 1 (PD-1) inhibition with pembrolizumab in patients with advanced non-small-cell lung cancer enrolled in a phase 1 study. We also sought to define and validate an expression level of the PD-1 ligand 1 (PD-L1) that is associated with the likelihood of clinical benefit. METHODS: We assigned 495 patients receiving pembrolizumab (at a dose of either 2 mg or 10 mg per kilogram of body weight every 3 weeks or 10 mg per kilogram every 2 weeks) to either a training group (182 patients) or a validation group (313 patients). We assessed PD-L1 expression in tumor samples using immunohistochemical analysis, with results reported as the percentage of neoplastic cells with staining for membranous PD-L1 (proportion score). Response was assessed every 9 weeks by central review. RESULTS: Common side effects that were attributed to pembrolizumab were fatigue, pruritus, and decreased appetite, with no clear difference according to dose or schedule. Among all the patients, the objective response rate was 19.4%, and the median duration of response was 12.5 months. The median duration of progression-free survival was 3.7 months, and the median duration of overall survival was 12.0 months. PD-L1 expression in at least 50% of tumor cells was selected as the cutoff from the training group. Among patients with a proportion score of at least 50% in the validation group, the response rate was 45.2%. Among all the patients with a proportion score of at least 50%, median progression-free survival was 6.3 months; median overall survival was not reached. CONCLUSIONS: Pembrolizumab had an acceptable side-effect profile and showed antitumor activity in patients with advanced non-small-cell lung cancer. PD-L1 expression in at least 50% of tumor cells correlated with improved efficacy of pembrolizumab. (Funded by Merck; KEYNOTE-001 ClinicalTrials.gov number, NCT01295827.).

Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a randomised controlled trial.

BACKGROUND: Despite recent advances in the treatment of advanced non-small-cell lung cancer, there remains a need for effective treatments for progressive disease. We assessed the efficacy of pembrolizumab for patients with previously treated, PD-L1-positive, advanced non-small-cell lung cancer. METHODS: We did this randomised, open-label, phase 2/3 study at 202 academic medical centres in 24 countries. Patients with previously treated non-small-cell lung cancer with PD-L1 expression on at least 1% of tumour cells were randomly assigned (1:1:1) in blocks of six per stratum with an interactive voice-response system to receive pembrolizumab 2 mg/kg, pembrolizumab 10 mg/kg, or docetaxel 75 mg/m(2) every 3 weeks. The primary endpoints were overall survival and progression-free survival both in the total population and in patients with PD-L1 expression on at least 50% of tumour cells. We used a threshold for significance of p<0.00825 (one-sided) for the analysis of overall survival and a threshold of p<0.001 for progression-free survival. This trial is registered at ClinicalTrials.gov, number NCT01905657. FINDINGS: Between Aug 28, 2013, and Feb 27, 2015, we enrolled 1034 patients: 345 allocated to pembrolizumab 2 mg/kg, 346 allocated to pembrolizumab 10 mg/kg, and 343 allocated to docetaxel. By Sept 30, 2015, 521 patients had died. In the total population, median overall survival was 10.4 months with pembrolizumab 2 mg/kg, 12.7 months with pembrolizumab 10 mg/kg, and 8.5 months with docetaxel. Overall survival was significantly longer for pembrolizumab 2 mg/kg versus docetaxel (hazard ratio [HR] 0.71, 95% CI 0.58-0.88; p=0.0008) and for pembrolizumab 10 mg/kg versus docetaxel (0.61, 0.49-0.75; p<0.0001). Median progression-free survival was 3.9 months with pembrolizumab 2 mg/kg, 4.0 months with pembrolizumab 10 mg/kg, and 4.0 months with docetaxel, with no significant difference for pembrolizumab 2 mg/kg versus docetaxel (0.88, 0.74-1.05; p=0.07) or for pembrolizumab 10 mg/kg versus docetaxel (HR 0.79, 95% CI 0.66-0.94; p=0.004). Among patients with at least 50% of tumour cells expressing PD-L1, overall survival was significantly longer with pembrolizumab 2 mg/kg than with docetaxel (median 14.9 months vs 8.2 months; HR 0.54, 95% CI 0.38-0.77; p=0.0002) and with pembrolizumab 10 mg/kg than with docetaxel (17.3 months vs 8.2 months; 0.50, 0.36-0.70; p<0.0001). Likewise, for this patient population, progression-free survival was significantly longer with pembrolizumab 2 mg/kg than with docetaxel (median 5.0 months vs 4.1 months; HR 0.59, 95% CI 0.44-0.78; p=0.0001) and with pembrolizumab 10 mg/kg than with docetaxel (5.2 months vs 4.1 months; 0.59, 0.45-0.78; p<0.0001). Grade 3-5 treatment-related adverse events were less common with pembrolizumab than with docetaxel (43 [13%] of 339 patients given 2 mg/kg, 55 [16%] of 343 given 10 mg/kg, and 109 [35%] of 309 given docetaxel). INTERPRETATION: Pembrolizumab prolongs overall survival and has a favourable benefit-to-risk profile in patients with previously treated, PD-L1-positive, advanced non-small-cell lung cancer. These data establish pembrolizumab as a new treatment option for this population and validate the use of PD-L1 selection. FUNDING: Merck & Co.

The F box protein Fbx6 regulates Chk1 stability and cellular sensitivity to replication stress.

ATR and Chk1 are two key protein kinases in the replication checkpoint. Activation of ATR-Chk1 has been extensively investigated, but checkpoint termination and replication fork restart are less well understood. Here, we report that DNA damage not only activates Chk1, but also exposes a degron-like region at the carboxyl terminus of Chk1 to an Fbx6-containing SCF (Skp1-Cul1-F box) E3 ligase, which mediates the ubiquitination and degradation of Chk1 and, in turn, terminates the checkpoint. The protein levels of Chk1 and Fbx6 showed an inverse correlation in both cultured cancer cells and in human breast tumor tissues. Further, we show that low levels of Fbx6 and consequent impairment of replication stress-induced Chk1 degradation are associated with cancer cell resistance to the chemotherapeutic agent, camptothecin. We propose that Fbx6-dependent Chk1 degradation contributes to S phase checkpoint termination and that a defect in this mechanism might increase tumor cell resistance to certain anticancer drugs.

Computational and bioinformatics frameworks for next-generation whole exome and genome sequencing.

It has become increasingly apparent that one of the major hurdles in the genomic age will be the bioinformatics challenges of next-generation sequencing. We provide an overview of a general framework of bioinformatics analysis. For each of the three stages of (1) alignment, (2) variant calling, and (3) filtering and annotation, we describe the analysis required and survey the different software packages that are used. Furthermore, we discuss possible future developments as data sources grow and highlight opportunities for new bioinformatics tools to be developed.

Quantitative in situ analysis of beta-catenin expression in breast cancer shows decreased expression is associated with poor outcome.

The role of beta-catenin in breast cancer and its prognostic value is controversial. The prognostic value had been assessed previously in a series of nonquantitative immunohistochemical studies with conflicting results. In efforts to clarify the relationship between beta-catenin protein expression and breast cancer prognosis, we have assessed a retrospective 600 case cohort of breast cancer tumors from the Yale Pathology archives on tissue microarrays. They were assessed using automated quantitative analysis (AQUA) with a series of array-embedded cell lines for which the beta-catenin concentration was standardized by an ELISA assay. The expression levels of the standard clinical markers HER2, estrogen receptor (ER), progesterone receptor (PR), and Ki-67 were also assessed on the same cohort. X-tile software was used to select optimal protein concentration cutpoints and to evaluate the outcome using a training set and a validation set. We found that low-level expression of membranous beta-catenin is associated with significantly worse outcome (38% versus 76%, 10-year survival, validation set log-rank P = 0.0016). Multivariate analysis of this marker, assessed in a proportional hazards model with tumor size, age, node status, nuclear grade, ER, PR, HER2, and Ki-67, is still highly significant with a hazard ratio of 6.8 (P < 0.0001, 95% confidence interval, 3.1-15.1). These results suggest that loss of beta-catenin expression at the membrane, as assessed by objective quantitative analysis methods, may be useful as a prognostic marker or may be part of a useful algorithm for prognosis in breast cancer.

Retrospective Molecular Epidemiology Study of PD-L1 Expression in Patients with EGFR-Mutant Non-small Cell Lung Cancer.

PURPOSE: Data are limited on programmed death ligand 1 (PD-L1) expression in epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: We retrospectively evaluated the relationship between PD-L1 expression and recurrence-free survival (RFS) and overall survival in 319 patients with EGFR-mutant NSCLC who were treated at Samsung Medical Center from 2006 to 2014. Membranous PD-L1 expression on tumor cells was measured using the PD-L1 IHC 22C3 pharmDx antibody and reported as tumor proportion score (TPS). Kaplan-Meier methods, log-rank test, and Cox proportional hazards models were used for survival analysis. RESULTS: All patients had ≥1 EGFR mutation-54% in exon 19 and 39% in exon 21. Overall, 51% of patients had PD-L1-positive tumors. The prevalence of PD-L1 positivity was higher among patients with stages II-IV versus stage I disease (64% vs. 44%) and among patients with other EGFR mutations (75%) than with L858R mutation (39%) or exon 19 deletion (52%). PD-L1 positivity was associated with shorter RFS, with an adjusted hazard ratio of 1.52 (95% confidence interval [CI], 0.81 to 2.84; median, 18 months) for the PD-L1 TPS ≥ 50% group, 1.51 (95% CI, 1.02 to 2.21; median, 31 months) for the PD-L1 TPS 1%-49% group, and 1.51 (95% CI, 1.05 to 2.18) for the combined PD-L1-positive groups (TPS ≥ 1%) compared with the PD-L1-negative group (median, 35 months). CONCLUSION: PD-L1 expression is associated with disease stage and type of EGFR mutation. PD-L1 positivity might be associated with worse RFS among patients with surgically treated EGFR-mutant NSCLC.

Classification of breast cancer using genetic algorithms and tissue microarrays.

PURPOSE: A multitude of breast cancer mRNA profiling studies has stratified breast cancer and defined gene sets that correlate with outcome. However, the number of genes used to predict patient outcome or define tumor subtypes by RNA expression studies is variable, nonoverlapping, and generally requires specialized technologies that are beyond those used in the routine pathology laboratory. It would be ideal if the familiarity and streamlined nature of immunohistochemistry could be combined with the rigorously quantitative and highly specific properties of nucleic acid-based analysis to predict patient outcome. EXPERIMENTAL DESIGN: We have used AQUA-based objective quantitative analysis of tissue microarrays toward the goal of discovery of a minimal number of markers with maximal prognostic or predictive value that can be applied to the conventional formalin-fixed, paraffin-embedded tissue section. RESULTS: The minimal discovered multiplexed set of tissue biomarkers was GATA3, NAT1, and estrogen receptor. Genetic algorithms were then applied after division of our cohort into a training set of 223 breast cancer patients to discover a prospectively applicable solution that can define a subset of patients with 5-year survival of 96%. This algorithm was then validated on an internal validation set (n=223, 5-year survival=95.8%) and further validated on an independent cohort from Sweden, which showed 5-year survival of 92.7% (n=149). CONCLUSIONS: With further validation, this test has both the familiarity and specificity for widespread use in management of breast cancer. More generally, this work illustrates the potential for multiplexed biomarker discovery on the tissue microarray platform.

Programmed Death Ligand 1 Expression in Paired Non-Small Cell Lung Cancer Tumor Samples.

BACKGROUND: Programmed death ligand 1 (PD-L1) expression may predict response to anti-programmed death 1 (anti-PD-1) or anti-PD-L1 treatment. There is limited information on changes in PD-L1 expression over time in patients with non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Eligible patients with NSCLC who received surgery or underwent biopsy at Samsung Medical Center, Seoul, Republic of Korea, and Aarhus University Hospital, Aarhus, Denmark, between February 2004 and April 2012 were included. PD-L1 expression in paired tumor tissue samples from the same patients at different dates and lesions was measured using a laboratory-developed prototype immunohistochemistry assay (22C3 antibody). PD-L1 positivity was defined as tumor cell membrane positivity in ≥ 1% of tumor cells (proportion score). Concordance of PD-L1 expression was analyzed by treating proportion score as categoric or continuous variables. RESULTS: Ninety-one patients were included in the analysis. The median interval between the 2 tumor collection dates was 20 months, with 91% of paired samples collected > 3 months apart. The concordance rate for PD-L1 classification between paired samples was 67% (95% confidence interval, 57%-77%). When treating the immunohistochemistry proportional score as a continuous variable, a significant correlation of PD-L1 expression was observed between the paired samples (Pearson correlation coefficient, 0.61; P < .001). CONCLUSION: There are good correlations of PD-L1 expression from paired NSCLC samples. For patients whose PD-L1 status is negative, it may be valuable to obtain additional tissue samples for retesting PD-L1 expression when anti-PD-1 immunotherapy is considered.

Quantitative analysis of breast cancer tissue microarrays shows that both high and normal levels of HER2 expression are associated with poor outcome.

Using a tissue microarray cohort of 300 breast cancers and 84 samples of normal breast epithelium, we analyzed HER2/neu expression and compared traditional clinical (manual) scoring with a recently developed system for the quantitative measurement of immunohistochemical stains (AQUA). As expected, both methods identified a population (10-15%) of high-HER2-expressing tumors with poor 30-year disease-related survival. Using AQUA analysis, we found that normal epithelium expresses a low but detectable level of HER2 and that 17.5% of tumors exhibit similar low-level HER2 expression. This low group was not definable by manual scoring. Surprisingly, HER2-normal tumors were as aggressive as HER2-overexpressing tumors. Our studies suggest that in situ quantitative measurement of HER2 stratifies breast tumors into three expression levels: normal, intermediate, and high, where both normal and high levels are associated with a worse outcome.

Automated quantitative analysis of tissue microarrays reveals an association between high Bcl-2 expression and improved outcome in melanoma.

The addition of B-cell lymphoma 2 (Bcl-2) antisense to dacarbazine in the treatment of metastatic melanoma demonstrates improved response rates and progression-free survival when compared with dacarbazine alone. Studies on small cohorts of melanoma patients have shown variability in Bcl-2 expression (60%-96% positive). We performed quantitative analysis of Bcl-2 expression in a large patient cohort to assess the association with outcome. Tissue microarrays containing intact melanoma specimens representing 402 patients (339 with associated survival data) were analyzed with our AQUA system for automated quantitative analysis. Automated, quantitative analysis uses S100 to define pixels as melanoma (tumor mask) within the array spot and measures intensity of Bcl-2 expression using a Cy5 conjugated antibody within the mask. A continuous index score is generated, which is directly proportional to the number of molecules per unit area. Scores were divided into quartiles and correlated with clinical variables. High Bcl-2 expression was associated with better outcome in the entire cohort and among metastatic specimens only (P = 0.004 and P = 0.015, respectively). Expression was higher in primary than in metastatic specimens (P < 0.0001). There was no association between Bcl-2 expression and Breslow depth or Clark level. The diverse results within the literature may be due to use of small cohorts or variability in staining technique. These results suggest studies are needed to evaluate the association between quantitative assessment of Bcl-2 expression and response to Bcl-2 targeting therapy toward the goal of improved response rates to these drugs.

Tissue microarray analysis of hepatocyte growth factor/Met pathway components reveals a role for Met, matriptase, and hepatocyte growth factor activator inhibitor 1 in the progression of node-negative breast cancer.

Numerous studies have demonstrated that overexpression of Met, the hepatocyte growth factor(HGF) receptor, plays an important role in tumorigenesis. Met activation can either occur through ligand-independent or -dependent mechanisms, both of which are mediated by a series of proteases and modulators. We studied the protein expression of several components of the HGF/Met pathway on a cohort of 330 node-negative breast carcinomas using a tissue microarray annotated with 30-year, disease-specific patient follow-up data. We examined HGF, matriptase (an activator of HGF expressed on mammary epithelial cell surfaces), HAI-I (the cognate inhibitor of matriptase), and the Met receptor itself. Our studies demonstrate tight correlation between the expression of HGF, matriptase, and Met in breast carcinoma. High-level expression of Met, matriptase, and HAI-I were associated with poor patient outcome. Met and HAI-I showed independent prognostic value when compared with traditional breast markers in a multivariate analysis. Intriguingly, antibodies against the intracellular but not the extracellular domain of Met were prognostic, suggesting that overexpression of the cytoplasmic-tail of Met, perhaps through cleavage or truncating mutation, may play an important role in breast cancer progression.

Development of a Companion Diagnostic PD-L1 Immunohistochemistry Assay for Pembrolizumab Therapy in Non-Small-cell Lung Cancer.

A companion diagnostic assay was codeveloped by Dako for pembrolizumab non-small-cell lung cancer clinical trials to detect PD-L1 expression by immunohistochemistry (IHC). This automated IHC assay has been analytically verified and validated using Dako's autostainer Link 48 and 22C3 mouse anti-PD-L1 monoclonal antibody to detect the PD-L1 expression in formalin-fixed paraffin-embedded human tumor tissue specimens. The PD-L1 22C3 IHC assay was optimized for high sensitivity and specificity. Repeatability and reproducibility studies were conducted at Dako and at 3 Clinical Laboratory Improvement Amendments certified laboratories during assay development. The studies included: intersite and intrasite, interobserver and intraobserver, interinstrument, interoperator, interday, and interlot, and intraday and intrarun. All precision studies performed at Dako and external laboratories achieved >85% point-estimate agreements for all 3 agreement types (negative, positive, and overall). A clinical cutoff (tumor proportion score ≥50%) of PD-L1 expression was determined and evaluated through a phase 1 clinical trial (KEYNOTE-001) for advanced non-small-cell lung cancer patients treated with pembrolizumab. The treatment effect of pembrolizumab in the 61 subjects who had a tumor PD-L1 of tumor proportion score ≥50% was substantial, with an overall response rate of 41% (95% confidence interval, 28.6-54.3) as compared with 20.6% (95% confidence interval, 15.5-26.5) observed in the 223 subjects irrespective of PD-L1 status. PD-L1 IHC 22C3 pharmDx is a sensitive, precise, and robust companion diagnostic assay, which will facilitate safe and effective use for pembrolizumab in cancer patients.

[Letter to the Editor] The need for improved education and training in research antibody usage and validation practices.

Address correspondence to Leonard P. Freedman, Global Biological Standards Institute, 1020 19th Street, NW, Suite 550, Washington, DC, 20036. E-mail: lfreedman@gbsi.org.

PD-L1 Expression and Survival among Patients with Advanced Non-Small Cell Lung Cancer Treated with Chemotherapy.

BACKGROUND: Recent clinical trial results have suggested that programmed cell death ligand 1 (PD-L1) expression measured by immunohistochemistry may predict response to anti-programmed cell death 1 (PD-1) therapy. Results on the association between PD-L1 expression and survival among patients with advanced non-small cell lung cancer (NSCLC) treated with chemotherapy are inconsistent. MATERIAL AND METHODS: We evaluated the relationship between PD-L1 expression and overall survival (OS) among 204 patients with advanced NSCLC treated at Aarhus University Hospital, Aarhus, Denmark, from 2007 to 2012. PD-L1 expression was measured using a prototype immunohistochemistry assay with the anti-PD-L1 22C3 antibody (Merck). PD-L1 strong positivity and weak positivity were defined to be traceable to the clinical trial version of the assay. RESULTS: Twenty-five percent of patients had PD-L1 strong-positive tumors, and 50% had PD-L1 weak-positive tumors. No statistically significant association was found between PD-L1 expression and survival; adjusted hazard ratio of 1.34 (95% confidence interval, 0.88-2.03; median OS, 9.0 months) for the PD-L1 strong-positive group and 1.07 (0.74-1.55; median OS, 9.8 months) for the PD-L1 weak-positive group compared with the PD-L1-negative group (median OS, 7.5 months). No association was seen between PD-L1 expression and OS when PD-L1 expression levels were stratified by median or tertiles. CONCLUSIONS: In concordance with previous studies, we found PD-L1 measured by immunohistochemistry to be frequently expressed in patients with advanced NSCLC. However, PD-L1 expression is not a strong prognostic marker in patients with advanced NSCLC treated with chemotherapy.

Development of a Prototype Immunohistochemistry Assay to Measure Programmed Death Ligand-1 Expression in Tumor Tissue.

CONTEXT: - With the abundance of therapeutics targeted against programmed death receptor-1 and its ligand (PD-L1) that are currently approved or in clinical development, there is interest in identifying those patients most likely to respond to these drugs. Expression of PD-L1 may be an indicator of an initial and robust inflammatory response to the presence of tumor cells. Therefore, tumors that express PD-L1 may be the most likely to respond to therapies that interrupt the negative feedback mechanism that leads to PD-L1 upregulation. OBJECTIVE: - To develop a prototype immunohistochemistry assay using the anti-PD-L1 antibody clone 22C3. DESIGN: - The assay was developed and optimized using commercially available reagents and archival tumor-bank tissue. RESULTS: - The optimized immunohistochemistry method had high precision and reproducibility. Using the prototype assay in 142 non-small cell lung cancer and 79 melanoma archival tumor-bank tissue samples, PD-L1 staining was observed at the plasma membrane of nucleated tumor and nontumor cells and, in some cases, as a distinct lichenoid pattern at the tumor-stroma border. Using a preliminary scoring method, 56% (80 of 142) of non-small cell lung cancer and 53% (42 of 79) of melanoma samples were defined as PD-L1+ based on a modified H-score of 1 or more or the presence of a distinctive staining pattern at the tumor-stroma interface. CONCLUSIONS: - The immunohistochemistry assay using the anti-PD-L1 antibody 22C3 merits further investigation in clinical trials and prevalence assessments to further understand the prognostic and predictive value of PD-L1 expression in cancer.

Development of a Companion Diagnostic for Pembrolizumab in Non-Small Cell Lung Cancer Using Immunohistochemistry for Programmed Death Ligand-1.

Context .- Programmed death ligand-1 (PD-L1) expression by tumors may enable them to avoid immunosurveillance. Objective .- To develop a PD-L1 immunohistochemical assay using the 22C3 anti-PD-L1 murine monoclonal antibody on the Dako platform as a possible companion diagnostic for pembrolizumab in patients with non-small cell lung cancer. Design .- Tumor samples from 146 patients with non-small cell lung cancer treated with pembrolizumab in KEYNOTE-001 and for whom response data were available were scored according to their staining intensity by a single pathologist using 4 methods: percentage of tumor cells staining at any intensity (PS1), moderate/strong intensity (PS2), strong intensity (PS3), and H-score (PS1 + PS2 + PS3). The cutoff score for predicting response to pembrolizumab was determined using receiver operating characteristic analysis. Progression-free and overall survival were assessed in patients with measurable disease per Response Evaluation Criteria in Solid Tumors, version 1.1 (n = 146). Results .- The 4 scoring methods assessed performed similarly; PS1 with a 50% cutoff score is the simplest and easiest method to implement in practice. Response to pembrolizumab was observed in 19 of 44 patients (43%) with a PS1 score of 50% or higher and 8 of 102 patients (8%) with PS1 lower than 50% (odds ratio, 8.93). Median progression-free and overall survival was 4.0 months and not yet reached, respectively, for patients with a PS1 of 50% or higher, and 2.1 and 6.1 months, respectively, for those with PS1 lower than 50%. Conclusion .- The PD-L1 immunohistochemical assay shows the potential for enrichment of trial populations and as a companion diagnostic tool in non-small cell lung cancer.

Prognostic Significance of PD-L1 in Patients with Non-Small Cell Lung Cancer: A Large Cohort Study of Surgically Resected Cases.

INTRODUCTION: The aim of our analysis was to evaluate the prognostic effect of programmed cell death ligand-1 (PD-L1) expression in patients with non-small cell lung cancer (NSCLC). METHODS: PD-L1 expression among 1070 surgically resected NSCLC specimens was evaluated by immunohistochemical analysis. Data were analyzed using Cox proportional hazard models adjusting for age, sex, smoking status, histologic type, stage, and performance status. RESULTS: Sixty-eight patients (6%) were strongly PD-L1 positive and 410 (38%) were weakly PD-L1 positive. A significantly higher prevalence of PD-L1 positivity was observed among patients with squamous cell carcinoma and among stage IIIB and IV patients. PD-L1 expression may be associated with poorer overall survival, with an adjusted hazard ratio of 1.56 (95% confidence interval [CI]: 1.08-2.26, p = 0.02) for strong PD-L1 positivity, 1.18 (95% CI: 0.96-1.46; p = 0.12) for weak PD-L1 positivity, and 1.23 (95% CI: 1.00-1.51; p = 0.05) for the combined strongly and weakly positive groups compared with PD-L1 negativity. Negative prognostic effect of PD-L1 expression was not statistically significant after adjustment for postoperative chemotherapy or radiotherapy. Similar results were observed for progression-free survival. Among stage I patients, the disease recurrence rate was higher in the PD-L1-positive versus in the PD-L1-negative group (48% versus 27%, p < 0.001), with an adjusted hazard ratio for disease-free survival of 2.01 (95% CI, 1.08-3.73; p = 0.03) for strong PD-L1 positivity and 1.57 (95% CI, 1.17-2.11; p = 0.003) for weak PD-L1 positivity compared with PD-L1 negativity. CONCLUSIONS: Tumor PD-L1 expression may be associated with poor prognosis in patients with NSCLC, although its significance weakens when postoperative therapy is considered.

Programmed Death-Ligand 1 Expression and Response to the Anti-Programmed Death 1 Antibody Pembrolizumab in Melanoma.

Purpose Expression of programmed death-ligand 1 (PD-L1) is a potential predictive marker for response and outcome after treatment with anti-programmed death 1 (PD-1). This study explored the relationship between anti-PD-1 activity and PD-L1 expression in patients with advanced melanoma who were treated with pembrolizumab in the phase Ib KEYNOTE-001 study (clinical trial information: NCT01295827). Patients and Methods Six hundred fifty-five patients received pembrolizumab10 mg/kg once every 2 weeks or once every 3 weeks, or 2 mg/kg once every 3 weeks. Tumor response was assessed every 12 weeks per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 by independent central review. Primary outcome was objective response rate. Secondary outcomes included progression-free survival (PFS) and overall survival (OS). Membranous PD-L1 expression in tumor and tumor-associated immune cells was assessed by a clinical trial immunohistochemistry assay (22C3 antibody) and scored on a unique melanoma (MEL) scale of 0 to 5 by one of three pathologists who were blinded to clinical outcome; a score ≥ 2 (membranous staining in ≥ 1% of cells) was considered positive. Results Of 451 patients with evaluable PD-L1 expression, 344 (76%) had PD-L1-positive tumors. Demographic and staging variables were equally distributed among PD-L1-positive and -negative patients. An association between higher MEL score and higher response rate and longer PFS (hazard ratio, 0.76; 95% CI, 0.71 to 0.82) and OS (hazard ratio, 0.76; 95% CI, 0.69 to 0.83) was observed ( P < .001 for each). Objective response rate was 8%, 12%, 22%, 43%, 57%, and 53% for MEL 0, 1, 2, 3, 4, and 5, respectively. Conclusion PD-L1 expression in pretreatment tumor biopsy samples was correlated with response rate, PFS, and OS; however, patients with PD-L1-negative tumors may also achieve durable responses.

Antitumor Activity of Pembrolizumab in Biomarker-Unselected Patients With Recurrent and/or Metastatic Head and Neck Squamous Cell Carcinoma: Results From the Phase Ib KEYNOTE-012 Expansion Cohort.

Purpose Treatment with pembrolizumab, an anti-programmed death-1 antibody, at 10 mg/kg administered once every 2 weeks, displayed durable antitumor activity in programmed death-ligand 1 (PD-L1) -positive recurrent and/or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) in the KEYNOTE-012 trial. Results from the expansion cohort, in which patients with HNSCC, irrespective of biomarker status, received a fixed dose of pembrolizumab at a less frequent dosing schedule, are reported. Patients and Methods Patients with R/M HNSCC, irrespective of PD-L1 or human papillomavirus status, received pembrolizumab 200 mg intravenously once every 3 weeks. Imaging was performed every 8 weeks. Primary end points were overall response rate (ORR) per central imaging vendor (Response Evaluation Criteria in Solid Tumors v1.1) and safety. Secondary end points included progression-free survival, overall survival, and association of response and PD-L1 expression. Patients who received one or more doses of pembrolizumab were included in analyses. Results Of 132 patients enrolled, median age was 60 years (range, 25 to 84 years), 83% were male, and 57% received two or more lines of therapy for R/M disease. ORR was 18% (95% CI, 12 to 26) by central imaging vendor and 20% (95% CI, 13 to 28) by investigator review. Median duration of response was not reached (range, ≥ 2 to ≥ 11 months). Six-month progression-free survival and overall survival rates were 23% and 59%, respectively. By using tumor and immune cells, a statistically significant increase in ORR was observed for PD-L1-positive versus -negative patients (22% v 4%; P = .021). Treatment-related adverse events of any grade and grade ≥ 3 events occurred in 62% and 9% of patients, respectively. Conclusion Fixed-dose pembrolizumab 200 mg administered once every 3 weeks was well tolerated and yielded a clinically meaningful ORR with evidence of durable responses, which supports further development of this regimen in patients with advanced HNSCC.

X-tile: a new bio-informatics tool for biomarker assessment and outcome-based cut-point optimization.

The ability to parse tumors into subsets based on biomarker expression has many clinical applications; however, there is no global way to visualize the best cut-points for creating such divisions. We have developed a graphical method, the X-tile plot that illustrates the presence of substantial tumor subpopulations and shows the robustness of the relationship between a biomarker and outcome by construction of a two dimensional projection of every possible subpopulation. We validate X-tile plots by examining the expression of several established prognostic markers (human epidermal growth factor receptor-2, estrogen receptor, p53 expression, patient age, tumor size, and node number) in cohorts of breast cancer patients and show how X-tile plots of each marker predict population subsets rooted in the known biology of their expression.