Local Pentraxin-2 Deficit Is a Feature of Intestinal Fibrosis in Crohn's Disease.

BACKGROUND: Pentraxin-2 (PTX-2) is a homo-pentameric plasma protein showing evidence of antifibrotic activity in Phase 2 clinical trials in idiopathic pulmonary fibrosis (IPF). Whether PTX-2 plays a role in other fibrotic diseases, including intestinal fibrosis which commonly occurs in inflammatory bowel disease (IBD), remains unknown. AIMS: This study aimed to qualitatively and quantitatively assess PTX-2 expression in fibrostenotic Crohn's disease (FCD) and determine whether expression is correlated with postsurgical restenosis. METHODS: Immunohistochemistry was performed in histologic sections of small bowel resected from patients with fibrostenotic Crohn's disease (FCD), comparing strictured segments with adjacent surgical margins from the same patient. Ileal resections from patients without inflammatory bowel disease were examined as controls. RESULTS: PTX-2 signal was analyzed in 18 patients with FCD and 15 patients without IBD and localized predominantly to submucosal vasculature, including arterial subendothelium and internal elastic lamina, and perivascular connective tissue. PTX-2 signal in the surgical margins from patients with FCD strictures (where tissue architecture was normal) was consistently lower than non-IBD samples. Fibrostenotic regions showed increased PTX-2 signal relative to surgical margins from the same patient in 14/15 paired samples. Submucosal/mural PTX-2 signal in fibrostenotic tissue was lower in patients who subsequently experienced re-stenosis (P = 0.015). CONCLUSIONS: This exploratory study is the first analysis of PTX-2 within the intestine, and demonstrates that PTX-2 signal is reduced in the architecturally normal bowel of patients with FCD. Lower submucosal PTX-2 levels in patients with re-stenosis raises the possibility of a protective role of PTX-2 in intestinal fibrosis.

Synthetic Antigen Controls for Immunohistochemistry.

Immunohistochemistry (IHC) assays provide valuable insights into protein expression patterns, the reliable interpretation of which requires well-characterized positive and negative control samples. Because appropriate tissue or cell line controls are not always available, a simple method to create synthetic IHC controls may be beneficial. Such a method is described here. It is adaptable to various antigen types, including proteins, peptides, or oligonucleotides, in a wide range of concentrations. This protocol explains the steps necessary to create synthetic antigen controls, using as an example a peptide from the human erythroblastic oncogene B2 (ERBB2/HER2) intracellular domain (ICD) recognized by a variety of diagnostically relevant antibodies. Serial dilutions of the HER2 ICD peptide in bovine serum albumin (BSA) solution are mixed with formaldehyde and heated for 10 min at 85 °C to solidify and cross-link the peptide/BSA mixture. The resulting gel can be processed, sectioned, and stained like a tissue, yielding a series of samples of known antigen concentrations spanning a wide range of staining intensities. This simple protocol is consistent with routine histology lab procedures. The method requires only that the user have a sufficient quantity of the desired antigen. Recombinant proteins, protein domains, or linear peptides that encode relevant epitopes may be synthesized locally or commercially. Laboratories generating in-house antibodies can reserve aliquots of the immunizing antigen as the synthetic control target. The opportunity to create well-defined positive controls across a wide range of concentrations allows users to assess intra- and inter-laboratory assay performance, gain insight into the dynamic range and linearity of their assays, and optimize assay conditions for their particular experimental goals.

BCL2 Expression in First-Line Diffuse Large B-Cell Lymphoma Identifies a Patient Population With Poor Prognosis.

INTRODUCTION: The prognostic value of B-cell lymphoma 2 (BCL2) expression in de novo diffuse large B-cell lymphoma (DLBCL) treated with immunochemotherapy is of interest to define a target patient population for clinical development of BCL2 inhibitors. We aimed to develop a reproducible immunohistochemistry algorithm and assay to determine BCL2 protein expression and assess the prognostic value of BCL2 in newly diagnosed DLBCL cohorts. PATIENTS AND METHODS: The prospectively defined algorithm incorporated BCL2 staining intensity and percentage of BCL2-positive cells. Functionally relevant cutoffs were based on the sensitivity of lymphoma cell lines to venetoclax. This assay was highly reproducible across laboratories. The prognostic impact of BCL2 expression was assessed in DLBCL patients from the phase 3 MAIN (n = 230) and GOYA (n = 366) trials, and a population-based registry (n = 310). RESULTS: Approximately 50% of tumors were BCL2 positive, with a higher frequency in high International Prognostic Index (IPI) and activated B-cell-like DLBCL subgroups. BCL2 expression was associated with poorer progression-free survival in the MAIN study (hazard ratio [HR], 1.66; 95% confidence interval [CI], 0.81-3.40; multivariate Cox regression adjusted for IPI and cell of origin). This trend was confirmed in the GOYA and registry cohorts in adjusted multivariate analyses (GOYA: HR, 1.72; 95% CI, 1.05-2.82; registry: HR, 1.89; 95% CI, 1.29-2.78). Patients with BCL2 immunohistochemistry-positive and IPI-high disease had the poorest prognosis: 3-year progression-free survival rates were 51% (GOYA) and 37% (registry). CONCLUSION: Findings support use of our BCL2 immunohistochemistry scoring system and assay to select patients with BCL2-positive tumors for future studies.

Synthetic Antigen Gels as Practical Controls for Standardized and Quantitative Immunohistochemistry.

Optimization and standardization of immunohistochemistry (IHC) protocols within and between laboratories requires reproducible positive and negative control samples. In many situations, suitable tissue or cell line controls are not available. We demonstrate here a method to incorporate target antigens into synthetic protein gels that can serve as IHC controls. The method can use peptides, protein domains, or whole proteins as antigens, and is compatible with a variety of fixation protocols. The resulting gels can be used to create tissue microarrays (TMAs) with a range of antigen concentrations that can be used to objectively quantify and calibrate chromogenic, fluorescent, or mass spectrometry-based IHC protocols. The method offers an opportunity to objectively quantify IHC staining results, and to optimize and standardize IHC protocols within and between laboratories. (J Histochem Cytochem 58:XXX-XXX, 2019).

A cell identity switch allows residual BCC to survive Hedgehog pathway inhibition.

Despite the efficacy of Hedgehog pathway inhibitors in the treatment of basal cell carcinoma (BCC)1, residual disease persists in some patients and may contribute to relapse when treatment is discontinued2. Here, to study the effect of the Smoothened inhibitor vismodegib on tumour clearance, we have used a Ptch1-Trp53 mouse model of BCC3 and found that mice treated with vismodegib harbour quiescent residual tumours that regrow upon cessation of treatment. Profiling experiments revealed that residual BCCs initiate a transcriptional program that closely resembles that of stem cells of the interfollicular epidermis and isthmus, whereas untreated BCCs are more similar to the hair follicle bulge. This cell identity switch was enabled by a mostly permissive chromatin state accompanied by rapid Wnt pathway activation and reprogramming of super enhancers to drive activation of key transcription factors involved in cellular identity. Accordingly, treatment of BCC with both vismodegib and a Wnt pathway inhibitor reduced the residual tumour burden and enhanced differentiation. Our study identifies a resistance mechanism in which tumour cells evade treatment by adopting an alternative identity that does not rely on the original oncogenic driver for survival.

Monitoring and Targeting Anti-VEGF Induced Hypoxia within the Viable Tumor by 19F-MRI and Multispectral Analysis.

The effect of anti-angiogenic agents on tumor oxygenation has been in question for a number of years, where both increases and decreases in tumor pO2 have been observed. This dichotomy in results may be explained by the role of vessel normalization in the response of tumors to anti-angiogenic therapy, where anti-angiogenic therapies may initially improve both the structure and the function of tumor vessels, but more sustained or potent anti-angiogenic treatments will produce an anti-vascular response, producing a more hypoxic environment. The first goal of this study was to employ multispectral (MS) 19F-MRI to noninvasively quantify viable tumor pO2 and evaluate the ability of a high dose of an antibody to vascular endothelial growth factor (VEGF) to produce a strong and prolonged anti-vascular response that results in significant tumor hypoxia. The second goal of this study was to target the anti-VEGF induced hypoxic tumor micro-environment with an agent, tirapazamine (TPZ), which has been designed to target hypoxic regions of tumors. These goals have been successfully met, where an antibody that blocks both murine and human VEGF-A (B20.4.1.1) was found by MS 19F-MRI to produce a strong anti-vascular response and reduce viable tumor pO2 in an HM-7 xenograft model. TPZ was then employed to target the anti-VEGF-induced hypoxic region. The combination of anti-VEGF and TPZ strongly suppressed HM-7 tumor growth and was superior to control and both monotherapies. This study provides evidence that clinical trials combining anti-vascular agents with hypoxia-activated prodrugs should be considered to improved efficacy in cancer patients.

Phase I First-in-Human Study of Venetoclax in Patients With Relapsed or Refractory Non-Hodgkin Lymphoma.

Purpose B-cell leukemia/lymphoma-2 (BCL-2) overexpression is common in many non-Hodgkin lymphoma (NHL) subtypes. A phase I trial in patients with NHL was conducted to determine safety, pharmacokinetics, and efficacy of venetoclax, a selective, potent, orally bioavailable BCL-2 inhibitor. Patients and Methods A total of 106 patients with relapsed or refractory NHL received venetoclax once daily until progressive disease or unacceptable toxicity at target doses from 200 to 1,200 mg in dose-escalation and safety expansion cohorts. Treatment commenced with a 3-week dose ramp-up period for most patients in dose-escalation cohorts and for all patients in safety expansion. Results NHL subtypes included mantle cell lymphoma (MCL; n = 28), follicular lymphoma (FL; n = 29), diffuse large B-cell lymphoma (DLBCL; n = 34), DLBCL arising from chronic lymphocytic leukemia (Richter transformation; n = 7), Waldenström macroglobulinemia (n = 4), and marginal zone lymphoma (n = 3). Venetoclax was generally well tolerated. Clinical tumor lysis syndrome was not observed, whereas laboratory tumor lysis syndrome was documented in three patients. Treatment-emergent adverse events were reported in 103 patients (97%), a majority of which were grade 1 to 2 in severity. Grade 3 to 4 events were reported in 59 patients (56%), and the most common were hematologic, including anemia (15%), neutropenia (11%), and thrombocytopenia (9%). Overall response rate was 44% (MCL, 75%; FL, 38%; DLBCL, 18%). Estimated median progression-free survival was 6 months (MCL, 14 months; FL, 11 months; DLBCL, 1 month). Conclusion Selective targeting of BCL-2 with venetoclax was well tolerated, and single-agent activity varied among NHL subtypes. We determined 1,200 mg to be the recommended single-agent dose for future studies in FL and DLBCL, with 800 mg being sufficient to consistently achieve durable response in MCL. Additional investigations including combination therapy to augment response rates and durability are ongoing.

Phase I Study of GDC-0425, a Checkpoint Kinase 1 Inhibitor, in Combination with Gemcitabine in Patients with Refractory Solid Tumors.

Purpose: Chk1 inhibition potentiates DNA-damaging chemotherapy by overriding cell-cycle arrest and genome repair. This phase I study evaluated the Chk1 inhibitor GDC-0425 given in combination with gemcitabine to patients with advanced solid tumors.Experimental Design: Patients received GDC-0425 alone for a 1-week lead-in followed by 21-day cycles of gemcitabine plus GDC-0425. Gemcitabine was initially administered at 750 mg/m2 (Arm A), then increased to 1,000 mg/m2 (Arm B), on days 1 and 8 in a 3 + 3 + 3 dose escalation to establish maximum tolerated dose (MTD). GDC-0425 was initially administered daily for three consecutive days; however, dosing was abbreviated to a single day on the basis of pharmacokinetics and tolerability. TP53 mutations were evaluated in archival tumor tissue. On-treatment tumor biopsies underwent pharmacodynamic biomarker analyses.Results: Forty patients were treated with GDC-0425. The MTD of GDC-0425 was 60 mg when administered approximately 24 hours after gemcitabine 1,000 mg/m2 Dose-limiting toxicities included thrombocytopenia (n = 5), neutropenia (n = 4), dyspnea, nausea, pyrexia, syncope, and increased alanine aminotransferase (n = 1 each). Common related adverse events were nausea (48%); anemia, neutropenia, vomiting (45% each); fatigue (43%); pyrexia (40%); and thrombocytopenia (35%). The GDC-0425 half-life was approximately 15 hours. There were two confirmed partial responses in patients with triple-negative breast cancer (TP53-mutated) and melanoma (n = 1 each) and one unconfirmed partial response in a patient with cancer of unknown primary origin.Conclusions: Chk1 inhibition with GDC-0425 in combination with gemcitabine was tolerated with manageable bone marrow suppression. The observed preliminary clinical activity warrants further investigation of this chemopotentiation strategy. Clin Cancer Res; 23(10); 2423-32. ©2016 AACR.

A Phase I Dose-Escalation Study Evaluating the Safety Tolerability and Pharmacokinetics of CUDC-427, a Potent, Oral, Monovalent IAP Antagonist, in Patients with Refractory Solid Tumors.

PURPOSE: To determine the dose-limiting toxicities (DLT), adverse events (AE), pharmacokinetics, and preliminary evidence of antitumor activity of CUDC-427 (formerly GDC-0917), a selective antagonist of inhibitor of apoptosis (IAP) proteins. EXPERIMENTAL DESIGN: Patients with advanced solid malignancies were treated with escalating doses of CUDC-427 orally on a daily 14-day on/7-day off schedule in 21-day cycles using a modified continuous reassessment method design. Blood samples were assayed to determine the pharmacokinetic properties, pharmacodynamic alterations of cellular IAP levels in peripheral blood mononuclear cells (PBMC), and monocyte chemoattractant protein-1 (MCP-1) levels. RESULTS: Forty-two patients received 119 cycles of CUDC-427. Overall, the most common treatment-related toxicities were fatigue, nausea, vomiting, and rash. One DLT (grade 3 fatigue) occurred in a patient at 450 mg dose level during cycle 1, and 5 patients experienced AEs related to CUDC-427 that led to discontinuation and included grade 3 pruritus, and fatigue, and grade 2 drug hypersensitivity, pneumonitis, rash, and QT prolongation. The maximum planned dose of 600 mg orally daily for 2 weeks was reached, which allometrically scaled to exceed the IC90 in preclinical xenograft studies. Significant decreases in cIAP-1 levels in PBMCs were observed in all patients 6 hours after initial dosing. Responses included durable complete responses in one patient with ovarian cancer and one patient with MALT lymphoma. CONCLUSIONS: CUDC-427 can be administered safely at doses up to 600 mg daily for 14 days every 3 weeks. The absence of severe toxicities, inhibition of cIAP-1 in PBMC, and antitumor activity warrant further studies. Clin Cancer Res; 22(18); 4567-73. ©2016 AACR.

Expression Profile of BCL-2, BCL-XL, and MCL-1 Predicts Pharmacological Response to the BCL-2 Selective Antagonist Venetoclax in Multiple Myeloma Models.

BCL-2 family proteins dictate survival of human multiple myeloma cells, making them attractive drug targets. Indeed, multiple myeloma cells are sensitive to antagonists that selectively target prosurvival proteins such as BCL-2/BCL-XL (ABT-737 and ABT-263/navitoclax) or BCL-2 only (ABT-199/GDC-0199/venetoclax). Resistance to these three drugs is mediated by expression of MCL-1. However, given the selectivity profile of venetoclax it is unclear whether coexpression of BCL-XL also affects antitumor responses to venetoclax in multiple myeloma. In multiple myeloma cell lines (n = 21), BCL-2 is expressed but sensitivity to venetoclax correlated with high BCL-2 and low BCL-XL or MCL-1 expression. Multiple myeloma cells that coexpress BCL-2 and BCL-XL were resistant to venetoclax but sensitive to a BCL-XL-selective inhibitor (A-1155463). Multiple myeloma xenograft models that coexpressed BCL-XL or MCL-1 with BCL-2 were also resistant to venetoclax. Resistance to venetoclax was mitigated by cotreatment with bortezomib in xenografts that coexpressed BCL-2 and MCL-1 due to upregulation of NOXA, a proapoptotic factor that neutralizes MCL-1. In contrast, xenografts that expressed BCL-XL, MCL-1, and BCL-2 were more sensitive to the combination of bortezomib with a BCL-XL selective inhibitor (A-1331852) but not with venetoclax cotreatment when compared with monotherapies. IHC of multiple myeloma patient bone marrow biopsies and aspirates (n = 95) revealed high levels of BCL-2 and BCL-XL in 62% and 43% of evaluable samples, respectively, while 34% were characterized as BCL-2(High)/BCL-XL (Low) In addition to MCL-1, our data suggest that BCL-XL may also be a potential resistance factor to venetoclax monotherapy and in combination with bortezomib. Mol Cancer Ther; 15(5); 1132-44. ©2016 AACR.

Patients With Proneural Glioblastoma May Derive Overall Survival Benefit From the Addition of Bevacizumab to First-Line Radiotherapy and Temozolomide: Retrospective Analysis of the AVAglio Trial.

PURPOSE: The AVAglio (Avastin in Glioblastoma) and RTOG-0825 randomized, placebo-controlled phase III trials in newly diagnosed glioblastoma reported prolonged progression-free survival (PFS), but not overall survival (OS), with the addition of bevacizumab to radiotherapy plus temozolomide. To establish whether certain patient subgroups derived an OS benefit from the addition of bevacizumab to first-line standard-of-care therapy, AVAglio patients were retrospectively evaluated for molecular subtype, and bevacizumab efficacy was assessed for each patient subgroup. PATIENTS AND METHODS: A total of 349 pretreatment specimens (bevacizumab arm, n = 171; placebo arm, n = 178) from AVAglio patients (total, N = 921) were available for biomarker analysis. Samples were profiled for gene expression and isocitrate dehydrogenase 1 (IDH1) mutation status and classified into previously identified molecular subtypes. PFS and OS were assessed within each subtype. RESULTS: A multivariable analysis accounting for prognostic covariates revealed that bevacizumab conferred a significant OS advantage versus placebo for patients with proneural IDH1 wild-type tumors (17.1 v 12.8 months, respectively; hazard ratio, 0.43; 95% CI, 0.26 to 0.73; P = .002). This analysis also revealed an interaction between the proneural subtype biomarker and treatment arm (P = .023). The group of patients with mesenchymal and proneural tumors derived a PFS benefit from bevacizumab compared with placebo; however, this translated to an OS benefit in the proneural subset only. CONCLUSION: Retrospective analysis of AVAglio data suggests that patients with IDH1 wild-type proneural glioblastoma may derive an OS benefit from first-line bevacizumab treatment. The predictive value of the proneural subtype observed in AVAglio should be validated in an independent data set.

A resource for cell line authentication, annotation and quality control.

Cell line misidentification, contamination and poor annotation affect scientific reproducibility. Here we outline simple measures to detect or avoid cross-contamination, present a framework for cell line annotation linked to short tandem repeat and single nucleotide polymorphism profiles, and provide a catalogue of synonymous cell lines. This resource will enable our community to eradicate the use of misidentified lines and generate credible cell-based data.

Mapping in vivo tumor oxygenation within viable tumor by 19F-MRI and multispectral analysis.

Quantifying oxygenation in viable tumor remains a major obstacle toward a better understanding of the tumor micro-environment and improving treatment strategies. Current techniques are often complicated by tumor heterogeneity. Herein, a novel in vivo approach that combines (19)F magnetic resonance imaging ((19)F-MRI) R 1 mapping with diffusion-based multispectral (MS) analysis is introduced. This approach restricts the partial pressure of oxygen (pO2) measurements to viable tumor, the tissue of therapeutic interest. The technique exhibited sufficient sensitivity to detect a breathing gas challenge in a xenograft tumor model, and the hypoxic region measured by MS (19)F-MRI was strongly correlated with histologic estimates of hypoxia. This approach was then applied to address the effects of antivascular agents on tumor oxygenation, which is a research question that is still under debate. The technique was used to monitor longitudinal pO2 changes in response to an antibody to vascular endothelial growth factor (B20.4.1.1) and a selective dual phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor (GDC-0980). GDC-0980 reduced viable tumor pO2 during a 3-day treatment period, and a significant reduction was also produced by B20.4.1.1. Overall, this method provides an unprecedented view of viable tumor pO2 and contributes to a greater understanding of the effects of antivascular therapies on the tumor's microenvironment.

PTEN loss mitigates the response of medulloblastoma to Hedgehog pathway inhibition.

Medulloblastoma is a cancer of the cerebellum, for which there is currently no approved targeted therapy. Recent transcriptomics approaches have demonstrated that medulloblastoma is composed of molecularly distinct subgroups, one of which is characterized by activation of the Hedgehog pathway, which in mouse models is sufficient to drive medulloblastoma development. There is thus considerable interest in targeting the Hedgehog pathway for therapeutic benefit in medulloblastoma, particularly given the recent approval of the Hedgehog pathway inhibitor vismodegib for metastatic and locally advanced basal cell carcinoma. Like other molecularly targeted therapies, however, there have been reports of acquired resistance to vismodegib, driven by secondary Hedgehog pathway mutations and potentially by activation of the phosphatidylinositol 3-kinase (PI3K) pathway. Given that acquired resistance to vismodegib may occur as a result of inappropriate PI3K pathway activation, we asked if loss of the PI3K pathway regulator, phosphatase and tensin homologue (Pten), which has been reported to occur in patients within the Hedgehog subgroup, would constitute a mechanism of innate resistance to vismodegib in Hedgehog-driven medulloblastoma. We find that Hedgehog pathway inhibition successfully restrains growth of Pten-deficient medulloblastoma in this mouse model, but does not drive tumor regression, as it does in Pten-wild-type medulloblastoma. Combined inhibition of the Hedgehog and PI3K pathways may lead to superior antitumor activity in PTEN-deficient medulloblastoma in the clinic.

An interleukin-17-mediated paracrine network promotes tumor resistance to anti-angiogenic therapy.

Although angiogenesis inhibitors have provided substantial clinical benefit as cancer therapeutics, their use is limited by resistance to their therapeutic effects. While ample evidence indicates that such resistance can be influenced by the tumor microenvironment, the underlying mechanisms remain incompletely understood. Here, we have uncovered a paracrine signaling network between the adaptive and innate immune systems that is associated with resistance in multiple tumor models: lymphoma, lung and colon. Tumor-infiltrating T helper type 17 (T(H)17) cells and interleukin-17 (IL-17) induced the expression of granulocyte colony-stimulating factor (G-CSF) through nuclear factor κB (NF-κB) and extracellular-related kinase (ERK) signaling, leading to immature myeloid-cell mobilization and recruitment into the tumor microenvironment. The occurrence of T(H)17 cells and Bv8-positive granulocytes was also observed in clinical tumor specimens. Tumors resistant to treatment with antibodies to VEGF were rendered sensitive in IL-17 receptor (IL-17R)-knockout hosts deficient in T(H)17 effector function. Furthermore, pharmacological blockade of T(H)17 cell function sensitized resistant tumors to therapy with antibodies to VEGF. These findings indicate that IL-17 promotes tumor resistance to VEGF inhibition, suggesting that immunomodulatory strategies could improve the efficacy of anti-angiogenic therapy.

Oncogenic RAS pathway activation promotes resistance to anti-VEGF therapy through G-CSF-induced neutrophil recruitment.

Granulocyte-colony stimulating factor (G-CSF) promotes mobilization of CD11b(+)Gr1(+) myeloid cells and has been implicated in resistance to anti-VEGF therapy in mouse models. High G-CSF production has been associated with a poor prognosis in cancer patients. Here we show that activation of the RAS/MEK/ERK pathway regulates G-CSF expression through the Ets transcription factor. Several growth factors induced G-CSF expression by a MEK-dependent mechanism. Inhibition of G-CSF release with a MEK inhibitor markedly reduced G-CSF production in vitro and synergized with anti-VEGF antibodies to reduce CD11b(+)Ly6G(+) neutrophil mobilization and tumor growth and led to increased survival in animal models of cancer, including a genetically engineered mouse model of pancreatic adenocarcinoma. Analysis of biopsies from pancreatic cancer patients revealed increased phospho-MEK, G-CSF, and Ets expression and enhanced neutrophil recruitment compared with normal pancreata. These results provide insights into G-CSF regulation and on the mechanism of action of MEK inhibitors and point to unique anticancer strategies.

Differential drug class-specific metastatic effects following treatment with a panel of angiogenesis inhibitors.

Inhibiting angiogenesis has become an important therapeutic strategy for cancer treatment but, like other current targeted therapies, benefits experienced for late-stage cancers can be curtailed by inherent refractoriness or by acquired drug resistance, requiring a need for better mechanistic understanding of such effects. Numerous preclinical studies have demonstrated that VEGF pathway inhibitors suppress primary tumour growth and metastasis. However, it has been recently reported that short-term VEGF and VEGFR inhibition can paradoxically accelerate tumour invasiveness and metastasis in certain models. Here we comprehensively compare the effects of both antibody and small molecule receptor tyrosine kinase (RTK) inhibitors targeting the VEGF-VEGFR pathway, using short-term therapy in various mouse models of metastasis. Our findings demonstrate that antibody inhibition of VEGF pathway molecules does not promote metastasis, in contrast to selected small molecule RTK inhibitors at elevated-therapeutic drug dosages. In particular, a multi-targeted RTK inhibitor, sunitinib, which most profoundly potentiated metastasis, also increased lung vascular permeability and promoted tumour cell extravasation. Mechanistically, sunitinib, but not anti-VEGF treatment, attenuated endothelial barrier function in culture and caused a global inhibition of protein tyrosine phosphorylation, including molecules important for maintaining endothelial cell-cell junctions. Together these findings indicate that, rather than a specific consequence of inhibiting the VEGF signalling pathway, pharmacological inhibitors of the VEGF pathway can have dose- and drug class-dependent side-effects on the host vasculature. These findings also advocate for the continued identification of mechanisms of resistance to anti-angiogenics and for therapy development to overcome it.

Navitoclax (ABT-263) reduces Bcl-x(L)-mediated chemoresistance in ovarian cancer models.

To examine the potential of combining Bcl-2 family inhibitors with chemotherapy in ovarian cancer, we evaluated a panel of 27 ovarian cancer cell lines for response to the combination of navitoclax (formerly ABT-263) and paclitaxel or gemcitabine. The majority of cell lines exhibited a greater than additive response to either combination, as determined by the Bliss independence model, and more than 50% of the ovarian cell lines exhibited strong synergy for the navitoclax/paclitaxel combination. To identify biomarkers for tumors likely to respond to this combination, we evaluated the protein levels of intrinsic apoptosis pathway components. Bcl-x(L) seems necessary, but not sufficient, for navitoclax/paclitaxel synergy in vitro, suggesting that exclusion of patients whose tumors have low or undetectable Bcl-x(L) would enrich for patients responsive to the combination. We evaluated Bcl-x(L) levels in ovarian cancer tumor tissue from 40 patients (20 taxane responsive and 20 with poor response to taxane) and found that patients with high Bcl-x(L) were less sensitive to taxane treatment (10 of 12) Bcl-x(L) positive patients, P = 0.014). These data support the use of navitoclax in combination with taxane-based therapy in ovarian cancer patients with high levels of Bcl-x(L).

Evidence for sequenced molecular evolution of IDH1 mutant glioblastoma from a distinct cell of origin.

PURPOSE: Mutation in isocitrate dehydrogenase 1 (IDH1) at R132 (IDH1(R132MUT)) is frequent in low-grade diffuse gliomas and, within glioblastoma (GBM), has been proposed as a marker for GBMs that arise by transformation from lower-grade gliomas, regardless of clinical history. To determine how GBMs arising with IDH1(R132MUT) differ from other GBMs, we undertook a comprehensive comparison of patients presenting clinically with primary GBM as a function of IDH1(R132) mutation status. PATIENTS AND METHODS: In all, 618 treatment-naive primary GBMs and 235 lower-grade diffuse gliomas were sequenced for IDH1(R132) and analyzed for demographic, radiographic, anatomic, histologic, genomic, epigenetic, and transcriptional characteristics. RESULTS: Investigation revealed a constellation of features that distinguishes IDH1(R132MUT) GBMs from other GBMs (including frontal location and lesser extent of contrast enhancement and necrosis), relates them to lower-grade IDH1(R132MUT) gliomas, and supports the concept that IDH1(R132MUT) gliomas arise from a neural precursor population that is spatially and temporally restricted in the brain. The observed patterns of DNA sequence, methylation, and copy number alterations support a model of ordered molecular evolution of IDH1(R132MUT) GBM in which the appearance of mutant IDH1 protein is an initial event, followed by production of p53 mutant protein, and finally by copy number alterations of PTEN and EGFR. CONCLUSION: Although histologically similar, GBMs arising with and without IDH1(R132MUT) appear to represent distinct disease entities that arise from separate cell types of origin as the result of largely nonoverlapping sets of molecular events. Optimal clinical management should account for the distinction between these GBM disease subtypes.