Carboplatin-induced upregulation of pan ß-tubulin and class III ß-tubulin is implicated in acquired resistance and cross-resistance of ovarian cancer.

Resistance to platinum- and taxane-based chemotherapy represents a major obstacle to long-term survival in ovarian cancer (OC) patients. Here, we studied the interplay between acquired carboplatin (CBP) resistance using two OC cell models, MES-OV CBP and SK-OV-3 CBP, and non-P-glycoprotein-mediated cross-resistance to paclitaxel (TAX) observed only in MES-OV CBP cells. Decreased platination, mesenchymal-like phenotype, and increased expression of α- and γ-tubulin were observed in both drug-resistant variants compared with parental cells. Both variants revealed increased protein expression of class III ß-tubulin (TUBB3) but differences in TUBB3 branching and nuclear morphology. Transient silencing of TUBB3 sensitized MES-OV CBP cells to TAX, and surprisingly also to CBP. This phenomenon was not observed in the SK-OV-3 CBP variant, probably due to the compensation by other ß-tubulin isotypes. Reduced TUBB3 levels in MES-OV CBP cells affected DNA repair protein trafficking and increased whole-cell platination level. Furthermore, TUBB3 depletion augmented therapeutic efficiency in additional OC cells, showing vice versa drug-resistant pattern, lacking ß-tubulin isotype compensation visible at the level of total ß-tubulin (TUBB) in vitro and ex vivo. In summary, the level of TUBB in OC should be considered together with TUBB3 in therapy response prediction.

Decreased levels of baseline and drug-induced tubulin polymerisation are hallmarks of resistance to taxanes in ovarian cancer cells and are associated with epithelial-to-mesenchymal transition.

BACKGROUND: ABCB1 expression is uncommon in ovarian cancers in the clinical setting so we investigated non-MDR mechanisms of resistance to taxanes. METHODS: We established eight taxane-resistant variants from the human ovarian carcinoma cell lines A2780/1A9, ES-2, MES-OV and OVCAR-3 by selection with paclitaxel or docetaxel, with counter-selection by the transport inhibitor valspodar. RESULTS: Non-MDR taxane resistance was associated with reduced intracellular taxane content compared to parental controls, and cross-resistance to other microtubule stabilising drugs. Collateral sensitivity to depolymerising agents (vinca alkaloids and colchicine) was observed with increased intracellular vinblastine. These variants exhibited marked decreases in basal tubulin polymer and in tubulin polymerisation in response to taxane exposure. TUBB3 content was increased in 6 of the 8 variants. We profiled gene expression of the parental lines and resistant variants, and identified a transcriptomic signature with two highly significant networks built around FN1 and CDKN1A that are associated with cell adhesion, cell-to-cell signalling, and cell cycle regulation. miR-200 family members miR-200b and miR-200c were downregulated in resistant cells, associated with epithelial to mesenchymal transition (EMT), with increased VIM, FN1, MMP2 and/or MMP9. CONCLUSIONS: These alterations may serve as biomarkers for predicting taxane effectiveness in ovarian cancer and should be considered as therapeutic targets.

Cancer Control in Central and Eastern Europe: Current Situation and Recommendations for Improvement.

: The incidence of many cancers is higher in Western European (WE) countries, but mortality is frequently higher in Central and Eastern European (CEE) countries. A panel of oncology leaders from CEE countries participating in the South Eastern European Research Oncology Group (SEEROG) was formed in 2015, aiming to analyze the current status and trends of oncology care in CEE and to propose recommendations leading to improved care and outcomes. The SEEROG panel, meeting during the 11th Central European Oncology Congress, proposed the following: (a) national cancer control plans (NCCPs) required in all CEE countries, defining priorities in cancer care, including finance allocation considering limited health care budgets; (b) national cancer registries, describing in detail epidemiological trends; (c) efforts to strengthen comprehensive cancer centers; (d) that multidisciplinary care should be mandated by the NCCPs; (e) that smaller hospitals should be connected to multidisciplinary tumor boards via the Internet, providing access to specialized expertise; (f) nationwide primary prevention programs targeting smoking, obesity, and alcohol consumption and centrally evaluated secondary prevention programs for cervical, colorectal, and breast cancers; (g) prioritize education for all involved in cancer care, including oncology nurses, general practitioners, and palliative care providers; (h) establish outpatient care in day hospitals to reduce costs associated with the current inpatient model of care in CEE countries and to improve patients' quality of life; (i) long-term pharmacoeconomic evaluations of new therapies in CEE countries; (j) increase national oncology budgets in view of the higher mortality rates in CEE compared with WE countries; and (k) CEE countries urgently need help from the European Union to increase and monitor overall investment in cancer care. IMPLICATIONS FOR PRACTICE: Significant differences in cancer incidence and mortality have been observed between European countries. While the incidence of many cancer types is higher in Western European (WE) countries, the mortality is generally higher in Central and Eastern Europe (CEE). The primary purpose of this review was to describe the current status and trends of oncology care in the CEE region, to raise awareness among physicians, regulators, and payers, and to propose the most needed changes in order to make the oncology care in CEE closer to the WE standards.

A first-in-human phase I study to evaluate the MEK1/2 inhibitor, cobimetinib, administered daily in patients with advanced solid tumors.

Objective Cobimetinib, a MEK1/2 inhibitor, was administered to patients with advanced solid tumors to assess safety, pharmacokinetics, pharmacodynamics, and anti-tumor activity. Methods For dose-escalation, a 3 + 3 design was used. Oral cobimetinib was administered once daily on a 21-day on/7-day off (21/7) or a 14-day on/14-day off (14/14) schedule. Serial plasma samples were collected for pharmacokinetic (PK) analysis on Day 1 and at steady state. In expansion stages, patients with RAS or RAF mutant tumors were treated at the maximum tolerated dose (MTD) of the 21/7 or 14/14 schedule. Results Ninety-seven patients received cobimetinib. In the 21/7 dose escalation, 36 patients enrolled in 8 cohorts (0.05 mg/kg-80 mg). Dose-limiting toxicities (DLTs) were Grade 4 hepatic encephalopathy, Grade 3 diarrhea, and Grade 3 rash. In the 14/14 dose escalation, 20 patients enrolled in 4 cohorts (60-125 mg). DLTs were Grade 3 rash and Grade 3 blurred vision associated with presence of reversible subretinal fluid. The MTD was 60 mg on 21/7 schedule and 100 mg on 14/14 schedule. Cobimetinib PK showed dose-proportional increases in exposure. The most frequent adverse events attributed to cobimetinib were diarrhea, rash, fatigue, edema, nausea, and vomiting. In patients treated at the 60-mg (21/7) or 100-mg (14/14) dose, one unconfirmed complete response and 6 confirmed partial responses were observed. All responses occurred in melanoma patients; 6 harbored the BRAF(V600E) mutation. Conclusions Cobimetinib is generally well tolerated and durable responses were observed in BRAF(V600E) mutant melanoma patients. Evaluation of cobimetinib in combination with other therapies is ongoing.

NFKBIA deletion in glioblastomas.

BACKGROUND: Amplification and activating mutations of the epidermal growth factor receptor (EGFR) oncogene are molecular hallmarks of glioblastomas. We hypothesized that deletion of NFKBIA (encoding nuclear factor of κ-light polypeptide gene enhancer in B-cells inhibitor-α), an inhibitor of the EGFR-signaling pathway, promotes tumorigenesis in glioblastomas that do not have alterations of EGFR. METHODS: We analyzed 790 human glioblastomas for deletions, mutations, or expression of NFKBIA and EGFR. We studied the tumor-suppressor activity of NFKBIA in tumor-cell culture. We compared the molecular results with the outcome of glioblastoma in 570 affected persons. RESULTS: NFKBIA is often deleted but not mutated in glioblastomas; most deletions occur in nonclassical subtypes of the disease. Deletion of NFKBIA and amplification of EGFR show a pattern of mutual exclusivity. Restoration of the expression of NFKBIA attenuated the malignant phenotype and increased the vulnerability to chemotherapy of cells cultured from tumors with NFKBIA deletion; it also reduced the viability of cells with EGFR amplification but not of cells with normal gene dosages of both NFKBIA and EGFR. Deletion and low expression of NFKBIA were associated with unfavorable outcomes. Patients who had tumors with NFKBIA deletion had outcomes that were similar to those in patients with tumors harboring EGFR amplification. These outcomes were poor as compared with the outcomes in patients with tumors that had normal gene dosages of NFKBIA and EGFR. A two-gene model that was based on expression of NFKBIA and O(6)-methylguanine DNA methyltransferase was strongly associated with the clinical course of the disease. CONCLUSIONS: Deletion of NFKBIA has an effect that is similar to the effect of EGFR amplification in the pathogenesis of glioblastoma and is associated with comparatively short survival.

Continuous 72-h infusion of zosuquidar with chemotherapy in patients with newly diagnosed acute myeloid leukemia stratified for leukemic blast P-glycoprotein phenotype.

PURPOSE: To evaluate the safety, tolerability, pharmacodynamics (PD), and potential efficacy of zosuquidar (Zos) in combination with daunorubicin and cytarabine in elderly patients with newly diagnosed acute myeloid leukemia (AML). METHODS: Patients with AML (N = 106) were treated with Zos as a 72-h continuous intravenous (CIV) infusion along with chemotherapy. Leukemic blasts from the patients were assessed for P-glycoprotein (P-gp) function using ex vivo bioassays for screening and PD analyses. Patient outcomes were categorized according to primary (N = 56) and secondary (N = 50) AML cohorts (pAML and sAML, respectively) and stratified into P-gp-high and P-gp-low subgroups. RESULTS: Patients with P-gp-high blasts exhibited comparable overall remission rates (ORR) to those with P-gp-low blasts in both the pAML and sAML cohorts. The P-gp-high and P-gp-low subgroups in the pAML cohort exhibited similar overall survival (OS). Patients with sAML and P-gp-high blasts exhibited significantly better OS than those in the P-gp-low subgroup. PD analyses revealed that Zos infusion provided 82 h of uninterrupted effective ≥ 90% inhibition of P-gp functional activity in leukemic blasts. CONCLUSIONS: These observations provide evidence of Zos efficacy with the 72-h CIV infusion approach. The similarity of ORR in the P-gp-high and P-gp-low subgroups is consistent with Zos-mediated neutralization of P-gp as verified by PD analyses. The bioassay identified sAML patients most likely to respond favorably to Zos co-therapy indicating feasibility as a Zos companion diagnostic. A follow-up placebo-controlled trial is needed to verify these promising results. GOV IDENTIFIER: NCT00129168; First posted on August 11, 2005.

A clinical trial of zosuquidar plus gemtuzumab ozogamicin (GO) in relapsed or refractory acute myeloid leukemia (RR AML): evidence of efficacy based on leukemic blast P-glycoprotein functional phenotype.

PURPOSE: To evaluate safety, tolerability, potential efficacy, and pharmacodynamics (PD) of zosuquidar (Zos) in combination with gemtuzumab ozogamicin (GO) in elderly patients with relapsed or refractory (RR) acute myeloid leukemia (AML). METHODS: Patients with RR AML (N = 41) were treated with Zos as a 48-h continuous intravenous infusion initiated 4 h prior to a 2-h infusion of GO on days 1 and 15. P-glycoprotein (P-gp) status of the patients' leukemic blasts and PD determinations were assessed with ex vivo bioassays. Patient outcomes were analyzed for the total cohort and as stratified into P-gp-positive (P-gp +) and P-gp-negative (P-gp‒) subgroups. RESULTS: The eligible cohort exhibited a 34% overall remission rate (ORR), a composite of patients that exhibited complete remission (CR), CR with incomplete platelet recovery, or morphologic remission. Patients with 1st relapsed disease exhibited 40% ORR. P-gp phenotype did not significantly predict ORR. However, the P-gp + subgroup exhibited a greater median overall survival (OS) of 6.0 months vs. 1.8 months for patients in the P-gp‒ subgroup (p = 0.01). PD analyses revealed 90-95% inhibition of blast P-gp function during Zos infusion. Treatment related toxicities were observed and resolved with decrease or discontinued Zos or GO dosages. CONCLUSIONS: Zos plus GO elicited appreciable ORR for an elderly patient population with RR AML. The greater OS of the P-gp + subgroup vs. the P-gp‒ subgroup suggests that patients with P-gp + leukemic blasts were being more effectively targeted by GO with Zos co-therapy. The poorer OS of the P-gp‒ subgroup suggests activity of Zos-insensitive multidrug resistant mechanisms. GOV IDENTIFIER: NCT00233909; First posted October 06, 2005.

A Phase I Trial of the ABCB1 Inhibitor, Oral Valspodar, in Combination With Paclitaxel in Patients With Advanced Solid Tumors.

OBJECTIVES: Multidrug resistance mediated by P-glycoprotein is a potential obstacle to cancer treatment. This phase 1 trial determined the safety of paclitaxel with valspodar, a P-glycoprotein inhibitor, in patients with advanced solid tumors. METHODS: Patients were treated with single-agent paclitaxel Q3W 175 mg/m 2 (or 135 mg/m 2 if heavily pretreated) as a 3-hour infusion. If their disease was stable (SD) or progressive (PD), paclitaxel at 30% (52.5 mg/m 2 ), 40% (70 mg/m 2 ), or 50% (87.5 mg/m 2 ) of 175 mg/m 2 (full dose) was administered with valspodar 5 mg/kg orally 4 times daily for 12 doses. Pharmacokinetic sampling (PK) for paclitaxel and valspodar was performed during single-agent and combination therapy. RESULTS: Sixteen patients had SD/PD after one cycle of paclitaxel and then received paclitaxel at 30% (n=3), 40% (n=3), and 50% (n=10) with valspodar. Hematologic adverse events (AEs) including myelosuppression at paclitaxel 40% were comparable to those of full-dose paclitaxel. Non-hematologic AEs consisted of reversible hepatic (hyperbilirubinemia and transaminitis) and neurologic AEs (ataxia and paresthesias). Eleven patients experienced SD with a median of 12.7 weeks (range, 5.4 to 36.0), 4 patients progressed, and 1 was inevaluable. Reduced dose paclitaxel with valspodar resulted in lower plasma peak concentrations of paclitaxel; otherwise, concentrations were similar to single-agent paclitaxel. CONCLUSION: Paclitaxel at 70 mg/m 2 was administered safely with valspodar. Limited efficacy in hematologic and solid tumors resulted in discontinuation of its clinical development and other transporter inhibitors. Recently, the development of ATP-binding cassette transporter inhibitors has been reconsidered to mitigate resistance to antibody-drug conjugates.

A phase I trial of vandetanib combined with capecitabine, oxaliplatin and bevacizumab for the first-line treatment of metastatic colorectal cancer.

BACKGROUND: Vandetanib is a tyrosine kinase inhibitor of both the vascular endothelial growth factor (VEGFR) and epidermal growth factor (EGFR) receptors. The primary objectives of this study were to determine the maximum tolerated dose of vandetanib with capecitabine and oxaliplatin, without and with bevacizumab, for the first line treatment of metastatic colorectal cancer (mCRC), and to define the dose limiting toxicities. MATERIALS AND METHODS: Three cohorts of patients were studied, with capecitabine at 1,000 mg/m(2) twice daily p.o. on days 1-14 of a 3 week cycle, with oxaliplatin i.v. at 130 mg/m(2) on day 1. Vandetanib dosing was 100 mg/day in cohort 1 and 300 mg/day in cohorts 2 and 3. Bevacizumab was added in cohort 3 at 7.5 mg/kg i.v. on day 1 every 3 weeks. RESULTS: Thirteen patients were enrolled and received from one to eight cycles per patient. Grade 4 dermatitis developed in one patient in the first cohort, and the cohort was expanded to six patients with no further dose limiting toxicities (DLT). The second cohort of 3 patients was well tolerated. The third cohort resulted in grade 3 diarrhea, requiring several days of hospitalization and i.v. hydration, in 3 of the 4 patients. Given the severity and duration of diarrhea, each of these was considered a DLT, and therefore cohort 3 was considered to be above the maximum tolerated dose. Six of the 13 patients achieved a partial or complete remission (46%). The time to progression ranged from 2 to 14 months. CONCLUSIONS: Vandetanib at doses of 100 mg and 300 mg daily in combination with capecitabine and oxaliplatin was well tolerated. However, the addition of bevacizumab resulted in severe diarrhea in three out of four patients. Bevacizumab was not well tolerated with vandetanib and XELOX in combination.

Phase I trial of ixabepilone administered as three oral doses each separated by 6 hours every 3 weeks in patients with advanced solid tumors.

BACKGROUND: Ixabepilone, which stabilizes microtubules, has low susceptibility to drug resistance mediated by P-glycoprotein or ßIII-tubulin. MATERIALS AND METHODS: This study was designed to determine the maximum tolerated dose (MTD) of oral ixabepilone when administered every 6 h for three doses, every 3 weeks, to patients with refractory advanced cancers. Eighteen patients were treated with escalating doses of ixabepilone: three at cohort 1 (30 mg/dose; 90 mg on Day 1), nine at cohort 2 (40 mg/dose; 120 mg on Day 1), and six at cohort 3 (50 mg/dose; 150 mg on Day 1). Serial plasma samples were collected during cycle 1 for pharmacokinetic (PK) measurements. RESULTS: Of the 18 treated patients, eight were male and ten were female. The median age was 59 years, and most had an excellent performance status (KPS 90-100; 61%). There were two dose limiting toxicities (DLT): Grade 4 febrile neutropenia at the 120 mg dose and Grade 4 neutropenic sepsis at the 150 mg dose. Because of the severity and duration of neutropenic sepsis at level 3, level 2 (120 mg) was defined as the MTD and this cohort was expanded to nine patients. High inter-individual variability in plasma drug concentrations was observed during the study, with particularly high levels in two patients with DLT. CONCLUSIONS: On the basis of this safety profile, the MTD of oral ixabepilone was defined as 120 mg given as three 40 mg doses each separated by 6 h on Day 1 of a 3-week cycle. However, the PK variability observed makes further development of this oral formulation unlikely.

Clinical trial designs for testing biomarker-based personalized therapies.

BACKGROUND: Advances in molecular therapeutics in the past decade have opened up new possibilities for treating cancer patients with personalized therapies, using biomarkers to determine which treatments are most likely to benefit them, but there are difficulties and unresolved issues in the development and validation of biomarker-based personalized therapies. We develop a new clinical trial design to address some of these issues. The goal is to capture the strengths of the frequentist and Bayesian approaches to address this problem in the recent literature and to circumvent their limitations. METHODS: We use generalized likelihood ratio tests of the intersection null and enriched strategy null hypotheses to derive a novel clinical trial design for the problem of advancing promising biomarker-guided strategies toward eventual validation. We also investigate the usefulness of adaptive randomization (AR) and futility stopping proposed in the recent literature. RESULTS: Simulation studies demonstrate the advantages of testing both the narrowly focused enriched strategy null hypothesis related to validating a proposed strategy and the intersection null hypothesis that can accommodate to a potentially successful strategy. AR and early termination of ineffective treatments offer increased probability of receiving the preferred treatment and better response rates for patients in the trial, at the expense of more complicated inference under small-to-moderate total sample sizes and some reduction in power. LIMITATIONS: The binary response used in the development phase may not be a reliable indicator of treatment benefit on long-term clinical outcomes. In the proposed design, the biomarker-guided strategy (BGS) is not compared to 'standard of care', such as physician's choice that may be informed by patient characteristics. Therefore, a positive result does not imply superiority of the BGS to 'standard of care'. The proposed design and tests are valid asymptotically. Simulations are used to examine small-to-moderate sample properties. CONCLUSION: Innovative clinical trial designs are needed to address the difficulties and issues in the development and validation of biomarker-based personalized therapies. The article shows the advantages of using likelihood inference and interim analysis to meet the challenges in the sample size needed and in the constantly evolving biomarker landscape and genomic and proteomic technologies.

Safety, tolerability and efficacy of agonist anti-CD27 antibody (varlilumab) administered in combination with anti-PD-1 (nivolumab) in advanced solid tumors.

BACKGROUND: Phase 1/2 dose-escalation and expansion study evaluating varlilumab, a fully human agonist anti-CD27 mAb, with nivolumab in anti-PD-1/L1 naïve, refractory solid tumors. METHODS: Phase 1 evaluated the safety of varlilumab (0.1-10 mg/kg) with nivolumab (3 mg/kg) administered once every 2 weeks. Phase 2 evaluated varlilumab regimens (3 mg/kg once every 2 weeks, 3 mg/kg once every 12 weeks, and 0.3 mg/kg once every 4 weeks) with nivolumab 240 mg once every 2 weeks in tumor-specific cohorts. Primary objective was safety; key clinical endpoints included objective response rate (ORR) and overall survival rate at 12 months (OS12) (glioblastoma (GBM) only). Exploratory objectives included determination of effects on peripheral blood and intratumoral immune signatures. RESULTS: 175 patients were enrolled (36 in phase 1 and 139 in phase 2). Phase 1 dose-escalation proceeded to the highest varlilumab dose level without determining a maximum tolerated dose. In phase 2, ORR were ovarian 12.5%, squamous cell carcinoma of the head and neck 12.5%, colorectal cancer 5%, and renal cell carcinoma 0%; GBM OS12 was 40.9%. Increased tumor PD-L1 and intratumoral T cell infiltration were observed in ovarian cancer patients, with increases of ≥5% associated with better progression-free survival. The most common treatment related adverse events were fatigue (18%), pruritus (16%), and rash (15%). CONCLUSION: Varlilumab and nivolumab were well tolerated, without significant toxicity beyond that expected for each agent alone. Clinical activity was observed in patients that are typically refractory to anti-PD-1 therapy, however, overall was not greater than expected for nivolumab monotherapy. Treatment was associated with proinflammatory changes in the tumor microenvironment, particularly in ovarian cancer where the changes were associated with better clinical outcomes. TRIAL REGISTRATION NUMBER: NCT02335918.

Phase I trial of oblimersen (Genasense®) and gemcitabine in refractory and advanced malignancies.

BACKGROUND: Overexpression of Bcl-2 is associated with worse prognosis for a number of cancer types. The present study was designed to determine the maximum tolerated dose (MTD) of oblimersen (antisense Bcl-2) and gemcitabine when administered to patients with refractory malignancies. MATERIALS AND METHODS: Sixteen patients with advanced solid tumors refractory to standard therapies were treated with escalating doses of oblimersen continuous, 120-h intravenous infusion given every 14 days, with a fixed-dose-rate intravenous infusion of gemcitabine administered on day 5 of each cycle. Serial plasma samples were collected to calculate the pharmacokinetics of oblimersen and gemcitabine, and also to measure the effect of oblimersen on Bcl-2 expression. RESULTS: 7 women and 9 men, median age 55 years (range 35-74 years), received a 5-day infusion of oblimersen at dose levels of 5 mg/kg/day (n = 4) or 7 mg/kg/day (n = 12). On the 5th day of the infusion, gemcitabine was given at 10 mg/m(2)/h for a total dose of 1,000 mg/m(2) (n = 7; cohorts I and II), 1,200 mg/m(2) (n = 3; cohort III), or 1,500 mg/m(2) (n = 6; cohort IV). Edema was the dose-limiting toxicity (DLT), necessitating expansion of cohort IV. No subsequent DLTs were noted. Thus, the maximum planned doses were well tolerated, and a formal MTD was not determined. Most hematologic toxicities were grade 1 or 2. There was low-grade fatigue, nausea/vomiting, and myalgias/arthralgias. Oblimersen C(ss) and AUC increased in relation to the dose escalation, but gemcitabine triphosphate levels did not correlate well with dose. There were no objective responses, though 5 patients had stable disease. A >75% reduction in Bcl-2 expression in peripheral blood mononuclear leucocytes was seen more frequently in patients who achieved stable disease than in progressing patients. CONCLUSIONS: The maximal planned dose levels of oblimersen and gemcitabine in combination were well tolerated. Only one DLT (edema) occurred. There was a correlation between Bcl-2 reduction and stable disease. The recommended doses of the drugs for future studies are 7 mg/kg/day of oblimersen on days 1-5, and gemcitabine 1,500 mg/m(2) on day 5, every two weeks.

Phase I study of valspodar (PSC-833) with mitoxantrone and etoposide in refractory and relapsed pediatric acute leukemia: a report from the Children's Oncology Group.

BACKGROUND: Valspodar, a non-immunosuppressive analog of cylosporine, is a potent P-glycoprotein (MDR1) inhibitor. As MDR1-mediated efflux of chemotherapeutic agents from leukemic blasts may contribute to drug resistance, a phase 1 study of valspodar combined with mitoxantrone and etoposide in pediatric patients with relapsed or refractory leukemias was performed. PROCEDURE: Patients received a valspodar-loading dose (2 mg/kg) followed by a 5-day continuous valspodar infusion (8, 10, 12.5, or 15 mg/kg/day) combined with lower than standard doses of mitoxantrone and etoposide. The valspodar dose was escalated using a standard 3 + 3 phase I design. RESULTS: Twenty-one patients were evaluable for toxicity and 20 for response. The maximum tolerated dose (MTD) of valspodar was 12.5 mg/kg/day, combined with 50% dose-reduced mitoxantrone and etoposide. The clearance of mitoxantrone and etoposide was decreased by 64% and 60%, respectively, when combined with valspodar. Dose-limiting toxicities included stomatitis, ataxia, and bone marrow aplasia. Three of 11 patients with acute lymphoblastic leukemia (ALL) had complete responses while no patient with acute myeloid leukemia (AML) had an objective response. In vitro studies demonstrated P-glycoprotein expression on the blasts of 5 of 14 patients, although only 1 had inhibition of rhodamine efflux by valspodar. CONCLUSIONS: While this regimen was tolerable, responses in this heavily pretreated population were limited to a subset of patients with ALL.

Genomic stability at the coding regions of the multidrug transporter gene ABCB1: insights into the development of alternative drug resistance mechanisms in human leukemia cells.

AIM: Despite considerable efforts to reverse clinical multidrug resistance (MDR), targeting the predominant multidrug transporter ABCB1/P-glycoprotein (P-gp) using small molecule inhibitors has been unsuccessful, possibly due to the emergence of alternative drug resistance mechanisms. However, the non-specific P-gp inhibitor cyclosporine (CsA) showed significant clinical benefits in patients with acute myeloid leukemia (AML), which likely represents the only proof-of-principle clinical trial using several generations of MDR inhibitors. Nevertheless, the mutational mechanisms that may underlie unsuccessful MDR modulation by CsA are not elucidated because of the absence of CsA-relevant cellular models. In this study, our aims were to establish CsA-resistant leukemia models and to examine the presence or absence of ABCB1 exonic mutations in these models as well as in diverse types of human cancer samples including AMLs. METHODS: Drug-resistant lines were established by stepwise drug co-selection and characterized by drug sensitivity assay, rhodamine-123 accumulation, [3H]-labeled drug export, ABCB1 cDNA sequencing, and RNase protection assay. The genomic stability of the ABCB1 coding regions was evaluated by exome sequencing analysis of variant allele frequencies in human populations. Moreover, the mutational spectrum of ABCB1 was further assessed in diverse types of cancer samples including AMLs in the Cancer Genome Atlas (TCGA) at the National Cancer Institute. RESULTS: We report the development of two erythroleukemia variants, RVC and RDC, which were derived by stepwise co-selection of K562/R7 drug-resistant leukemia cells with the etoposide-CsA and doxorubicin-CsA drug combinations, respectively. Interestingly, both RVC and RDC cell lines, which retained P-gp expression, showed altered multidrug-resistant phenotypes that were resistant to CsA modulation. Strikingly, no mutations were found in the ABCB1 coding regions in these variant cells even under long-term stringent drug selection. Genomically, ABCB1 displayed relatively low variant allele frequencies in human populations when compared with several ABC superfamily members. Moreover, ABCB1 also exhibited a very low mutational frequency in AMLs compared with all types of human cancer. In addition, we found that CsA played a role in undermining the selection of highly drug-resistant cells via induction of low-level and unstable drug resistance. CONCLUSION: Our data indicate that ABCB1 coding regions are genomically stable and relatively resistant to drug-induced mutations. Non-ABCB1 mutational mechanisms are responsible for the drug-resistant phenotypes in both RVC and RDC cell lines, which are also prevalent in clinical AML patients. Accordingly, we propose several relevant models that account for the development of alternative drug resistance mechanisms in the absence of ABCB1 mutations.

Safety and activity of varlilumab, a novel and first-in-class agonist anti-CD27 antibody, for hematologic malignancies.

CD27, a costimulatory molecule on T cells, induces intracellular signals mediating cellular activation, proliferation, effector function, and cell survival on binding to its ligand, CD70. Varlilumab, a novel, first-in-class, agonist immunoglobulin G1 anti-CD27 antibody, mediates antitumor immunity and direct killing of CD27+ tumor cells in animal models. This first-in-human, dose-escalation, and expansion study evaluated varlilumab in patients with hematologic malignancies. Primary objectives were to assess safety and the maximum tolerated and optimal biologic doses of varlilumab. Secondary objectives were to evaluate pharmacokinetics, pharmacodynamics, immunogenicity, and antitumor activity. In a 3 + 3 dose-escalation design, 30 patients with B-cell (n = 25) or T-cell (n = 5) malignancies received varlilumab (0.1, 0.3, 1, 3, or 10 mg/kg IV) as a single dose with a 28-day observation period, followed by weekly dosing (4 doses per cycle, up to 5 cycles, depending on tumor response). In an expansion cohort, 4 additional patients with Hodgkin lymphoma received varlilumab at 0.3 mg/kg every 3 weeks (4 doses per cycle, up to 5 cycles). No dose-limiting toxicities were observed. Treatment-related adverse events, generally grade 1 to 2, included fatigue, decreased appetite, anemia, diarrhea, and headache. Exposure was linear and dose-proportional across dose groups and resulted in increases in proinflammatory cytokines and soluble CD27. One patient with stage IV Hodgkin lymphoma experienced a complete response and remained in remission at >33 months with no further anticancer therapy. These data support further investigation of varlilumab for hematologic malignancies, particularly in combination approaches targeting nonredundant immune regulating pathways. This trial was registered at www.clinicaltrials.gov as #NCT01460134.

A phase II study of gefitinib, 5-fluorouracil, leucovorin, and oxaliplatin in previously untreated patients with metastatic colorectal cancer.

PURPOSE: We investigated the gefitinib, 5-fluorouracil (5-FU), leucovorin and oxaliplatin (IFOX) regimen as first-line therapy in patients with metastatic colorectal cancer. EXPERIMENTAL DESIGN: Eligible patients had stage IV colorectal adenocarcinoma, and had not received prior chemotherapy for metastatic disease. Each cycle consisted of 14 days. Cycle 1 consisted of oxaliplatin, leucovorin, and 5-FU (FOLFOX-4). All subsequent cycles consisted of FOLFOX-4 with gefitinib at 500 mg orally daily throughout the 14-day cycle. RESULTS: Forty-five patients were enrolled and were assessable for toxicity. Forty-three patients were assessable for response. Thirty-one of the 43 patients (72%) had either a complete or partial response by the Response Evaluation Criteria in Solid Tumors. Median overall survival was 20.5 months. Median time to progression was 9.3 months. Commonly encountered grade 3 or 4 toxicities included diarrhea in 67% of patients and neutropenia in 60%. Grade 2 acneiform skin rash typical of gefitinib occurred in 60% of patients. CONCLUSIONS: IFOX is an active first-line regimen in patients with metastatic colorectal adenocarcinoma, showing higher response rates but also increased toxicities compared with FOLFOX-4 alone in a similar patient population.

A network model of a cooperative genetic landscape in brain tumors.

CONTEXT: Gliomas, particularly glioblastomas, are among the deadliest of human tumors. Gliomas emerge through the accumulation of recurrent chromosomal alterations, some of which target yet-to-be-discovered cancer genes. A persistent question concerns the biological basis for the coselection of these alterations during gliomagenesis. OBJECTIVES: To describe a network model of a cooperative genetic landscape in gliomas and to evaluate its clinical relevance. DESIGN, SETTING, AND PATIENTS: Multidimensional genomic profiles and clinical profiles of 501 patients with gliomas (45 tumors in an initial discovery set collected between 2001 and 2004 and 456 tumors in validation sets made public between 2006 and 2008) from multiple academic centers in the United States and The Cancer Genome Atlas Pilot Project (TCGA). MAIN OUTCOME MEASURES: Identification of genes with coincident genetic alterations, correlated gene dosage and gene expression, and multiple functional interactions; association between those genes and patient survival. RESULTS: Gliomas select for a nonrandom genetic landscape-a consistent pattern of chromosomal alterations-that involves altered regions ("territories") on chromosomes 1p, 7, 8q, 9p, 10, 12q, 13q, 19q, 20, and 22q (false-discovery rate-corrected P<.05). A network model shows that these territories harbor genes with putative synergistic, tumor-promoting relationships. The coalteration of the most interactive of these genes in glioblastoma is associated with unfavorable patient survival. A multigene risk scoring model based on 7 landscape genes (POLD2, CYCS, MYC, AKR1C3, YME1L1, ANXA7, and PDCD4) is associated with the duration of overall survival in 189 glioblastoma samples from TCGA (global log-rank P = .02 comparing 3 survival curves for patients with 0-2, 3-4, and 5-7 dosage-altered genes). Groups of patients with 0 to 2 (low-risk group) and 5 to 7 (high-risk group) dosage-altered genes experienced 49.24 and 79.56 deaths per 100 person-years (hazard ratio [HR], 1.63; 95% confidence interval [CI], 1.10-2.40; Cox regression model P = .02), respectively. These associations with survival are validated using gene expression data in 3 independent glioma studies, comprising 76 (global log-rank P = .003; 47.89 vs 15.13 deaths per 100 person-years for high risk vs low risk; Cox model HR, 3.04; 95% CI, 1.49-6.20; P = .002) and 70 (global log-rank P = .008; 83.43 vs 16.14 deaths per 100 person-years for high risk vs low risk; HR, 3.86; 95% CI, 1.59-9.35; P = .003) high-grade gliomas and 191 glioblastomas (global log-rank P = .002; 83.23 vs 34.16 deaths per 100 person-years for high risk vs low risk; HR, 2.27; 95% CI, 1.44-3.58; P<.001). CONCLUSIONS: The alteration of multiple networking genes by recurrent chromosomal aberrations in gliomas deregulates critical signaling pathways through multiple, cooperative mechanisms. These mutations, which are likely due to nonrandom selection of a distinct genetic landscape during gliomagenesis, are associated with patient prognosis.

Monosomy of chromosome 10 associated with dysregulation of epidermal growth factor signaling in glioblastomas.

CONTEXT: Glioblastomas--uniformly fatal brain tumors--often have both monosomy of chromosome 10 and gains of the epidermal growth factor receptor (EGFR) gene locus on chromosome 7, an association for which the mechanism is poorly understood. OBJECTIVES: To assess whether coselection of EGFR gains on 7p12 and monosomy 10 in glioblastomas promotes tumorigenic epidermal growth factor (EGF) signaling through loss of the annexin A7 (ANXA7) gene on 10q21.1-q21.2 and whether ANXA7 acts as a tumor suppressor gene by regulating EGFR in glioblastomas. DESIGN, SETTING, AND PATIENTS: Multidimensional analysis of gene, coding sequence, promoter methylation, messenger RNA (mRNA) transcript, protein data for ANXA7 (and EGFR), and clinical patient data profiles of 543 high-grade gliomas from US medical centers and The Cancer Genome Atlas pilot project (made public 2006-2008; and unpublished, tumors collected 2001-2008). Functional analyses using LN229 and U87 glioblastoma cells. MAIN OUTCOME MEASURES: Associations among ANXA7 gene dosage, coding sequence, promoter methylation, mRNA transcript, and protein expression. Effect of ANXA7 haploinsufficiency on EGFR signaling and patient survival. Joint effects of loss of ANXA7 and gain of EGFR expression on tumorigenesis. RESULTS: Heterozygous ANXA7 gene deletion is associated with significant loss of ANXA7 mRNA transcript expression (P = 1 x 10(-15); linear regression) and a reduction (mean [SEM]) of 91.5% (2.3%) of ANXA7 protein expression compared with ANXA7 wild-type glioblastomas (P = .004; unpaired t test). ANXA7 loss of function stabilizes the EGFR protein (72%-744% increase in EGFR protein abundance) and augments EGFR transforming signaling in glioblastoma cells. ANXA7 haploinsufficiency doubles tumorigenic potential of glioblastoma cells, and combined ANXA7 knockdown and EGFR overexpression promotes tumorigenicity synergistically. The heterozygous loss of ANXA7 in approximately 75% of glioblastomas in the The Cancer Genome Atlas plus infrequency of ANXA7 mutation (approximately 6% of tumors) indicates its role as a haploinsufficiency gene. ANXA7 mRNA transcript expression, dichotomized at the median, associates with patient survival in 191 glioblastomas (log-rank P = .008; hazard ratio [HR], 0.667; 95% confidence interval [CI], 0.493-0.902; 46.9 vs 74.8 deaths/100 person-years for high vs low ANXA7 mRNA expression) and with a separate group of 180 high-grade gliomas (log-rank P = .00003; HR, 0.476; 95% CI, 0.333-0.680; 21.8 vs 50.0 deaths/100 person-years for high vs low ANXA7 mRNA expression). Deletion of the ANXA7 gene associates with poor patient survival in 189 glioblastomas (log-rank P = .042; HR, 0.686; 95% CI, 0.476-0.989; 54.0 vs 80.1 deaths/100 person-years for wild-type ANXA7 vs ANXA7 deletion). CONCLUSION: Haploinsufficiency of the tumor suppressor ANXA7 due to monosomy of chromosome 10 provides a clinically relevant mechanism to augment EGFR signaling in glioblastomas beyond that resulting from amplification of the EGFR gene.

The Syk inhibitor R406 is a modulator of P-glycoprotein (ABCB1)-mediated multidrug resistance.

In a previously published study, higher levels of spleen tyrosine kinase (Syk) were observed in recurrent post-chemotherapy ovarian cancers compared to primary tumors. Syk inhibition was found to stabilize microtubules and potentiate paclitaxel activity in cellular models of taxane-resistant ovarian cancers. We further studied the effects of Syk inhibition on paclitaxel activity in Syk(+) ovarian cancer cell models and in variants selected for taxane resistance. Syk inhibition was accomplished using RNAi and by exposure to the small molecule competitive inhibitor R406, the active metabolite of fostamatinib. Exposure to R406 or to a SYK-specific pool of siRNAs did not alter taxane activity in the OVCAR-3 cell line, which has the most Syk content in our panel of nine human ovarian cancer cell lines. However, treatment with R406 sensitised the multidrug resistant (MDR) variants MES-SA/Dx5 and SK-OV-3/TR to paclitaxel in a dose-dependent manner resulting from the inhibition of the ABCB1/P-glycoprotein (P-gp) drug transporter. These observations are Syk-independent since both MDR cell models are Syk negative. R406 modulated resistance to other known P-gp substrates, and we observed orthovanadate-sensitive ATPase stimulation resulting from treatment with R406. These data indicate that the chemo-sensitizing effect of R406 in taxane-resistant cells previously reported was not associated with Syk but resulted from the modulation of P-gp-mediated MDR.