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Clinical Cancer Research

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Clinical Cancer Research

Murine mAb 225 was effective against the EGFR tyrosine kinase and inhibited tumor growth in preclinical studies. A phase I trial showed safety, tumor localization, and satisfactory pharmacokinetics. Human:murine chimeric C225 retained biologic activity, which was essential for the conduct of subsequent combination therapy trials and eventual regulatory approval. Clin Cancer Res; 21(2); 227–9. ©2015 AACR.

See related article by Goldstein et al., Clin Cancer Res 1995;1(11) November 1995;1311–8

Asparaginases develop innovative "tumor starvation" conditions for all antileukemia treatments; however, administrations are limited by the toxicities of this drug. Patients exhibiting moderate toxicity have optimal treatment outcomes. Certain asparagine synthetase polymorphisms may contribute to severe host toxicities in divergent subsets of patients, whereas others do not. Clinical correlations should be evaluated. Clin Cancer Res; 21(2); 230–2. ©2014 AACR.

See related article by Tanfous et al., p. 329

Kinetochores are large protein structures assembled on centromeric DNA during mitosis that bind to microtubules of the mitotic spindle to orchestrate and power chromosome movements. Deregulation of kinetochore–microtubule (KT–MT) attachments has been implicated in driving chromosome instability and cancer evolution; however, the nature and source of KT–MT attachment defects in cancer cells remain largely unknown. Here, we highlight recent findings suggesting that oncogene-driven changes in kinetochore regulation occur in glioblastoma multiforme (GBM) and possibly other cancers exhibiting chromosome instability, giving rise to novel therapeutic opportunities. In particular, we consider the GLE2p-binding sequence domains of BubR1 and the newly discovered BuGZ, two kinetochore-associated proteins, as candidate therapeutic targets for GBM. Clin Cancer Res; 21(2); 233–9. ©2014 AACR.

Acute myelogenous leukemia stem cells (AML–LSC) give rise to the leukemic bulk population and maintain disease. Relapse can arise from residual LSCs that have distinct sensitivity and dependencies when compared with the AML bulk. AML–LSCs are driven by genetic and epigenomic changes, and these alterations influence prognosis and clonal selection. Therapies targeting these molecular aberrations have been developed and show promising responses in advanced clinical trials; however, so far success with LSCs has been limited. Besides the genetic diversity, AML–LSCs are critically influenced by the microenvironment, and a third crucial aspect has recently come to the fore: A group of evolutionarily conserved signaling pathways such as canonical Wnt signaling, Notch signaling, or the Hedgehog pathway can be essential for maintenance of AML–LSC but may be redundant for normal hematopoietic stem cells. In addition, early reports suggest also regulators of cell polarity may also influence hematopoietic stem cells and AML biology. Interactions between these pathways have been investigated recently and suggest a network of signaling pathways involved in regulation of self-renewal and response to oncogenic stress. Here, we review how recent discoveries on regulation of AML–LSC-relevant evolutionarily conserved pathways may open opportunities for novel treatment approaches eradicating residual disease. Clin Cancer Res; 21(2); 240–8. ©2015 AACR.

Tumors exhibit genomic and phenotypic heterogeneity, which has prognostic significance and may influence response to therapy. Imaging can quantify the spatial variation in architecture and function of individual tumors through quantifying basic biophysical parameters such as CT density or MRI signal relaxation rate; through measurements of blood flow, hypoxia, metabolism, cell death, and other phenotypic features; and through mapping the spatial distribution of biochemical pathways and cell signaling networks using PET, MRI, and other emerging molecular imaging techniques. These methods can establish whether one tumor is more or less heterogeneous than another and can identify subregions with differing biology. In this article, we review the image analysis methods currently used to quantify spatial heterogeneity within tumors. We discuss how analysis of intratumor heterogeneity can provide benefit over more simple biomarkers such as tumor size and average function. We consider how imaging methods can be integrated with genomic and pathology data, instead of being developed in isolation. Finally, we identify the challenges that must be overcome before measurements of intratumoral heterogeneity can be used routinely to guide patient care. Clin Cancer Res; 21(2); 249–57. ©2014 AACR.

Purpose: Tumor necrosis factor (TNF)–like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible molecule 14 (Fn14) are a ligand–receptor pair frequently overexpressed in solid tumors. TWEAK:Fn14 signaling regulates multiple oncogenic processes through MAPK, AKT, and NFB pathway activation. A phase I study of RG7212, a humanized anti-TWEAK IgG1 monoclonal antibody, was conducted in patients with advanced solid tumors expressing Fn14.

Experimental Design: Dose escalations, over a 200- to 7,200-mg range, were performed with patients enrolled in weekly (QW), bi-weekly (Q2W), or every-three-week (Q3W) schedules. Primary objectives included determination of dose and safety profile. Secondary endpoints included assessments related to inhibition of TWEAK:Fn14 signaling, tumor proliferation, tumor immune cell infiltration, and pharmacokinetics.

Results: In 192 treatment cycles administered to 54 patients, RG7212 was well-tolerated with no dose-limiting toxicities observed. More than 95% of related adverse events were limited to grade 1/2. Pharmacokinetics were dose proportional for all cohorts, with a t1/2 of 11 to 12 days. Pharmacodynamic changes included clearance of free and total TWEAK ligand and reductions in tumor Ki-67 and TRAF1. A patient with BRAF wild-type melanoma who received 36 weeks of RG7212 therapy had tumor regression and pharmacodynamic changes consistent with antitumor effects. Fifteen patients (28%) received 16 or more weeks of RG7212 treatment.

Conclusion: RG7212 demonstrated excellent tolerability and favorable pharmacokinetics. Pharmacodynamic endpoints were consistent with reduced TWEAK:Fn14 signaling. Tumor regression was observed and prolonged stable disease was demonstrated in multiple heavily pretreated patients with solid tumors. These encouraging results support further study of RG7212. Clin Cancer Res; 21(2); 258–66. ©2014 AACR.

Purpose: A phase I trial of AT9283 (a multitargeted inhibitor of Aurora kinases A and B) was conducted in children and adolescents with solid tumors, to identify maximum-tolerated dose (MTD), safety, efficacy, pharmacokinetics, and pharmacodynamic (PD) activity.

Experimental Design: AT9283 was administered as a 72-hour continuous intravenous infusion every 3 weeks. A rolling-six design, explored six dose levels (7, 9, 11.5, 14.5, 18.5, and 23 mg/m2/d). Pharmacokinetic and PD assessments, included inhibition of phospho-histone 3 (pHH3) in paired skin punch biopsies.

Results: Thirty-three patients were evaluable for toxicity. There were six dose-limiting toxicities and the MTD was 18.5 mg/m2/d. Most common drug-related toxicities were hematologic (neutropenia, anemia, and thrombocytopenia in 36.4%, 18.2%, and 21.2% of patients), which were grade ≥3 in 30.3%, 6.1%, and 3% of patients. Nonhematologic toxicities included fatigue, infections, febrile neutropenia and ALT elevation. One patient with central nervous system–primitive neuroectodermal tumor (CNS-PNET) achieved a partial response after 16 cycles and 3 cases were stable for four or more cycles. Plasma concentrations were comparable with those in adults at the same dose level, clearance was similar although half-life was shorter (4.9 ± 1.5 hours, compared with 8.4 ± 3.7 hours in adults). Inhibition of Aurora kinase B was shown by reduction in pHH3 in 17 of 18 patients treated at ≥11.5 mg/m2/d.

Conclusion: AT9283 was well tolerated in children and adolescents with solid tumors with manageable hematologic toxicity. Target inhibition was demonstrated. Disease stabilization was documented in intracranial and extracranial pediatric solid tumors and a phase II dose determined. Clin Cancer Res; 21(2); 267–73. ©2014 AACR.

Purpose: The CC chemokine receptor 4 (CCR4) is expressed on malignant T cells in cutaneous T-cell lymphoma (CTCL) as well as on regulatory T cells (Treg). When mogamulizumab, a defucosylated monoclonal antibody, binds to CCR4, it induces antibody-dependent cellular cytotoxicity against CCR4+ malignant T cells. The goal of this study was to determine the effect of mogamulizumab on CCR4+ Tregs in patients with CTCL.

Experimental Design: Peripheral blood of 24 patients with CTCL participating in a phase I/II trial was analyzed for CCR4 expression on different T-cell subsets by flow cytometry, before and after one course of mogamulizumab. The number and function of natural killer (NK) cells were also analyzed. Lesional biopsies were examined for CCR4, Foxp3, and CD16 expression by immunohistochemistry.

Results: Malignant T cells in peripheral blood were 20.8%–100% positive for CCR4 at baseline. Fourteen patients who achieved a response in blood had high baseline CCR4 expression on malignant T cells. Tregs in blood were 58.6% to 100% positive for CCR4 at baseline and showed decreased numbers and CCR4 expression after treatment. CD8+ T cells in blood were 3.2% to 23.2% positive for CCR4 at baseline and showed limited reduction of CCR4 expression with increased percentages of CD8+ T cells after treatment. Of 14 patients tested for NK cells in blood, 10 showed increased percentages after treatment. Four of 6 patients tested showed increased NK cell cytotoxicity. Sixteen of 18 patients who had CCR4+ lymphocytes in baseline lesions showed decreased numbers after treatment.

Conclusions: Mogamulizumab reduces levels of CCR4+ malignant T cells and also CCR4+ Tregs in patients with CTCL, which may in turn improve immune profiles. Clin Cancer Res; 21(2); 274–85. ©2014 AACR.

Purpose: WHO grade 2 low-grade gliomas (LGG) with high risk factors for recurrence are mostly lethal despite current treatments. We conducted a phase I study to evaluate the safety and immunogenicity of subcutaneous vaccinations with synthetic peptides for glioma-associated antigen (GAA) epitopes in HLA-A2+ adults with high-risk LGGs in the following three cohorts: (i) patients without prior progression, chemotherapy, or radiotherapy (RT); (ii) patients without prior progression or chemotherapy but with prior RT; and (iii) recurrent patients.

Experimental Design: GAAs were IL13Rα2, EphA2, WT1, and Survivin. Synthetic peptides were emulsified in Montanide-ISA-51 and given every 3 weeks for eight courses with intramuscular injections of poly-ICLC, followed by q12 week booster vaccines.

Results: Cohorts 1, 2, and 3 enrolled 12, 1, and 10 patients, respectively. No regimen-limiting toxicity was encountered except for one case with grade 3 fever, fatigue, and mood disturbance (cohort 1). ELISPOT assays demonstrated robust IFN responses against at least three of the four GAA epitopes in 10 and 4 cases of cohorts 1 and 3, respectively. Cohort 1 patients demonstrated significantly higher IFN responses than cohort 3 patients. Median progression-free survival (PFS) periods since the first vaccine are 17 months in cohort 1 (range, 10–47+) and 12 months in cohort 3 (range, 3–41+). The only patient with large astrocytoma in cohort 2 has been progression-free for more than 67 months since diagnosis.

Conclusion: The current regimen is well tolerated and induces robust GAA-specific responses in WHO grade 2 glioma patients. These results warrant further evaluations of this approach. Clin Cancer Res; 21(2); 286–94. ©2014 AACR.

Purpose: Gastrointestinal stromal tumor (GIST) is a relatively rare tumor that is treated with targeted therapies in advanced stages. Randomized clinical trials (RCT) often require long follow-up and large sample sizes to evaluate overall survival (OS), the gold-standard measure of treatment efficacy. However, changes in therapy following disease progression may complicate survival assessments. Establishing surrogate endpoints may facilitate the drug approval and availability of new efficacious treatments; however, no published studies have investigated this topic in unresectable and/or metastatic GIST.

Experimental Design: A systematic literature review identified 14 RCTs and five observational studies of sufficient methodologic quality published between January 1995 and December 2013 (29 treatment arms; 2,189 patients). Weighted linear regression was used to evaluate the relation between median OS and median progression-free survival (PFS) for all arms combined and stratified by treatment line, treatment type, and quality score.

Results: Median OS and PFS were positively related with a correlation of 0.91. The association was still moderate (correlation 0.72) after eliminating four influential data points. In stratified analyses, correlation of OS and PFS was greater in later lines of therapy (first line = 0.52; second line = 0.80; third- and later-line = 0.70) and imatinib showed a stronger association (0.91) than other evaluated treatments (–0.26 to 0.69).

Conclusion: This analysis identified a strong relationship between median OS and PFS, especially in later lines of therapy. Findings suggest that PFS could serve as a surrogate marker for OS; however, analyses of patient-level data are needed to establish its validity in GIST. Clin Cancer Res; 21(2); 295–302. ©2014 AACR.

Purpose: To determine the safety and toxicities of sequential MMC (mitomycin C) + BCG (bacillus Calmette–Guérin) in patients with non–muscle-invasive bladder cancer (NMIBC) and explore evidence for potentiation of BCG activity by MMC.

Experimental Design: A 3 + 3 phase I dose-escalation trial of six weekly treatments was conducted in patients with NMIBC. MMC (10, 20, or 40 mg) was instilled intravesically for 30 minutes, followed by a 10-minute washout with gentle saline irrigation and then instillation of BCG (half or full strength) for 2 hours. Urine cytokines were monitored and compared with levels in a control cohort receiving BCG only. Murine experiments were carried out as described previously.

Results: Twelve patients completed therapy, including 3 patients receiving full doses. The regimen was well tolerated with no treatment-related dose-limiting toxicities. Urinary frequency and urgency, and fatigue were common. Eleven (91.7%) patients were free of disease at a mean (range) follow-up of 21.4 (8.4–27.0) months. Median posttreatment urine concentrations of IL2, IL8, IL10, and TNFα increased over the 6-week treatment period. A greater increase in posttreatment urinary IL8 during the 6-week period was observed in patients receiving MMC + BCG compared with patients receiving BCG monotherapy. In mice, intravesical MMC + BCG skewed tumor-associated macrophages (TAM) toward a beneficial M1 phenotype.

Conclusions: Instillation of sequential MMC + BCG is safe tolerable up to 40-mg MMC plus full-strength BCG. This approach could provide improved antitumor activity over BCG monotherapy by augmenting beneficial M1 TAMs. Clin Cancer Res; 21(2); 303–11. ©2014 AACR.

Purpose: The peptides derived from ideal cancer–testis antigens, including LY6K, CDCA1, and IMP3 (identified using genome-wide cDNA microarray analyses), were used in immunotherapy for head and neck squamous cell cancer (HNSCC). In this trial, we analyzed the immune response to and safety and efficacy of vaccine therapy.

Experimental Design: A total of 37 patients with advanced HNSCC were enrolled in this trial of peptide vaccine therapy, and the OS, PFS, and immunologic response were evaluated using enzyme-linked ImmunoSpot (ELISPOT) and pentamer assays. The peptides were subcutaneously administered weekly with IFA. The primary endpoints were evaluated on the basis of differences between HLA-A*2402-positive [A24(+)] patients treated with peptide vaccine therapy and –negative [A24(–)] patients treated without peptide vaccine therapy among those with advanced HNSCC.

Results: Our cancer vaccine therapy was well tolerated. The OS of the A24(+) vaccinated group (n = 37) was statistically significantly longer than that of the A24(–) group (n = 18) and median survival time (MST) was 4.9 versus 3.5 months, respectively; P < 0.05. One of the patients exhibited a complete response. In the A24(+) vaccinated group, the ELISPOT assay identified LY6K-, CDCA1-, and IMP3-specific CTL responses in 85.7%, 64.3%, and 42.9% of the patients, respectively. The patients showing LY6K- and CDCA1-specific CTL responses demonstrated a longer OS than those without CTL induction. Moreover, the patients exhibiting CTL induction for multiple peptides demonstrated better clinical responses.

Conclusions: The immune response induced by this vaccine may improve the prognosis of patients with advanced HNSCC. Clin Cancer Res; 21(2); 312–21. ©2014 AACR.

Purpose: Paclitaxel, a widely used chemotherapeutic drug, can cause peripheral neuropathies leading to dose reductions and treatment suspensions and decreasing the quality of life of patients. It has been suggested that genetic variants altering paclitaxel pharmacokinetics increase neuropathy risk, but the major causes of interindividual differences in susceptibility to paclitaxel toxicity remain unexplained. We carried out a whole-exome sequencing (WES) study to identify genetic susceptibility variants associated with paclitaxel neuropathy.

Experimental Design: Blood samples from 8 patients with severe paclitaxel-induced peripheral neuropathy were selected for WES. An independent cohort of 228 cancer patients with complete paclitaxel neuropathy data was used for variant screening by DHPLC and association analysis. HEK293 cells were used for heterologous expression and characterization of two novel CYP3A4 enzymes.

Results: WES revealed 2 patients with rare CYP3A4 variants, a premature stop codon (CYP3A4*20 allele) and a novel missense variant (CYP3A4*25, p.P389S) causing reduced enzyme expression. Screening for CYP3A4 variants in the independent cohort revealed three additional CYP3A4*20 carriers, and two patients with missense variants exhibiting diminished enzyme activity (CYP3A4*8 and the novel CYP3A4*27 allele, p.L475V). Relative to CYP3A4 wild-type patients, those carrying CYP3A4 defective variants had more severe neuropathy (2- and 1.3-fold higher risk of neuropathy for loss-of-function and missense variants, respectively, P = 0.045) and higher probability of neuropathy-induced paclitaxel treatment modifications (7- and 3-fold higher risk for loss-of-function and missense variants, respectively, P = 5.9 x 10–5).

Conclusion: This is the first description of a genetic marker associated with paclitaxel treatment modifications caused by neuropathy. CYP3A4 defective variants may provide a basis for paclitaxel treatment individualization. Clin Cancer Res; 21(2); 322–8. ©2014 AACR.

Purpose: Asparaginase (ASNase) is a standard and critical component in the therapy of childhood acute lymphoblastic leukemia (ALL), but it is also associated with several toxicities.

Experimental design: We recently reported the results of an association study between ASNase pathway genes and event-free survival (EFS) in childhood patients with ALL. The same polymorphisms were interrogated here in relation to allergies, pancreatitis, and thrombotic events following treatment with E. coli ASNase.

Results: Among patients of the discovery group, allergies, and pancreatitis were more frequent in individuals who are homozygous for the triple-repeat allele (3R) of the asparagine synthetase (ASNS) gene, resulting in remarkably higher risk of these toxicities associated with 3R3R genotype [OR for allergies, 14.6; 95% confidence interval (CI), 3.6–58.7; P < 0.0005 and OR for pancreatitis, 8.6; 95% CI, 2.0–37.3; P = 0.01]. In contrast, the ASNS haplotype *1 harboring double-repeat (2R) allele had protective effect against these adverse reactions (P ≤ 0.01). The same haplotype was previously reported to confer reduction in EFS. The risk effect of 3R3R genotype was not replicated in the validation cohort, whereas the protective effect of haplotype *1 against allergies was maintained (P ≤ 0.002). Analysis with additional polymorphisms in ASNS locus in lymphoblastoid cell lines showed that haplotype *1 is diversified in several subtypes of which one was associated with reduced in vitro sensitivity to ASNase (rs10486009, P = 0.01) possibly explaining an association seen in clinical setting.

Conclusions: This finding might have implication for treatment individualization in ALL and other cancers using asparagine depletion strategies. Clin Cancer Res; 21(2); 329–34. ©2014 AACR.

See related commentary by Avramis, p. 230

Purpose: We describe a noninvasive PET imaging method that monitors early therapeutic efficacy of BAY 87-2243, a novel small-molecule inhibitor of mitochondrial complex I as a function of hypoxia-inducible factor-1α (HIF1α) activity.

Experimental Design: Four PET tracers [18F-FDG, 18F-Fpp(RGD)2, 18F-FLT, and 18F-FAZA] were assessed for uptake into tumor xenografts of drug-responsive (H460, PC3) or drug-resistant (786-0) carcinoma cells. Mice were treated with BAY 87-2243 or vehicle. At each point, RNA from treated and vehicle H460 tumor xenografts (n = 3 each) was isolated and analyzed for target genes.

Results: Significant changes in uptake of 18F-FAZA, 18F-FLT, and 18F-Fpp(RGD)2 (P < 0.01) occurred with BAY 87-2243 treatment with 18F-FAZA being the most prominent. 18F-FDG uptake was unaffected. 18F-FAZA tumor uptake declined by 55% to 70% (1.21% ± 0.10%ID/g to 0.35 ± 0.1%ID/g; n = 6, vehicle vs. treatment) in both H460 (P < 0.001) and PC3 (P < 0.05) xenografts 1 to 3 days after drug administration. 18F-FAZA uptake in 786-0 xenografts was unaffected. Decline occurred before significant differences in tumor volume, thus suggesting 18F-FAZA decrease reflected early changes in tumor metabolism. BAY 87-2243 reduced expression of hypoxia-regulated genes CA IX, ANGPTL4, and EGLN-3 by 99%, 93%, and 83%, respectively (P < 0.001 for all), which corresponds with reduced 18F-FAZA uptake upon drug treatment. Heterogeneous expression of genes associated with glucose metabolism, vessel density, and proliferation was observed.

Conclusions: Our studies suggest suitability of 18F-FAZA-PET as an early pharmacodynamic monitor on the efficacy of anticancer agents that target the mitochondrial complex I and intratumor oxygen levels (e.g., BAY 87-2243). Clin Cancer Res; 21(2); 335–46. ©2014 AACR.

Purpose: SET is an endogenous PP2A inhibitor that might represent a novel molecular target for antitumor therapy. The aim of this study was to evaluate the molecular effects of SET deregulation and its potential clinical significance in metastatic colorectal cancer (mCRC).

Experimental Design: We studied the biologic effects of SET on cell growth, colonosphere formation, caspase activity, PP2A activation status, and sensitivity to oxaliplatin and FTY720 treatments. Moreover, we analyzed SET expression by immunostaining in 242 patients with mCRC.

Results: SET deregulation promotes cell growth and colonosphere formation and inhibits PP2A, thereby impairing its antitumor effects. Moreover, SET reduces sensitivity to oxaliplatin in colorectal cancer cell lines, which is restored after FTY720 treatment. SET overexpression was detected in 24.8% (60 of 242) of patients with mCRC and determined significantly shorter overall (8.6 vs. 27 months; P < 0.001) and progression-free survival (7.1 vs. 13.7 months; P < 0.001), and poor response to oxaliplatin-based chemotherapy (P = 0.004). Interestingly, its prognostic value was particularly evident in patients younger than 70 years and in those harboring KRAS mutations.

Conclusions: SET overexpression is a frequent event in mCRC that plays a potential oncogenic role associated with worse outcome and resistance to oxaliplatin. Moreover, this alteration defines a subgroup of patients who could benefit from therapies containing PP2A activators such as FTY720. Clin Cancer Res; 21(2); 347–56. ©2014 AACR.

Purpose: Activation of YAP1, a novel oncogene in the Hippo pathway, has been observed in many cancers, including colorectal cancer. We investigated whether activation of YAP1 is significantly associated with prognosis or treatment outcomes in colorectal cancer.

Experimental Design: A gene expression signature reflecting YAP1 activation was identified in colorectal cancer cells, and patients with colorectal cancer were stratified into two groups according to this signature: activated YAP1 colorectal cancer (AYCC) or inactivated YAP1 colorectal cancer (IYCC). Stratified patients in five test cohorts were evaluated to determine the effect of the signature on colorectal cancer prognosis and response to cetuximab treatment.

Results: The activated YAP1 signature was associated with poor prognosis for colorectal cancer in four independent patient cohorts with stage I–III disease (total n = 1,028). In a multivariate analysis, the impact of the YAP1 signature on disease-free survival was independent of other clinical variables [hazard ratio (HR), 1.63; 95% confidence interval (CI), 1.25–2.13; P < 0.001]. In patients with stage IV colorectal cancer and wild-type KRAS, IYCC patients had a better disease control rate and progression-free survival (PFS) after cetuximab monotherapy than did AYCC patients; however, in patients with KRAS mutations, PFS duration after cetuximab monotherapy was not different between IYCC and AYCC patients. In multivariate analysis, the effect of YAP1 activation on PFS was independent of KRAS mutation status and other clinical variables (HR, 1.82; 95% CI, 1.05–3.16; P = 0.03).

Conclusions: Activation of YAP1 is highly associated with poor prognosis for colorectal cancer and may be useful in identifying patients with metastatic colorectal cancer resistant to cetuximab. Clin Cancer Res; 21(2); 357–64. ©2014 AACR.

Purpose: VEGF receptor (VEGFR) kinases are important drug targets in oncology that affect function of systemic endothelial cells. To discover genetic markers that affect VEGFR inhibitor pharmacodynamics, we performed a genome-wide association study of serum soluble vascular VEGFR2 concentrations [sVEGFR2], a pharmacodynamic biomarker for VEGFR2 inhibitors.

Experimental Design: We conducted a genome-wide association study (GWAS) of [sVEGFR2] in 736 healthy Old Order Amish volunteers. Gene variants identified from the GWAS were genotyped serially in a cohort of 128 patients with advanced solid tumor with baseline [sVEGFR2] measurements, and in 121 patients with renal carcinoma with [sVEGFR2] measured before and during pazopanib therapy.

Results: rs34231037 (C482R) in KDR, the gene encoding sVEGFR2 was found to be highly associated with [sVEGFR2], explaining 23% of the variance (P = 2.7 x 10–37). Association of rs34231037 with [sVEGFR2] was replicated in 128 patients with cancer with comparable effect size (P = 0.025). Furthermore, rs34231037 was a significant predictor of changes in [sVEGFR2] in response to pazopanib (P = 0.01).

Conclusion: Our findings suggest that genome-wide analysis of phenotypes in healthy populations can expedite identification of candidate pharmacogenetic markers. Genotyping for germline variants in KDR may have clinical utility in identifying patients with cancer with unusual sensitivity to effects of VEGFR2 kinase inhibitors. Clin Cancer Res; 21(2); 365–72. ©2014 AACR.

Purpose: Hyperactivation of ERK1/2 MAPK (hMAPK) leads to loss of estrogen receptor (ER) expression and poor outcome in breast cancer. microRNAs (miRNA) play important regulatory roles and serve as biomarkers of disease. Here, we describe molecular, pathologic, and clinical outcome associations of an hMAPK–miRNA expression signature in breast cancer.

Experimental Design: An hMAPK–miRNA signature was identified, and associations of this signature with molecular and genetic alterations, gene expression, pathologic features, and clinical outcomes were determined in primary breast cancers from training data and validated using independent datasets. Univariate and multivariate analyses identified subsignatures associated with increased disease recurrence and poorer disease survival among ER-positive (ER+) patients, respectively.

Results: High-hMAPK–miRNA status significantly correlated with ER-negativity, enrichment for basal and HER2-subtypes, and reduced recurrence-free and disease-specific survival in publicly available datasets. A robust determination of a recurrence signature and a survival signature identified hMAPK–miRNAs commonly associated with poor clinical outcome, and specific subsets associated more closely with either disease recurrence or disease survival, especially among ER+ cancers of both luminal A and luminal B subtypes. Multivariate analyses indicated that these recurrence and survival signatures significantly associated with increased risk of disease-specific death and disease recurrence in ER+ cancer and ER+ cancers treated with hormone therapy.

Conclusions: We report an hMAPK–miRNA signature and two subsignatures derived from it that associate significantly with adverse clinical features, poor clinical outcome, and poor response to hormone therapy in breast cancer, thus identifying potential effectors of MAPK signaling, and novel predictive and prognostic biomarkers or therapeutic targets in breast cancer. Clin Cancer Res; 21(2); 373–85. ©2014 AACR.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal disease that develops relatively symptom-free and is therefore advanced at the time of diagnosis. The absence of early symptoms and effective treatments has created a critical need for identifying and developing new noninvasive biomarkers and therapeutic targets.

Experimental Design: We investigated the metabolism of a panel of PDAC cell lines in culture and noninvasively in vivo with 1H magnetic resonance spectroscopic imaging (MRSI) to identify noninvasive biomarkers and uncover potential metabolic targets.

Results: We observed elevated choline-containing compounds in the PDAC cell lines and tumors. These elevated choline-containing compounds were easily detected by increased total choline (tCho) in vivo, in spectroscopic images obtained from tumors. Principal component analysis of the spectral data identified additional differences in metabolites between immortalized human pancreatic cells and neoplastic PDAC cells. Molecular characterization revealed overexpression of choline kinase (Chk)-α, choline transporter 1 (CHT1), and choline transporter–like protein 1 (CTL1) in the PDAC cell lines and tumors.

Conclusions: Collectively, these data identify new metabolic characteristics of PDAC and reveal potential metabolic targets. Total choline detected with 1H MRSI may provide an intrinsic, imaging probe–independent biomarker to complement existing techniques in detecting PDAC. The expression of Chk-α, CHT1, and CTL1 may provide additional molecular markers in aspirated cytological samples. Clin Cancer Res; 21(2); 386–95. ©2014 AACR.

Purpose: Improved therapeutic approaches are needed for the treatment of pancreatic ductal adenocarcinoma (PDAC). As dual MEK and PI3K inhibition is presently being used in clinical trials for patients with PDAC, we sought to test the efficacy of combined targeting of these pathways in PDAC using both in vitro drug screens and genetically engineered mouse models (GEMM).

Experimental Design: We performed high-throughput screening of >500 human cancer cell lines (including 46 PDAC lines), for sensitivity to 50 clinically relevant compounds, including MEK and PI3K inhibitors. We tested the top hit in the screen, the MEK1/2 inhibitor, AZD6244, for efficacy alone or in combination with the PI3K inhibitors, BKM120 or GDC-0941, in a KrasG12D-driven GEMM that recapitulates the histopathogenesis of human PDAC.

Results: In vitro screens revealed that PDAC cell lines are relatively resistant to single-agent therapies. The response profile to the MEK1/2 inhibitor, AZD6244, was an outlier, showing the highest selective efficacy in PDAC. Although MEK inhibition alone was mainly cytostatic, apoptosis was induced when combined with PI3K inhibitors (BKM120 or GDC-0941). When tested in a PDAC GEMM and compared with the single agents or vehicle controls, the combination delayed tumor formation in the setting of prevention and extended survival when used to treat advanced tumors, although no durable responses were observed.

Conclusions: Our studies point to important contributions of MEK and PI3K signaling to PDAC pathogenesis and suggest that dual targeting of these pathways may provide benefit in some patients with PDAC. Clin Cancer Res; 21(2); 396–404. ©2014 AACR.

Purpose: Vaccinia virus has strong potential as a novel therapeutic agent for treatment of pancreatic cancer. We investigated whether arming vaccinia virus with interleukin-10 (IL10) could enhance the antitumor efficacy with the view that IL10 might dampen the host immunity to the virus, increasing viral persistence, thus maximizing the oncolytic effect and antitumor immunity associated with vaccinia virus.

Experimental Design: The antitumor efficacy of IL10-armed vaccinia virus (VVLTK-IL10) and control VVTK was assessed in pancreatic cancer cell lines, mice bearing subcutaneous pancreatic cancer tumors and a pancreatic cancer transgenic mouse model. Viral persistence within the tumors was examined and immune depletion experiments as well as immunophenotyping of splenocytes were carried out to dissect the functional mechanisms associated with the viral efficacy.

Results: Compared with unarmed VVLTK, VVLTK-IL10 had a similar level of cytotoxicity and replication in vitro in murine pancreatic cancer cell lines, but rendered a superior antitumor efficacy in the subcutaneous pancreatic cancer model and a K-ras-p53 mutant-transgenic pancreatic cancer model after systemic delivery, with induction of long-term antitumor immunity. The antitumor efficacy of VVLTK-IL10 was dependent on CD4+ and CD8+, but not NK cells. Clearance of VVLTK-IL10 was reduced at early time points compared with the control virus. Treatment with VVLTK-IL10 resulted in a reduction in virus-specific, but not tumor-specific CD8+ cells compared with VVLTK.

Conclusions: These results suggest that VVLTK-IL10 has strong potential as an antitumor therapeutic for pancreatic cancer. Clin Cancer Res; 21(2); 405–16. ©2014 AACR.

Purpose: Head and neck squamous cell carcinomas (HNSCC) are frequently drug resistant and have a mortality rate of 45%. We have previously shown that E2F7 may contribute to drug resistance in SCC cells. However, the mechanism and pathways involved remain unknown.

Experimental Design: We used transcriptomic profiling to identify candidate pathways that may contribute to E2F7-dependent resistance to anthracyclines. We then manipulated the activity/expression of the candidate pathway using overexpression, knockdown, and pharmacological inhibitors in in vitro and in vivo models of SCC to demonstrate causality. In addition, we examined the expression of E2F7 and a downstream effector in a tissue microarray (TMA) generated from HNSCC patient samples.

Results: E2F7-deficient keratinocytes were selectively sensitive to doxorubicin and this was reversed by overexpressing E2F7. Transcriptomic profiling identified Sphingosine kinase 1 (Sphk1) as a potential mediator of E2F7-dependent drug resistance. Knockdown and overexpression studies revealed that Sphk1 was a downstream target of E2F7. TMA studies showed that E2F7 overexpression correlated with Sphk1 overexpression in human HNSCC. Moreover, inhibition of Sphk1 by shRNA or the Sphk1-specific inhibitor, SK1-I (BML-EI411), enhanced the sensitivity of SCC cells to doxorubicin in vitro and in vivo. Furthermore, E2F7-induced doxorubicin resistance was mediated via Sphk1-dependent activation of AKT in vitro and in vivo.

Conclusion: We identify a novel drugable pathway in which E2F7 directly increases the transcription and activity of the Sphk1/S1P axis resulting in activation of AKT and subsequent drug resistance. Collectively, this novel combinatorial therapy can potentially be trialed in humans using existing agents. Clin Cancer Res; 21(2); 417–27. ©2014 AACR.

Purpose: Non–small cell lung cancer (NSCLC) is a leading cause of cancer-related death worldwide. Patients with NSCLC with EGFR-activating mutation benefit greatly by gefitinib, an EGFR tyrosine kinase inhibitor. However, acquired resistance limits its clinical use. Histone deacetylases (HDAC) are oncoproteins associated with cancer progression and drug resistance. Here, we disclosed that inhibition of HDAC1 induced protein phosphatase DUSP1 upregulation to overcome gefitinib-acquired resistance.

Experimental Design: The effect of HDAC1 inhibition restored gefitinib sensitivity was assessed by in vitro MTT and apoptotic assays, and in vivo xenograft and orthotopic lung cancer mouse models. Protein phosphatase array was used to detect DUSP1 expression. Immunohistochemical staining and quantitative PCR were used to analyze DUSP1 expression in clinical NSCLC specimens.

Results: Gefitinib-resistant NSCLC cells showed HDAC1 overexpression, and its knockdown sensitized resistant cells to gefitinib in vitro and in preclinical models through DUSP1 expression. Overexpression of DUSP1 in resistant cells restored gefitinib sensitivity by inhibiting EGFR signaling and inducing apoptosis, whereas its knockdown in sensitive cells conferred gefitinib resistance. A novel HDAC inhibitor, WJ-26210-2, in combination with gefitinib upregulated DUSP1 expression to exert in vitro and in vivo synergistic effect on inactivation of EGFR signaling, growth inhibition, and apoptosis. Clinically, high DUSP1 level was correlated with delayed emergence of gefitinib-acquired resistance.

Conclusions: Decreased DUSP1 might be a mechanism responsible for gefitinib resistance, and DUSP1 might be a biomarker for gefitinib efficacy. HDAC1 inhibition–induced DUSP1 upregulation could be a promising strategy to overcome gefitinib-acquired resistance. Clin Cancer Res; 21(2); 428–38. ©2015 AACR.

Purpose: New therapies are urgently needed for patients with acute myelogenous leukemia (AML). The novel NEDDylation inhibitor MLN4924 (pevonedistat) has demonstrated significant preclinical antileukemic activity and preliminary efficacy in patients with AML in a phase I trial. On the basis of its antimyeloid and DNA-damaging properties, we investigated the ability of MLN4924 to augment conventional cytarabine (ara-C) therapy.

Experimental Design: The effects of MLN4924/ara-C on viability, clonogenic survival, apoptosis, DNA damage, and relevant pharmacodynamic targets were determined. The efficacy and pharmacodynamics of MLN4924/ara-C were assessed in an AML xenograft model.

Results: Cotreatment of AML cell lines and primary patient specimens with MLN4924 and ara-C led to diminished clonogenic survival, increased apoptosis, and synergistic levels of DNA damage. RNAi demonstrated that stabilization of CDT-1, an event previously shown to mediate the DNA-damaging effects of MLN4924, was not a key regulator of sensitivity to the MLN4924/ara-C combination. Global metabolic profiling revealed that MLN4924 disrupts nucleotide metabolism and depletes intracellular nucleotide pools in AML cells. Subsequent experiments showed that MLN4924 promoted increased incorporation of ara-C into the DNA of AML cells. This effect as well as the therapeutic benefit of the MLN4924/ara-C combination was antagonized by supplementation with the nucleotide building block ribose. Coadministration of MLN4924 and ara-C to mice bearing FLT3-ITD+ AML xenografts stably inhibited disease progression and increased DNA damage in vivo.

Conclusions: Our findings provide strong rationale for clinical investigation of the MLN4924/ara-C combination and establish a new link between therapeutic inhibition of NEDDylation and alterations in nucleotide metabolism. Clin Cancer Res; 21(2); 439–47. ©2014 AACR.

Purpose: Cancer cells are highly dependent on folate metabolism, making them susceptible to drugs that inhibit folate receptor activities. Targeting overexpressed folate receptor alpha (FRα) in cancer cells offers a therapeutic opportunity. We investigated the functional mechanisms of MORAB-003 (farletuzumab), a humanized mAb against FRα, in ovarian cancer models.

Experimental Design: We first examined FRα expression in an array of human ovarian cancer cell lines and then assessed the in vivo effect of MORAB-003 on tumor growth and progression in several orthotopic mouse models of ovarian cancer derived from these cell lines. Molecular mechanisms of tumor cell death induced by MORAB-003 were investigated by cDNA and protein expression profiling analysis. Mechanistic studies were performed to determine the role of autophagy in MORAB-003–induced cell death.

Results: MORAB-003 significantly decreased tumor growth in the high-FRα IGROV1 and SKOV3ip1 models but not in the low-FRα A2780 model. MORAB-003 reduced proliferation, but had no significant effect on apoptosis. Protein expression and cDNA microarray analyses showed that MORAB-003 regulated an array of autophagy-related genes. It also significantly increased expression of LC3 isoform II and enriched autophagic vacuolization. Blocking autophagy with hydroxychloroquine or bafilomycin A1 reversed the growth inhibition induced by MORAB-003. In addition, alteration of FOLR1 gene copy number significantly correlated with shorter disease-free survival in patients with ovarian serous cancer.

Conclusions: MORAB-003 displays prominent antitumor activity in ovarian cancer models expressing FRα at high levels. Blockade of folate receptor by MORAB-003 induced sustained autophagy and suppressed cell proliferation. Clin Cancer Res; 21(2); 448–59. ©2014 AACR.

Purpose: Tumor hypoxia is one of the crucial microenvironments to promote therapy resistance (TR) in glioblastoma multiforme (GBM). Livin, a member of the family of inhibitor of apoptosis proteins, contributes antiapoptosis. However, the role of tumor hypoxia in Livin regulation and its impact on TR are unclear.

Experimental Design: Livin expression and apoptosis for tumor hypoxic cells derived from human glioblastoma xenografts or in vitro hypoxic stress-treated glioblastoma cells were determined by Western blotting, immunofluorescence imaging, and annexin V staining assay. The mechanism of hypoxia-induced Livin induction was investigated by chromatin immunoprecipitation assay and reporter assay. Genetic and pharmacologic manipulation of Livin was utilized to investigate the role of Livin on tumor hypoxia–induced TR in vitro or in vivo.

Results: The upregulation of Livin expression and downregulation of caspase activity were observed under cycling and chronic hypoxia in glioblastoma cells and xenografts, concomitant with increased TR to ionizing radiation and temozolomide. However, knockdown of Livin inhibited these effects. Moreover, hypoxia activated Livin transcription through the binding of hypoxia-inducible factor-1α to the Livin promoter. The targeted inhibition of Livin by the cell-permeable peptide (TAT-Lp15) in intracerebral glioblastoma-bearing mice demonstrated a synergistic suppression of tumor growth and increased the survival rate in standard-of-care treatment with radiation plus temozolomide.

Conclusions: These findings indicate a novel pathway that links upregulation of Livin to tumor hypoxia–induced TR in GBM and suggest that targeting Livin using cell-permeable peptide may be an effective therapeutic strategy for tumor microenvironment–induced TR. Clin Cancer Res; 21(2); 460–70. ©2014 AACR.

Purpose: To determine whether HERV-K envelope (ENV) protein could function as a tumor-associated antigen and elicit specific T-cell responses against autologous ovarian cancer cells.

Experimental Design: The expression of HERV-K transcripts and ENV protein, the presence of serum antibodies against HERV-K, reverse transcriptase (RT) activities, and cellular immune responses in primary ovarian cancer tissues and patient blood samples were analyzed and compared with samples from patients with benign ovarian diseases and normal female donors.

Results: Ovarian cancer cells in primary tumors and ascites expressed markers of cancer stem cells and markers of both mesenchymal and epithelial cells. Expression of HERV transcripts and HERV-K ENV protein and reverse transcriptase activities were higher in ovarian cancer compared with adjacent normal and benign tissues. The ovarian cancer patient plasma also had high reverse transcriptase activities and the ovarian cancer patient sera contained HERV-K immunoreactive antibodies. HERV-K–specific T cells generated from autologous dendritic cells pulsed with HERV-K ENV antigens exhibited phenotypes and functions consistent with a cellular immune response including T-cell proliferation, IFN production, and HERV-K–specific cytotoxic T lymphocyte (CTL) activity. Significantly higher CTL lysis of autologous tumor cells than of uninvolved normal cells was demonstrated in patients with ovarian cancer than patients with benign diseases and further enhanced lysis was observed if T regulatory cells were depleted.

Conclusion: Endogenous retroviral gene products in ovarian cancer may represent a potentially valuable new pool of tumor-associated antigens for targeting of therapeutic vaccines to ovarian cancer. Clin Cancer Res; 21(2); 471–83. ©2014 AACR.

Purpose: The early detection of lung cancer in heavy smokers by low-dose CT (LDCT) can reduce the mortality. However, LDCT screening increases the number of indeterminate solitary pulmonary nodules (SPN) in asymptomatic individuals, leading to overdiagnosis. Making a definitive preoperative diagnosis of malignant SPNs has been a clinical challenge. We have demonstrated that sputum miRNAs could provide potential biomarkers for lung cancer. Here, we aimed to develop sputum miRNA biomarkers for diagnosis of malignant SPNs.

Experimental Design: Using quantitative RT-PCR, we evaluated expressions of 13 sputum miRNAs, previously identified sputum miRNA signatures of lung cancer, in a training set of 122 patients with either malignant (n = 60) or benign SPNs (n = 62) to define a panel of biomarkers. We then validated the biomarker panel in an internal testing set of 136 patients with either malignant (n = 67) or benign SPNs (n = 69), and an external testing cohort of 155 patients with either malignant (n = 76) or benign SPNs (n = 79).

Results: In the training set, a panel of three miRNA biomarkers (miRs21, 31, and 210) was developed, producing 82.93% sensitivity and 87.84% specificity for identifying malignant SPNs. The sensitivity and specificity of the biomarkers in the two independent testing cohorts were 82.09% and 88.41%, 80.52% and 86.08%, respectively, confirming the diagnostic value.

Conclusions: Sputum miRNA biomarkers may improve LDCT screening for lung cancer in heavy smokers by preoperatively diagnosing malignant SPNs. Nevertheless, a prospective study in a large population to validate the biomarkers is needed. Clin Cancer Res; 21(2); 484–9. ©2015 AACR.