EVALUATING THE EFFECTS OF MONENSIN OVERDOSE IN DAIRY CATTLE

Monensin is approved as a feed additive by the FDA Center for Veterinary Medicine to increase milk production efficiency in lactating dairy cattle. To assess the effects of a gross error in mixing monensin into cattle feed, a 10-fold overdose was given for three consecutive days to naïve cows as well as cows previously dosed with monensin within the label range. Cows were evaluated during the overdose and for a subsequent 4 week observation period. Physiological variables were analyzed, including dry matter intake, body weight, body condition score, and serum chemistry profile. Production variables were analyzed, including milk yield and milk composition. Cows were blocked according to pre-treatment milk output, days in milk, and body condition. Results were analyzed using linear mixed model methodology with a baseline covariate. The study provided information for the veterinarian and the dairy farmer for determining whether an overdose may have occurred, for assessing the prognosis, and for deciding whether to continue feeding monensin immediately following an overdose

EGFR Gene Copy Number by FISH May Predict Outcome of Necitumumab in Squamous Lung Carcinomas: Analysis from the SQUIRE Study.

Necitumumab is a monoclonal antibody targeting EGFR. In the SQUIRE trial, the addition of necitumumab to chemotherapy for squamous cell lung cancer significantly improved overall survival (OS) (hazard ratio [HR] = 0.84); in a post hoc analysis, EGFR copy number gain determined by fluorescence in situ hybridization (FISH) showed a trend toward improved OS (HR = 0.70) and progression-free survival (PFS) (HR = 0.71) with the addition of necitumumab. We present the analysis of granular EGFR FISH data from SQUIRE to examine the potential predictive role of high polysomy and gene amplification, as both were included in the FISH-positive category. Available specimens from SQUIRE underwent FISH analysis in a central laboratory, and each sample was evaluated by using the Colorado EGFR scoring criteria. The correlation of granular FISH parameters with clinical outcomes was assessed. Samples were available for 557 of 1093 patients; 208 patients (37.3%) were FISH-positive, including 167 (30.0%) with high polysomy and 41 (7.4%) with gene amplification. In patients with high polysomy, the addition of necitumumab resulted in a statistically significant increase in PFS (6.08 versus 5.13 months [p = 0.044]) and nonstatistically significant increase in OS (12.6 versus 9.5 months [p = 0.133]); among patients with gene amplification, the addition of necitumumab did not significantly improve PFS (7.4 versus 5.6 months; [p = 0.334]) but did improve OS (14.8 versus 7.6 months; [p = 0.033]). EGFR copy number gain by FISH might have a role as a predictive biomarker for necitumumab in squamous cell lung cancer. In our opinion, these data encourage further studies to prospectively evaluate this potential biomarker

Brief Report: Randomized Phase 2 Studies of Checkpoint Inhibitors Alone or in Combination with Pegilodecakin in Patients with Metastatic Non-Small-Cell Lung Cancer (CYPRESS-1 and CYPRESS-2)

INTRODUCTION: Checkpoint inhibitors (CPI) have been approved to treat metastatic NSCLC. Pegilodecakin+CPI suggested promising efficacy in phase 1 IVY, providing rationale for randomized phase 2 trials CYPRESS-1 and CYPRESS-2. METHODS: CYPRESS-1 (N=101) and CYPRESS-2 (N=52) included ECOG 0-1, 1L/2L metastatic NSCLC, respectively, without known EGFR/ALK mutations. Patients were randomized 1:1; control arms received pembrolizumab (CYPRESS-1) or nivolumab (CYPRESS-2); experimental arms received pegilodecakin+CPI. Patients had PD-L1 tumor proportion score (TPS) ≥50% (CYPRESS-1) or 0-49% (CYPRESS-2). Primary endpoint was ORR per investigator. Secondary endpoints included PFS, OS, and safety. Exploratory endpoints included immune activation biomarkers. RESULTS: Median follow-up for CYPRESS-1 and CYPRESS-2 was 10.0 and 11.6 months, respectively. Results for pegilodecakin+pembrolizumab versus pembrolizumab were: ORR per investigator 47%vs.44% (Odds ratio:1.1;95%CI[0.5,2.5]); mPFS 6.3vs.6.1 months (HR:0.937;95%CI[0.541,1.625]); and mOS 16.3 months vs. not reached (HR:1.507;95%CI[0.708,3.209]). Results per blinded independent central review (BICR) were consistent. Treatment discontinuation rate due to AEs doubled in the experimental arm (32%vs.15%). Gr≥3 treatment related adverse events (TRAEs)(62%vs.19%) included anemia(20%vs.0%) and thrombocytopenia(12%vs.2%). Results for pegilodecakin+nivolumab versus nivolumab were: ORR per investigator 15%vs.12% (Odds ratio:1.2;95%CI[0.3,5.9]); mPFS 1.9vs.1.9 months (HR:1.006;95%CI[0.519,1.951]); and mOS 6.7vs.10.7 months (HR:1.871;95%CI[0.772,4.532]). Gr≥3 TRAEs (70.4%vs.16.7%) included anemia(40.7%vs.0%), fatigue(18%vs.0%), and thrombocytopenia(14.8%vs.0%). Biomarker data suggested activation of immunostimulatory signals of IL-10R pathway in pegilodecakin-containing arms. CONCLUSION: Despite evidence of biological effect in peripheral blood, adding pegilodecakin to CPI did not improve ORR, PFS, or OS, in 1L/2L NSCLC. Pegilodecakin+CPI demonstrated overall higher toxicity compared to CPI alone, leading to doubling of treatment discontinuation rate due to AEs

Phase II study of pemetrexed and cisplatin plus cetuximab followed by pemetrexed and cetuximab maintenance therapy in patients with advanced nonsquamous non-small cell lung cancer

Objectives: The aim was to determine if combined pemetrexed, cisplatin, and cetuximab was efficacious and safe as first-line treatment in advanced nonsquamous non-small cell lung cancer (NSCLC). Patients and methods: In this single-arm, multicenter clinical trial, patients with Stage IIIB/IV nonsquamous NSCLC received first-line therapy consisting of pemetrexed (500 mg/m(2)) and cisplatin (75 mg/m2) on Day 1 (21-day cycles) plus weekly cetuximab (400 mg/m(2) loading dose, then 250 mg/m(2)) for 4-6 cycles. Non-progressing patients received maintenance therapy consisting of pemetrexed and cetuximab as above until disease progression. All patients received vitamin supplementation, dexamethasone, and antihistamine prophylaxis. The primary endpoint was objective response rate (ORR). Secondary endpoints were progression-free survival (PFS), 1-year survival rate, translational research (TR) and safety. Results: Of the 113 patients receiving study drug, 109 were protocol-qualified. All patients completed >= 1 cycle of induction, and 51 (45%) and 49 (43%) patients completed >= 1 cycle of maintenance with pemetrexed and cetuximab, respectively. The ORR (n=109) was 38.5% (80% confidence interval [CI], 32.3-45.1%), all partial responses. Median PFS was 5.8 (80% CI, 4.4-6.7) months. One-year survival rate was 45% (80% CI, 39-51%). In exploratory analyses, there was some preliminary evidence of potential prognostic relationships with efficacy outcomes for epidermal growth factor receptor and thyroid transcription factor-1 protein expression, but not for KRAS mutation or for thymidylate synthase or folate receptor-alpha protein expression. Seventy-three (64.6%) patients had study drug-related Grade 3/4 adverse events (AEs). Drug-related serious AEs were reported in 31 (27.4%) patients. There were 3 (2.7%) potentially drug-related deaths on-study or within 30 days of follow up. Conclusion: Pemetrexed, cisplatin, and cetuximab appeared efficacious and tolerable in advanced nonsquamous NSCLC patients. The TR outcomes are hypothesis-generating given the study’s size and nonrandomized nature. (C) 2013 Elsevier Ireland Ltd. All rights reserved

Tumor Cell Expression of Vascular Endothelial Growth Factor Receptor 2 Is an Adverse Prognostic Factor in Patients with Squamous Cell Carcinoma of the Lung

<div><p>A robust immunohistochemical (IHC) assay for VEGFR2 was developed to investigate its utility for patient tailoring in clinical trials. The sensitivity, specificity, and selectivity of the IHC assay were established by siRNA knockdown, immunoblotting, mass spectrometry, and pre-absorption experiments. Characterization of the assay included screening a panel of multiple human cancer tissues and an independent cohort of non-small cell lung carcinoma (NSCLC, n = 118) characterized by TTF-1, p63, CK5/6, and CK7 IHC. VEGFR2 immunoreactivity was interpreted qualitatively (VEGFR2 positive/negative) in blood vessels and by semi-quantitative evaluation using H-scores in tumor cells (0–300). Associations were determined among combinations of VEGFR2 expression in blood vessels and tumor cells, and clinico-pathologic characteristics (age, sex, race, histologic subtype, disease stage) and overall survival using Kaplan-Meier analyses and appropriate statistical models. VEGFR2 expression both in blood vessels and in tumor cells in carcinomas of the lung, cervix, larynx, breast, and others was demonstrated. In the validation cohort, 99/118 (83.9%) NSCLC tissues expressed VEGFR2 in the blood vessels and 46/118 (39.0%) showed high tumor cell positivity (H-score ≥10). Vascular and tumor cell expression were inversely correlated (<i>p</i> = 0.0175). High tumor cell expression of VEGFR2 was associated with a 3.7-fold reduction in median overall survival in lung squamous-cell carcinoma (SCC, n = 25, <i>p</i> = 0.0134). The inverse correlation between vascular and tumor cell expression of VEGFR2 and the adverse prognosis associated with high VEGFR2 expression in immunohistochemically characterized pulmonary SCC are new findings with potential therapeutic implications. The robustness of this novel IHC assay will support further evaluation of its utility for patient tailoring in clinical trials of antiangiogenic agents.</p></div

Biomarker analyses in REGARD gastric/GEJ carcinoma patients treated with VEGFR2-targeted antibody ramucirumab

Background: Angiogenesis inhibition is an important strategy for cancer treatment. Ramucirumab, a human IgG1 monoclonal antibody that targets VEGF receptor 2 (VEGFR2), inhibits VEGF-A, -C, -D binding and endothelial cell proliferation. To attempt to identify prognostic and predictive biomarkers, retrospective analyses were used to assess tumour (HER2, VEGFR2) and serum (VEGF-C and -D, and soluble (s) VEGFR1 and 3) biomarkers in phase 3 REGARD patients with metastatic gastric/gastroesophageal junction carcinoma. Methods: A total of 152 out of 355 (43%) patients randomised to ramucirumab or placebo had ⩾1 evaluable biomarker result using VEGFR2 immunohistochemistry or HER2, immunohistochemistry or FISH, of blinded baseline tumour tissue samples. Serum samples (32 patients, 9%) were assayed for VEGF-C and -D, and sVEGFR1 and 3. Results: None of the biomarkers tested were associated with ramucirumab efficacy at a level of statistical significance. High VEGFR2 endothelial expression was associated with a non-significant prognostic trend toward shorter progression-free survival (high vs low HR=1.65, 95% CI=0.84,3.23). Treatment with ramucirumab was associated with a trend toward improved survival in both high (HR=0.69, 95% CI=0.38, 1.22) and low (HR=0.73, 95% CI=0.42, 1.26) VEGFR2 subgroups. The benefit associated with ramucirumab did not appear to differ by tumoural HER2 expression. Conclusions: REGARD exploratory analyses did not identify a strong potentially predictive biomarker of ramucirumab efficacy; however, statistical power was limited

VEGFR2 IHC is specific and selective against other VEGFR family members.

<p>The anti-VEGFR2 55B11 antibody specifically recognizes a unique epitope in the sequence HSDDTDTTVYSSEEA that is harbored within the c-terminal region. (A) HIS-tagged recombinant proteins corresponding to the intracellular domains of VEGFR1, VEGFR2, and VEGFR3 were incubated at 200-fold molar excess with 55B11 prior to IHC on serial sections of ductal carcinoma of the breast. VEGFR2 and not VEGFR1 or VEGFR3 pre-absorbed the antibody leading to negative immunoreactivity. (B) Recombinant 21 or 22-mer peptides corresponding to contiguous stretches of the immunogenic sequence of 55B11 were generated as indicated in the schematic (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080292#pone.0080292.s002" target="_blank">Table S2</a>). Each peptide was incubated at 200-fold molar excess with 55B11 prior to IHC on serial sections of pulmonary SCC. Pre-incubation with peptides 6 and 12 abolished immunoreactivity, while pre-incubation with all other peptides had no effect. The results shown for peptides 5 and 13 are representative of peptides 1–5, 7–11, and 13. (C) Recombinant 10 or 11-mer peptides corresponding to contiguous stretches of the consensus sequence of peptides 6 and 12 were generated as indicated in the schematic. Each peptide was incubated at 200-fold molar excess with 55B11 prior to IHC on serial sections of pulmonary SCC, a unique patient sample than in panel B. Pre-incubation with peptides 6 (data not shown), 12 and 15 abolished all immunoreactivity, while pre-incubation with peptides 14, 17 and 18 had no effect. Pre-incubation with peptide 16 showed an attenuated effect. All slides were counterstained with hematoxylin. Original magnification, ×200. Scale bars: 50 µm.</p

Histopathological subtypes and VEGFR2 IHC immunoreactivity in a multi-tissue screening TMA cohort, n = 50.

<p>Pos, immunopositivity.</p

Refinement of IHC assay parameters was integral to detect subcellular immunoreactivity in tumor cells.

<p>Representative immunoreactivity is shown on serial sections of a pulmonary SCC. Slides were subjected to heat induced epitope retrieval (HIER) conditions using buffers of differing pH, followed by an optimized IHC protocol using 55B11: primary antibody reagent negative control (A), Tris buffer (B), citrate buffer (C), and EDTA buffer (D). VEGFR2 immunoreactivity in endothelial cells lining blood vessels (black arrows) and membranous (black arrowheads), cytoplasmic, and nuclear compartments of malignant tumor cells was found to be most optimal with EDTA buffer (panel D). Slides were counterstained with hematoxylin. Original magnification ×200. Scale bar: 50 µm, applicable to all panels.</p