Personalized neoantigen vaccine and pembrolizumab in advanced hepatocellular carcinoma: a phase 1/2 trial.

Programmed cell death protein 1 (PD-1) inhibitors have modest efficacy as a monotherapy in hepatocellular carcinoma (HCC). A personalized therapeutic cancer vaccine (PTCV) may enhance responses to PD-1 inhibitors through the induction of tumor-specific immunity. We present results from a single-arm, open-label, phase 1/2 study of a DNA plasmid PTCV (GNOS-PV02) encoding up to 40 neoantigens coadministered with plasmid-encoded interleukin-12 plus pembrolizumab in patients with advanced HCC previously treated with a multityrosine kinase inhibitor. Safety and immunogenicity were assessed as primary endpoints, and treatment efficacy and feasibility were evaluated as secondary endpoints. The most common treatment-related adverse events were injection-site reactions, observed in 15 of 36 (41.6%) patients. No dose-limiting toxicities or treatment-related grade ≥3 events were observed. The objective response rate (modified intention-to-treat) per Response Evaluation Criteria in Solid Tumors 1.1 was 30.6% (11 of 36 patients), with 8.3% (3 of 36) of patients achieving a complete response. Clinical responses were associated with the number of neoantigens encoded in the vaccine. Neoantigen-specific T cell responses were confirmed in 19 of 22 (86.4%) evaluable patients by enzyme-linked immunosorbent spot assays. Multiparametric cellular profiling revealed active, proliferative and cytolytic vaccine-specific CD4+ and CD8+ effector T cells. T cell receptor ß-chain (TCRß) bulk sequencing results demonstrated vaccination-enriched T cell clone expansion and tumor infiltration. Single-cell analysis revealed posttreatment T cell clonal expansion of cytotoxic T cell phenotypes. TCR complementarity-determining region cloning of expanded T cell clones in the tumors following vaccination confirmed reactivity against vaccine-encoded neoantigens. Our results support the PTCV's mechanism of action based on the induction of antitumor T cells and show that a PTCV plus pembrolizumab has clinical activity in advanced HCC. ClinicalTrials.gov identifier: NCT04251117 .

Immunotherapy Targeting HPV16/18 Generates Potent Immune Responses in HPV-Associated Head and Neck Cancer.

PURPOSE: Clinical responses with programmed death (PD-1) receptor-directed antibodies occur in about 20% of patients with advanced head and neck squamous cell cancer (HNSCCa). Viral neoantigens, such as the E6/E7 proteins of HPV16/18, are attractive targets for therapeutic immunization and offer an immune activation strategy that may be complementary to PD-1 inhibition. PATIENTS AND METHODS: We report phase Ib/II safety, tolerability, and immunogenicity results of immunotherapy with MEDI0457 (DNA immunotherapy targeting HPV16/18 E6/E7 with IL12 encoding plasmids) delivered by electroporation with CELLECTRA constant current device. Twenty-two patients with locally advanced, p16+ HNSCCa received MEDI0457. RESULTS: MEDI0457 was associated with mild injection site reactions, but no treatment-related grade 3-5 adverse events (AE) were noted. Eighteen of 21 evaluable patients showed elevated antigen-specific T-cell activity by IFNγ ELISpot, and persistent cellular responses surpassing 100 spot-forming units (SFUs)/106 peripheral blood mononuclear cells (PBMCs) were noted out to 1 year. Induction of HPV-specific CD8+ T cells was observed. MEDI0457 shifted the CD8+/FoxP3+ ratio in 4 of 5 post immunotherapy tumor samples and increased the number of perforin+ immune infiltrates in all 5 patients. One patient developed metastatic disease and was treated with anti-PD-1 therapy with a rapid and durable complete response. Flow-cytometric analyses revealed induction of HPV16-specific PD-1+ CD8+ T cells that were not found prior to MEDI0547 (0% vs. 1.8%). CONCLUSIONS: These data demonstrate that MEDI0457 can generate durable HPV16/18 antigen-specific peripheral and tumor immune responses. This approach may be used as a complementary strategy to PD-1/PD-L1 inhibition in HPV-associated HNSCCa to improve therapeutic outcomes.

Sonic hedgehog pathway expression in non-small cell lung cancer.

OBJECTIVE: Activation of the hedgehog pathway is an important signaling mechanism crucial in embryogenesis and has strong links to carcinogenesis. This study investigates the expression of the Sonic hedgehog pathway molecules in non-small cell lung tumors as it relates to clinical outcome of various non-small cell lung cancers. METHODS: A tissue microarray with 81 samples from 42 patients with various non-small cell lung cancer histologies was examined without the aid of laser microdissection. All samples were stained with antibodies directed against Sonic hedgehog, Ptch-1, Smoothened, and Gli-1. RESULTS: Most of the tumor samples showed negative to weak expression of the pathway proteins (Sonic hedgehog, 38% negative to 20% weak; Ptch-1, 100% negative; Smoothened, 69% negative to 7% weak; Gli-1, 57% negative to 5% weak) compared with higher expression in normal lung epithelial cells. CONCLUSION: The same pathway expression did not correlate with clinical outcome. While our results do not provide any indication that the pathway molecules are correlated to overall patient survival possibly due to the limited sample size, our study shows minimum overexpression of Sonic hedgehog pathway in non-small cell lung cancer and this did not correlate clinically with patient outcome.

Anti-vascular endothelial growth factor treatment in combination with chemotherapy delays hematopoietic recovery due to decreased proliferation of bone marrow hematopoietic progenitor cells.

INTRODUCTION: Targeting of cancer by chemotherapy in combination with anti-vascular endothelial growth factor (VEGF) therapy has demonstrated not only the clinical efficacy but also a higher risk of serious hematologic complications including neutropenia. The purpose of the study was to elucidate the molecular mechanisms responsible for the development of neutropenia during the combination treatment. METHODS: Mouse model and in vitro studies were undertaken to determine the effect of interference with VEGF signaling by VEGF-specific agents or a multitargeted VEGF receptor (VEGFR) tyrosine kinase inhibitor on proliferation of hematopoietic progenitor cell (HPC) and repopulation of the hematopoietic compartment after myeloablation. RESULTS: The studies demonstrated that blockage of VEGFR1 or VEGFR2 signaling decreased HPC proliferation and impaired repopulation of the hematopoietic compartment after myelosuppression by slowing the progression of HPC through the cell cycle. The combination of cytotoxic drugs and VEGFR tyrosine kinase inhibitor had an additive inhibitory effect and decreased proliferation of HPC significantly stronger than either agent alone. CONCLUSIONS: Signaling through both VEGFR1 and VEGFR2 is required for normal reconstitution of the hematopoietic compartment after cytotoxic chemotherapy.

Impact of FDG PET/CT on delineation of the gross tumor volume for radiation planning in non-small-cell lung cancer.

PURPOSE: Computed tomography (CT) remains the gold standard for delineation of tumor volumes for radiotherapy (RT) planning. However, positron emission tomography (PET) overlay on CT has shown to impact the gross target volume (GTV), decrease intraobserver variability, and change the treatment planning in a significant number of patients. The objective of this study was to evaluate the influence and accuracy of FDG PET in GTV definition as a complementary modality to CT for patients with non-small-cell lung carcinoma at Vanderbilt University Medical Center. METHODS: Data from 11 consecutive patients with non-small-cell lung carcinoma, which were referred to FDG PET/CT for initial staging and RT planning were analyzed retrospectively. All patients had undergone routine staging using a RT noncontrasted CT. Both the RT CT and PET/CT images were acquired using standard protocols but with the patients positioned in the same RT immobilization devices. Both the CT and PET/CT images were transferred to the RT planning workstation for contouring. GTV, pathologic nodal and metastases volumes were first defined in the conventional manner based on RT CT. The FDG PET and RT CT planning image datasets were coregistered with the help of the transmission CT from PET/CT. FDG PET GTVs were determined by a team of radiation oncologists and nuclear physician with expertise in PET/CT, and displayed simultaneously with the CT contours. The RT CT and PET GTV were measured and the percent difference was calculated for the primary tumor, pathologic lymph nodes, and distant metastases. A difference of 15% was considered significant. RESULTS: The primary tumor GTV was decreased in 36% (n = 4) of patients by differentiating atelectasis and postobstructive pneumonia from tumor, and increased GTV in 27% (n = 3) of patients by detecting additional tumor burden. Increased nodal disease burden was detected in 18% (n = 2) of patients. The use of PET/CT changed treatment from curative to palliative by detecting distant metastasis in 27% (n = 3) of patients. CONCLUSIONS: Our results are consistent with the published data of PET/CT altering GTV in a significant number of patients, detecting tumor spread to additional lymph nodes and distant metastases. While these advantages support the use of PET/CT in RT planning, it remains unknown what impact this will have on patient outcomes.

Regulated cell death pathways: new twists in modulation of BCL2 family function.

A number of cell death pathways have been recognized. Though apoptosis and autophagy have been well characterized, programmed necrosis has recently received attention and may provide clinical alternatives to suppress resistant tumors. Necrosis is primarily characterized by large-scale permeabilization, swelling, and rupture of cell membranes and the release of pro-inflammatory cytokines. Traditionally, necrosis in cancer cells has been indicative of poor prognoses, as chronic inflammation was found to encourage tumor growth. Yet, many antitumor effects associated with necrosis have been discovered in certain settings, such as the formation of an effective antitumor immune response. In this way, finding ways to attenuate the pro-tumor effects of necrosis while engaging the antitumor pathways via drugs, radiation, and sensitization may prove valuable as a clinical focus for the future. We hypothesize that the use of Bcl-2 inhibitors may enhance necrotic death characterized by inflammation and antitumor immunity. In this article, we briefly review apoptosis and autophagy and reason how necrosis may be a suitable alternative therapeutic endpoint. We then highlight novel inhibitors of Bcl-2 that may provide clinical application of our hypothesis in the future.

A phase II study of celecoxib in combination with paclitaxel, carboplatin, and radiotherapy for patients with inoperable stage IIIA/B non-small cell lung cancer.

PURPOSE: Cyclooxygenase (COX)-2 up-regulation plays an important role in the pathogenesis of lung cancer. Selective COX-2 inhibitors have promoted chemosensitivity and radiosensitivity of tumor cells in preclinical trials. EXPERIMENTAL DESIGN: In a single-institution phase II study, we sought to determine the effectiveness of concurrent chemoradiation given with celecoxib and examined biomarkers to predict response to COX-2 inhibition. RESULTS: Seventeen patients with stage IIIA or IIIB non-small cell lung cancer (NSCLC) were enrolled in the study. All received 400 mg celecoxib twice daily continuously while on trial in addition to concurrent chemoradiation therapy with paclitaxel and carboplatin. Celecoxib was continued until disease progression. The overall objective response rate was 42.9%, and the median overall survival time was 203 days. In contrast to nonresponders, those patients with complete and partial responses had a significant decrease in the level of urinary 11alpha-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M), the major metabolite of prostaglandin E(2), after 1 week of celecoxib administration. Patients with very high levels of PGE-M before initiation of therapy also responded poorly to therapy. Serum vascular endothelial growth factor levels did not predict response or survival. CONCLUSION: The trial was terminated because it did not meet the predetermined goal of 80% overall response rate. In unselected patients, the addition of celecoxib to concurrent chemoradiotherapy with inoperable stage IIIA/B NSCLC does not improve survival. Urinary PGE-M is a promising biomarker for predicting response to COX-2 inhibition in NSCLC.

Thioredoxin-1 modulates transcription of cyclooxygenase-2 via hypoxia-inducible factor-1alpha in non-small cell lung cancer.

Hypoxic induction of gene expression occurs mainly via the hypoxia-inducible factor-1 (HIF-1) transcription factor and is a critical step in tumor growth. Cyclooxygenase-2 (COX-2) is commonly overexpressed in non-small cell lung cancer (NSCLC). In this study, we sought to determine the role of HIF-1 in the induction of COX-2 expression during hypoxia. Through sequence comparison of hypoxia-responsive genes, COX-2 promoter deletion analysis, and site-directed mutagenesis, we identified a hypoxia-responsive element within the COX-2 promoter that interacts with HIF-1alpha and underlies the mechanism of hypoxic activation of COX-2 in lung cancer cells. Proteomic analysis of NSCLC identified thioredoxin-1 as a redox protein overexpressed in NSCLC correlated with poor prognosis. We also show that thioredoxin-1 stabilizes HIF-1alpha to induce hypoxia-responsive genes under normoxic conditions. Our results identify two new mechanisms for regulation of COX-2 expression in NSCLC.

Targeting cyclooxygenase-2 in recurrent non-small cell lung cancer: a phase II trial of celecoxib and docetaxel.

Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in prostaglandin (PG) synthesis and is overexpressed in 70% to 90% of non-small cell lung cancers (NSCLC). Preclinical studies suggest inhibition of COX-2 can enhance the cytotoxic effect of docetaxel. To test this concept clinically, we administered celecoxib (400 mg p.o. twice daily) plus docetaxel (75 mg/m(2) every 3 weeks) to a cohort of patients with recurrent, previously treated NSCLC. Patients first received single agent celecoxib for 5 to 10 days to ascertain the effectiveness of COX-2 inhibition, which was determined by measuring pre- and post-celecoxib levels of urinary 11alpha-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M), the major metabolite of prostaglandin E(2) (PGE(2)). We enrolled 56 patients (35 men, 21 women; median age, 61 years). All patients had received at least one prior chemotherapy regimen. The overall response rate was 11% and median survival was 6 months, similar to that observed with docetaxel alone. Pre-celecoxib urinary PGE-M decreased from a mean level of 27.2 to 12.2 ng/mg Cr after 5 to 10 days of celecoxib (P = 0.001). When grouped by quartile, patients with the greatest proportional decline in urinary PGE-M levels experienced a longer survival compared to those with no change or an increase in PGE-M (14.8 versus 6.3 versus 5.0 months). Our data suggest that combining celecoxib with docetaxel using the doses and schedule employed does not improve survival in unselected patients with recurrent, previously treated NSCLC. However, in light of the apparent survival prolongation in the subset with a marked decline in urinary PGE-M levels, further investigation of strategies designed to decrease PGE(2) synthesis in NSCLC seems warranted.

Quantification of the major urinary metabolite of PGE2 by a liquid chromatographic/mass spectrometric assay: determination of cyclooxygenase-specific PGE2 synthesis in healthy humans and those with lung cancer.

Prostaglandin (PG)E2 is a major cyclooxygenase (COX) product that is important in human physiology and pathophysiology. Quantification of systemic PG production in humans is best assessed by measuring excreted urinary metabolites. Accurate and easy-to-perform assays to quantify the major urinary metabolite of PGE2, 11alpha-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M), do not exist. We now report the development of a robust and facile method to measure urinary PGE-M excretion in humans using stable isotope dilution techniques employing liquid chromatography/tandem mass spectrometry (LC/MS/MS). Concentrations of the metabolite in urine from healthy humans are nearly twofold greater in men than in women (10.4+/-1.5 vs. 6.0+/-0.7 ng/mg creatinine). Levels of PGE-M in healthy humans are suppressed significantly not only by the nonselective COX inhibitor ibuprofen but also by the COX-2 selective inhibitor rofecoxib, suggesting that the majority of PGE2 formed in vivo is derived from COX-2. Increased COX-2 expression and increased PGE2 production are associated with malignancy. Levels of PGE-M were found to be greatly increased in humans with unresectable non-small cell cancer of the lung, and this increase is dramatically reduced by administration of the COX-2 inhibitor celecoxib, implying that COX-2 contributes significantly to the overproduction of PGE2. In summary, quantification of PGE-M using LC/MS/MS provides a facile and accurate method to assess PGE2 formation in human physiological and pathophysiological processes.

Case-mixing effects on anagram solution.

The effects of mixed-case letters on anagram solution were investigated in 2 studies with college-aged participants. In Experiment 1, the participants attempted to solve anagrams printed in either mixed- or same-case letters. The results showed that mixed-case letters disrupted the solution process. To determine if this effect was a result of the inappropriate grouping of letters of the same case, the authors conducted Experiment 2 as a partial replication of Experiment 1. In Experiment 2, in some cases, uppercase letters in the mixed-case condition formed a word that was of a higher frequency than was the solution word. Once again mixed-case letters disrupted the solution process. However, the authors found no evidence for the hypothesis on the inappropriate grouping of letters as the disruption was independent of having an embedded word in the anagram.