First-line avelumab plus chemotherapy in patients with advanced solid tumors: results from the phase 1b/2 JAVELIN Chemotherapy Medley study.

INTRODUCTION: Chemotherapy can potentially enhance the activity of immune checkpoint inhibitors by promoting immune priming. The phase 1b/2 JAVELIN Chemotherapy Medley trial evaluated first-line avelumab + concurrent chemotherapy in patients with advanced urothelial carcinoma or nonsmall cell lung cancer (NSCLC). PATIENTS AND METHODS: Avelumab 800 mg or 1200 mg was administered continuously every 3 weeks (Q3W) with standard doses of cisplatin + gemcitabine in patients with urothelial carcinoma, or carboplatin + pemetrexed in patients with nonsquamous NSCLC. Dual primary endpoints were dose-limiting toxicity (DLT; phase 1b) and confirmed objective response (phase 1b/2). RESULTS: In phase 1b, urothelial carcinoma and NSCLC cohorts received avelumab 800 mg (n=13 and n=6, respectively) or 1200 mg (n=6 each) + chemotherapy. In evaluable patients with urothelial carcinoma treated with avelumab 800 mg or 1200 mg + chemotherapy, DLT occurred in 1/12 (8.3%) and 1/6 (16.7%), respectively; no DLT occurred in the NSCLC cohort. In phase 2, 35 additional patients with urothelial carcinoma received avelumab 1200 mg + chemotherapy. Across all treated patients, safety profiles were similar irrespective of avelumab dose. Objective response rates (95% confidence internal) with avelumab 800 mg or 1200 mg + chemotherapy, respectively, across phase 1b/2, were 53.8% (25.1-80.8) and 39.0% (24.2-55.5) in urothelial carcinoma, and 50.0% (11.8-88.2) and 33.3% (4.3-77.7) in NSCLC. CONCLUSIONS: Preliminary efficacy and safety findings with avelumab + chemotherapy in urothelial carcinoma and NSCLC were consistent with previous studies of similar combination regimens. Conclusions about clinical activity are limited by small patient numbers. CLINICALTRIALS: gov identifier, NCT03317496.

Crizotinib in Patients With MET-Amplified NSCLC.

INTRODUCTION: MET amplification is a rare, potentially actionable, primary oncogenic driver in patients with NSCLC. METHODS: The influence of MET amplification on the clinical activity of the ALK, ROS1, and MET inhibitor, crizotinib (250 mg twice daily), was examined in patients with NSCLC (NCT00585195) who were enrolled into high (≥4 MET-to-CEP7 ratio), medium (>2.2 to <4 MET-to-CEP7 ratio), or low (≥1.8 to ≤2.2 MET-to-CEP7 ratio) amplification categories. Retrospective next-generation sequencing profiling was performed on archival tumor tissue. End points included objective response rate (ORR), duration of response, and progression-free survival. RESULTS: A total of 38 patients with a MET-to-CEP7 ratio greater than or equal to 1.8 by local fluorescence in situ hybridization testing received crizotinib. All patients were response-assessable, among whom 21, 14, and 3 had high, medium, and low MET amplification, respectively. ORRs of 8 of 21 (38.1%), 2 of 14 (14.3%), and 1 of 3 (33.3%), median duration of response of 5.2, 3.8, and 12.2 months, and median progression-free survival values of 6.7, 1.9, and 1.8 months were observed for those with high, medium, and low MET amplification, respectively. MET amplification gene copy number greater than or equal to 6 was detected by next-generation sequencing in 15 of 19 (78.9%) analyzable patients. Of these 15 patients, objective responses were observed in six (40%), two of whom had concurrent MET exon 14 alterations. No responses were observed among five patients with concurrent KRAS, BRAF, or EGFR mutations. CONCLUSIONS: Patients with high-level, MET-amplified NSCLC responded to crizotinib with the highest ORR. Use of combined diagnostics for MET and other oncogenes may potentially identify patients most likely to respond to crizotinib.

Angiogenic and immunomodulatory biomarkers in axitinib-treated patients with advanced renal cell carcinoma.

Aim: Immunomodulatory mechanisms contributing to angiogenic inhibition in renal tumors are not well characterized. We report associations between efficacy and tumor-associated immune cells and mRNA/miRNA expression in patients from AXIS. Materials & methods: Immunohistochemistry (n = 52) and mRNA/miRNA expression analyses (n = 72) were performed on tumor samples. Results: In axitinib-treated patients, higher CXCR4 and TLR3 expression, respectively, was associated with longer progression-free survival (hazard ratio; 95% CI: 0.3; 0.1-0.8 and 0.4; 0.2-0.9) and showed interaction with treatment (p = 0.029 and p < 0.001); lower CCR7 expression was associated with objective response (odds ratio: 0.1; 95% CI: 0.01-1.0) and longer overall survival (hazard ratio: 3.9; 95% CI: 1.4-10.3). Conclusion: CCR7, CXCR4 and TLR3 expression levels may be prognostic/predictive of clinical benefit with axitinib. Clinical trial identifier: ClinicalTrials.gov NCT00678392.

Antitumor activity of crizotinib in lung cancers harboring a MET exon 14 alteration.

MET exon 14 alterations are oncogenic drivers of non-small-cell lung cancers (NSCLCs)1. These alterations are associated with increased MET activity and preclinical sensitivity to MET inhibition2. Crizotinib is a multikinase inhibitor with potent activity against MET3. The antitumor activity and safety of crizotinib were assessed in 69 patients with advanced NSCLCs harboring MET exon 14 alterations. Objective response rate was 32% (95% confidence interval (CI), 21-45) among 65 response-evaluable patients. Objective responses were observed independent of the molecular heterogeneity that characterizes these cancers and did not vary by splice-site region and mutation type of the MET exon 14 alteration, concurrent increased MET copy number or the detection of a MET exon 14 alteration in circulating tumor DNA. The median duration of response was 9.1 months (95% CI, 6.4-12.7). The median progression-free survival was 7.3 months (95% CI, 5.4-9.1). MET exon 14 alteration defines a molecular subgroup of NSCLCs for which MET inhibition with crizotinib is active. These results address an unmet need for targeted therapy in people with lung cancers with MET exon 14 alterations and adds to an expanding list of genomically driven therapies for oncogenic subsets of NSCLC.

Detecting Gene Rearrangements in Patient Populations Through a 2-Step Diagnostic Test Comprised of Rapid IHC Enrichment Followed by Sensitive Next-Generation Sequencing.

Targeted therapy combined with companion diagnostics has led to the advancement of next-generation sequencing (NGS) for detection of molecular alterations. However, using a diagnostic test to identify patient populations with low prevalence molecular alterations, such as gene rearrangements, poses efficiency, and cost challenges. To address this, we have developed a 2-step diagnostic test to identify NTRK1, NTRK2, NTRK3, ROS1, and ALK rearrangements in formalin-fixed paraffin-embedded clinical specimens. This test is comprised of immunohistochemistry screening using a pan-receptor tyrosine kinase cocktail of antibodies to identify samples expressing TrkA (encoded by NTRK1), TrkB (encoded by NTRK2), TrkC (encoded by NTRK3), ROS1, and ALK followed by an RNA-based anchored multiplex polymerase chain reaction NGS assay. We demonstrate that the NGS assay is accurate and reproducible in identification of gene rearrangements. Furthermore, implementation of an RNA quality control metric to assess the presence of amplifiable nucleic acid input material enables a measure of confidence when an NGS result is negative for gene rearrangements. Finally, we demonstrate that performing a pan-receptor tyrosine kinase immunohistochemistry staining enriches detection of the patient population for gene rearrangements from 4% to 9% and has a 100% negative predictive value. Together, this 2-step assay is an efficient method for detection of gene rearrangements in both clinical testing and studies of archival formalin-fixed paraffin-embedded specimens.

Spatiotemporal regulation of epithelial-mesenchymal transition is essential for squamous cell carcinoma metastasis.

Epithelial-mesenchymal transition (EMT) is implicated in converting stationary epithelial tumor cells into motile mesenchymal cells during metastasis. However, the involvement of EMT in metastasis is still controversial, due to the lack of a mesenchymal phenotype in human carcinoma metastases. Using a spontaneous squamous cell carcinoma mouse model, we show that activation of the EMT-inducing transcription factor Twist1 is sufficient to promote carcinoma cells to undergo EMT and disseminate into blood circulation. Importantly, in distant sites, turning off Twist1 to allow reversion of EMT is essential for disseminated tumor cells to proliferate and form metastases. Our study demonstrates in vivo the requirement of "reversible EMT" in tumor metastasis and may resolve the controversy on the importance of EMT in carcinoma metastasis.

A Src-Tks5 pathway is required for neural crest cell migration during embryonic development.

In the adult organism, cell migration is required for physiological processes such as angiogenesis and immune surveillance, as well as pathological events such as tumor metastasis. The adaptor protein and Src substrate Tks5 is necessary for cancer cell migration through extracellular matrix in vitro and tumorigenicity in vivo. However, a role for Tks5 during embryonic development, where cell migration is essential, has not been examined. We used morpholinos to reduce Tks5 expression in zebrafish embryos, and observed developmental defects, most prominently in neural crest-derived tissues such as craniofacial structures and pigmentation. The Tks5 morphant phenotype was rescued by expression of mammalian Tks5, but not by a variant of Tks5 in which the Src phosphorylation sites have been mutated. We further evaluated the role of Tks5 in neural crest cells and neural crest-derived tissues and found that loss of Tks5 impaired their ventral migration. Inhibition of Src family kinases also led to abnormal ventral patterning of neural crest cells and their derivatives. We confirmed that these effects were likely to be cell autonomous by shRNA-mediated knockdown of Tks5 in a murine neural crest stem cell line. Tks5 was required for neural crest cell migration in vitro, and both Src and Tks5 were required for the formation of actin-rich structures with similarity to podosomes. Additionally, we observed that neural crest cells formed Src-Tks5-dependent cell protrusions in 3-D culture conditions and in vivo. These results reveal an important and novel role for the Src-Tks5 pathway in neural crest cell migration during embryonic development. Furthermore, our data suggests that this pathway regulates neural crest cell migration through the generation of actin-rich pro-migratory structures, implying that similar mechanisms are used to control cell migration during embryogenesis and cancer metastasis.

The 'ins' and 'outs' of podosomes and invadopodia: characteristics, formation and function.

Podosomes and invadopodia are actin-based dynamic protrusions of the plasma membrane of metazoan cells that represent sites of attachment to - and degradation of - the extracellular matrix. The key proteins in these structures include the actin regulators cortactin and neural Wiskott-Aldrich syndrome protein (N-WASP), the adaptor proteins Tyr kinase substrate with four SH3 domains (TKS4) and Tyr kinase substrate with five SH3 domains (TKS5), and the metalloprotease membrane type 1 matrix metalloprotease (MT1MMP; also known as MMP14). Many cell types can produce these structures, including invasive cancer cells, vascular smooth muscle and endothelial cells, and immune cells such as macrophages and dendritic cells. Recently, progress has been made in our understanding of the regulatory and functional aspects of podosome and invadopodium biology and their role in human disease.

Activated STAT3 is a mediator and biomarker of VEGF endothelial activation.

STAT3 plays important roles in cell proliferation and survival signaling and is often constitutively activated in transformed cells. In this study, we examined STAT3 activation in endothelial cells (EC) during angiogenic activation and therapeutic angiogenesis inhibition. VEGF stimulation of cultured EC induced STAT3 phosphorylation by a VEGFR2- and Src-dependent mechanism. FGF2 but not PlGF also induced EC STAT3 activation in vitro. Activated STAT3 mediated VEGF induction of EC Bcl-2 and contributed to VEGF protection of EC from apoptosis. In vivo, p-STAT3 was absent by immunohistological staining in the vascular EC of most normal mouse organs but was present in the vessels of mouse and human tumors. Tumor vascular p-STAT3 increased as tumors were induced to overexpress VEGF, indicating that VEGF is an activator of EC p-STAT3 in vivo. Tumor vascular p-STAT3 decreased during angiogenesis inhibition by antagonists of VEGF-VEGFR signaling, VEGF Trap and SU5416, indicating that VEGF contributed to the EC STAT3 activation seen in the tumors prior to treatment and that p-STAT3 may be used to monitor therapy. These studies show that p-STAT3 is a mediator and biomarker of endothelial activation that reports VEGF-VEGFR2 activity and may be useful for studying the pharmacodynamics of targeted angiogenesis inhibitors.

Inhibition of tumor endothelial ERK activation, angiogenesis, and tumor growth by sorafenib (BAY43-9006).

Activation of the Raf-MEK-ERK signal transduction pathway in endothelial cells is required for angiogenesis. Raf is the kinase most efficiently inhibited by the multikinase inhibitor sorafenib, which has shown activity against certain human cancers in clinical trials. To understand the mechanisms underlying this activity, we studied how it controlled growth of K1735 murine melanomas. Therapy caused massive regional tumor cell death accompanied by severe tumor hypoxia, decreased microvessel density, increased percentage of pericyte-covered vessels, and increased caliber and decreased arborization of vessels. These signs of K1735 angiogenesis inhibition, along with its ability to inhibit Matrigel neovascularization, showed that sorafenib is an effective anti-angiogenic agent. Extracellular signal-regulated kinase (ERK) activation in tumor endothelial cells, revealed by immunostaining for phospho-ERK and CD34, was inhibited, whereas AKT activation, revealed by phospho-AKT immunostaining, was not inhibited in K1735 and two other tumor types treated with sorafenib. Treatment decreased endothelial but not tumor cell proliferation and increased both endothelial cell and tumor cell apoptosis. These data indicate that sorafenib's anti-tumor efficacy may be primarily attributable to angiogenesis inhibition resulting from its inhibition of Raf-MEK-ERK signaling in endothelial cells. Assessing endothelial cell ERK activation in tumor bio-psies may provide mechanistic insights into and allow monitoring of sorafenib's activity in patients in clinical trials.

Neovasculogenic therapy to augment perfusion and preserve viability in ischemic cardiomyopathy.

BACKGROUND: Ischemic cardiomyopathy is a global health concern with limited therapy. We recently described endogenous revascularization utilizing granulocyte-macrophage colony stimulating factor (GMCSF) to induce endothelial progenitor cell (EPC) production and intramyocardial stromal cell-derived factor-1alpha (SDF) as a specific EPC chemokine. The EPC-mediated neovascularization and enhancement of myocardial function was observed. In this study we examined the regional biologic mechanisms underlying this therapy. METHODS: Lewis rats underwent left anterior descending coronary artery (LAD) ligation and developed ischemic cardiomyopathy over 6 weeks. Three weeks after ligation, the animals received either subcutaneous GMCSF and intramyocardial SDF injections or saline injections as control. Six weeks after LAD ligation circulating EPC density was studied by flow cytometry. Quadruple immunofluorescent vessel staining for mature, proliferating vasculature was performed. Confocal angiography was utilized to identify fluorescein lectin-lined vessels to assess perfusion. Ischemia reversal was studied by measuring myocardial adenosine triphosphate (ATP) levels. Myocardial viability was assayed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling detection of apoptosis and quantitation of myofilament density. RESULTS: The GMCSF/SDF therapy enhanced circulating leukocyte (13.1 +/- 4.5 x 10(6) vs 3.1 +/- 0.5 x 10(6)/cc, p = 0.001, n = 6) and EPC (14.2 +/- 6.6 vs 2.2 +/- 2.1/cc, p = 0.001, n = 6) concentrations. Tetraimmunofluorescent labeling demonstrated enhanced stable vasculature with this therapy (39.2 +/- 8.1 vs 25.4 +/- 5.1%, p = 0.006, n = 7). Enhanced perfusion was shown by confocal microangiography of borderzone lectin-labeled vessels (28.2 +/- 5.4 vs 11.5 +/- 3.0 vessels/high power field [hpf], p = 0.00001, n = 10). Ischemia reversal was demonstrated by enhanced cellular ATP levels in the GMCSF/SDF borderzone myocardium (102.5 +/- 31.0 vs 26.9 +/- 4.1 nmol/g, p = 0.008, n = 5). Borderzone cardiomyocyte viability was noted by decreased apoptosis (3.2 +/- 1.4% vs 5.4 +/- 1.0%, p = 0.004, n = 10) and enhanced cardiomyocyte density (40.0 +/- 5.6 vs 27.0 +/- 6 myofilaments/hpf, p = 0.01, n=10). CONCLUSIONS: Endogenous revascularization for ischemic cardiomyopathy utilizing GMCSF EPC upregulation and SDF EPC chemokinesis upregulates circulating EPCs, enhances vascular stability, and augments myocardial function by enhancing perfusion, reversing cellular ischemia, and increasing cardiomyocyte viability.