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1.  Therapeutic Efficacy of an Fc-Enhanced TCR-Like Antibody to the Intracellular WT1 Oncoprotein 
Purpose
RMFPNAPYL (RMF), a WT1-derived CD8 T cell epitope presented by HLA-A*02:01, is a validated target for T-cell-based immunotherapy. We previously reported ESK1, a high avidity (Kd < 0.2nM), fully-human monoclonal antibody (mAb) specific for the WT1 RMF peptide/HLA-A*02:01 complex, which selectively bound and killed WT1+ and HLA-A*02:01+ leukemia and solid tumor cell lines.
Experimental Design
We engineered a second-generation mAb, ESKM, to have enhanced antibody dependent cell-mediated cytotoxicity (ADCC) function due to altered Fc glycosylation. ESKM was compared to native ESK1 in binding assays, in vitro ADCC assays, and mesothelioma and leukemia therapeutic models and pharmacokinetic studies in mice. ESKM toxicity was assessed in HLA-A*02:01+ transgenic mice.
Results
ESK antibodies mediated ADCC against hematopoietic and solid tumor cells at concentrations below 1µg/ml, but ESKM was about 5–10 fold more potent in vitro against multiple cancer cell lines. ESKM was more potent in vivo against JMN mesothelioma, and effective against SET2 AML and fresh ALL xenografts. ESKM had a shortened half-life (4.9 vs 6.5 days), but an identical biodistribution pattern in C57BL6/J mice. At therapeutic doses of ESKM, there was no difference in half-life or biodistribution in HLA-A*02:01+ transgenic mice compared to the parent strain. Importantly, therapeutic doses of ESKM in these mice caused no depletion of total WBCs or hematopoetic stem cells, or pathologic tissue damage.
Conclusions
The data provide proof of concept that an Fc-enhanced mAb can improve efficacy against a low-density, tumor-specific, peptide/MHC target, and support further development of this mAb against an important intracellular oncogenic protein.
doi:10.1158/1078-0432.CCR-13-2756
PMCID: PMC4119489  PMID: 24850840
2.  Galeterone Prevents Androgen Receptor Binding to Chromatin and Enhances Degradation of Mutant Androgen Receptor 
Purpose
Galeterone inhibits the enzyme CYP17A1 and is currently in phase 2 clinical trials for castration-resistant prostate cancer (CRPC). Galeterone is also a direct androgen receptor (AR) antagonist and may enhance AR degradation. This study was undertaken to determine the molecular basis for AR effects and their therapeutic potential.
Experimental Design
Effects of galeterone on AR expression and activities were examined in prostate cancer (PCa) cell lines.
Results
Similar to the AR antagonist enzalutamide, but in contrast to bicalutamide, galeterone did not induce binding of a constitutively active VP16-AR fusion protein to reporter genes and did not induce AR recruitment to endogenous androgen regulated genes based on chromatin immunoprecipitation. Galeterone at low micromolar concentrations that did not induce cellular stress responses enhanced AR protein degradation in LNCaP and C4-2 cells, which express a T878A mutant AR, but not in PCa cells expressing wildtype AR. Further transfection studies using stable LNCaP and PC3 cell lines ectopically expressing wildtype or T878A mutant ARs confirmed that galeterone selectively enhances degradation of the T878A mutant AR.
Conclusions
Similar to enzalutamide, galeterone may be effective as a direct AR antagonist in CRPC. It may be particularly effective against PCa cells with the T878A AR mutation, but may also enhance degradation of wildtype AR in vivo through a combination of direct and indirect mechanisms. Finally, these findings show that conformational changes in AR can markedly enhance its degradation, and thereby support efforts to develop further antagonists that enhance AR degradation.
doi:10.1158/1078-0432.CCR-14-0292
PMCID: PMC4119496  PMID: 24874833
3.  Genetic Modification of T Cells Redirected towards CS1 Enhances Eradication of Myeloma Cells 
Purpose
Our goal is to test if CS1 could be targeted by CAR T cells to treat MM.
Experimental Design
We generated a retroviral construct of a CS1-specific CAR and engineered primary human T cells expressing the CAR. We then tested the capacity of CS1-CAR T cells to eradicate human multiple myeloma tumor cells in vitro, ex vivo and in vivo using orthotopic MM xenograft mouse models.
Results
In vitro, compared to mock-transduced T cells, upon recognizing CS1 positive MM cells, CS1-CAR-tranduced T cells secreted more IFN-γ as well as IL-2, expressed higher levels of the activation marker CD69, showed higher capacity for degranulation, and displayed enhanced cytotoxicity. Ectopically forced expression of CS1 in MM cells with low CS1 expression enhanced recognition and killing by CAR T cells. Ex vivo, CS1-CAR T cells also showed similarly enhanced activities when responding to primary MM cells. More importantly, in orthotopic MM xenograft mouse models, adoptive transfer of human primary T cells expressing CS1-CAR efficiently suppressed the growth of human MM.1S and IM9 myeloma cells and significantly prolonged mouse survival.
Conclusions
CS1 is a promising antigen that can be targeted by CAR-expressing T cells for treatment of MM.
doi:10.1158/1078-0432.CCR-13-2510
PMCID: PMC4119545  PMID: 24677374
Chimeric antigen receptor; CS1; T cells; multiple myeloma; gene therapy
4.  CARTs on the road for myeloma 
Summary
Chimeric antigen receptors re-direct T cells to surface antigens. Discovery and validation of appropriate target antigens expands the possible indications for CAR-T cells. CS1 is expressed at high levels by multiple myeloma cells, but also to some extent on other lymphocytes. CS1 may be a viable target for CAR-T cells in multiple myeloma.
doi:10.1158/1078-0432.CCR-14-0721
PMCID: PMC4119563  PMID: 24919574
5.  Cisplatin-Induced Renal Injury is Independently Mediated by OCT2 and p53 
Purpose
Tubular secretion of cisplatin is abolished in mice deficient for the organic cation transporters Oct1 and Oct2 [Oct1/2(−/−) mice], and these animals are protected from severe cisplatin-induced kidney damage. Since tubular necrosis is not completely absent in Oct1/2(−/−) mice, we hypothesized that alternate pathways are involved in the observed injury.
Experimental Design
Studies were done in wildtype, Oct1/2(−/−), or p53-deficient animals, all on an FVB background, receiving i.p. cisplatin at 15 mg/kg. The cisplatin metabolites were analyzed using mass spectrometry, and gene expression was assessed using Affymetrix microarrays and RT-PCR arrays.
Results
KEGG pathway analyses on kidneys from mice exposed to cisplatin revealed that most significantly altered genes were associated with the p53 signaling network, including Cdnk1a and Mdm2, in both wildtype (P=2.40×10–11) and Oct1/2(−/−) mice (P=1.92×10-8). This was confirmed by demonstrating that homozygosity for a p53-null allele partially reduced renal tubular damage, while loss of p53 in Oct1/2(−/−) mice [p53(−/−)/Oct1/2(−/−)] completely abolished nephrotoxicity. We found that pifithrin-α, an inhibitor of p53-dependent transcriptional activation, inhibits Oct2 and can mimic the lack of nephrotoxicity observed in p53(−/−)/Oct1/2(−/−) mice.
Conclusions
These findings indicate that (i) the p53 pathway plays a crucial role in the kidney in response to cisplatin treatment and (ii) clinical exploration of OCT2 inhibitors may not lead to complete nephroprotection unless the p53 pathway is simultaneously antagonized.
doi:10.1158/1078-0432.CCR-14-0319
PMCID: PMC4119572  PMID: 24916697
Cisplatin; OCT2; p53; pifithrin-α; nephrotoxicity
6.  Tyrosine phosphoproteomics identified both co-drivers and co-targeting strategies for T790M-related EGFR-TKI resistance in non-small cell lung cancer 
Purpose
Irreversible EGFR-tyrosine kinase inhibitors (TKIs) are thought to be one strategy to overcome EGFR-TKI resistance induced by T790M gate-keeper mutations in non-small cell lung cancer (NSCLC), yet they display limited clinical efficacy. We hypothesized that additional resistance mechanisms that cooperate with T790M could be identified by profiling tyrosine phosphorylation in NSCLC cells with acquired resistance to reversible EGFR-TKI and harboring T790M.
Experimental Design
We profiled PC9 cells with TKI-sensitive EGFR mutation and paired EGFR-TKI-resistant PC9GR (gefitinib-resistant) cells with T790M using immunoaffinity purification of tyrosine-phosphorylated peptides and mass-spectrometry-based identification/quantification. Profiles of erlotinib perturbations were examined.
Results
We observed a large fraction of the tyrosine phosphoproteome was more abundant in PC9- and PC9GR-erlotinib treated cells, including phosphopeptides corresponding to MET, IGF, and AXL signaling. Activation of these receptor tyrosine kinases by growth factors could protect PC9GR cells against the irreversible EGFR-TKI afatinib. We identified a Src-family kinase (SFK) network as EGFR-independent and confirmed that neither erlotinib nor afatinib affected Src phosphorylation at the activation site. The SFK-inhibitor dasatinib plus afatinib abolished Src phosphorylation and completely suppressed downstream phosphorylated Akt and Erk. Dasatinib further enhanced anti-tumor activity of afatinib or T790M-selective EGFR-TKI (WZ4006) in proliferation and apoptosis assays in multiple NSCLC cell lines with T790M mediated resistance. This translated into tumor regression in PC9GR xenograft studies with combined afatinib and dasatinib.
Conclusions
Our results identified both co-drivers of resistance along with T790M and support further studies of irreversible or T790M-selective EGFR inhibitors combined with dasatinib in NSCLC patients with acquired T790M.
doi:10.1158/1078-0432.CCR-13-1559
PMCID: PMC4119578  PMID: 24919575
7.  Hepatic Uptake Transporters and Docetaxel Disposition in Mice 
doi:10.1158/1078-0432.CCR-14-0949
PMCID: PMC4127641  PMID: 24919571
Oatp1b2; docetaxel; pharmacokinetics
8.  Complex Disease–, Gene–, and Drug–Drug Interactions: Impacts of Renal Function, CYP2D6 Phenotype, and OCT2 Activity on Veliparib Pharmacokinetics 
Purpose
Veliparib, a poly (ADP-ribose) polymerase (PARP) inhibitor, undergoes renal excretion and liver metabolism. This study quantitatively assessed the interactions of veliparib with metabolizing enzyme (CYP2D6) and transporter (OCT2) in disease settings (renal impairment).
Experimental Design
Veliparib in vitro metabolism was examined in human liver microsomes and recombinant enzymes carrying wild-type CYP2D6 or functional defect variants (CYP2D6*10 and *4). Plasma pharmacokinetics were evaluated in 27 patients with cancer. A parent–metabolite joint population model was developed to characterize veliparib and metabolite (M8) pharmacokinetics and to identify patient factors influencing veliparib disposition. A physiologically based pharmacokinetic model integrated with a mechanistic kidney module was developed to quantitatively predict the individual and combined effects of renal function, CYP2D6 phenotype, and OCT2 activity on veliparib pharmacokinetics.
Results
In vitro intrinsic clearance of CYP2D6.1 and CYP2D6.10 for veliparib metabolism were 0.055 and 0.017 μL/min/pmol CYP, respectively. Population mean values for veliparib oral clearance and M8 clearance were 13.3 and 8.6 L/h, respectively. Creatinine clearance was identified as the significant covariate on veliparib oral clearance. Moderate renal impairment, CYP2D6 poor metabolizer, and co-administration of OCT2 inhibitor (cimetidine) increased veliparib steady-state exposure by 80%, 20%, and 30%, respectively. These factors collectively led to >2-fold increase in veliparib exposure.
Conclusions
Renal function (creatinine clearance) is a significant predictor for veliparib exposure in patients with cancer. Although a single factor (i.e., renal impairment, CYP2D6 deficiency, and reduced OCT2 activity) shows a moderate impact, they collectively could result in a significant and potentially clinically relevant increase in veliparib exposure.
doi:10.1158/1078-0432.CCR-14-0791
PMCID: PMC4151156  PMID: 24947923
9.  Germline mutation in BRCA1 or BRCA2 and ten-year survival for women diagnosed with epithelial ovarian cancer 
Purpose
To analyse the effect of germline mutations in BRCA1 and BRCA2 on mortality in ovarian cancer patients up to ten years after diagnosis.
Experimental Design
We used unpublished survival time data for 2,242 patients from two case-control studies and extended survival-time data for 4,314 patients from previously reported studies. All participants had been screened for deleterious germline mutations in BRCA1 and BRCA2. Survival time was analysed for the combined data using Cox proportional hazard models with BRCA1 and BRCA2 as time-varying covariates. Competing risks were analysed using Fine and Gray model.
Results
The combined 10-year overall survival was 30% (95% CI, 28%-31%) for non-carriers, 25% (95% CI, 22%-28%) for BRCA1 carriers, and 35% (95% CI, 30%-41%) for BRCA2 carriers. The hazard ratio for BRCA1 was 0.53 at time zero and increased over time becoming greater than one at ·4.8 years. For BRCA2, the hazard ratio was 0.42 at time zero and increased over time (predicted to become greater than one at 10.5 years). The results were similar when restricted to 3,202 patients with high-grade serous tumors, and to ovarian cancer specific mortality.
Conclusions
BRCA1/2 mutations are associated with better short-term survival, but this advantage decreases over time and, in BRCA1 carriers is eventually reversed. This may have important implications for therapy of both primary and relapsed disease and for analysis of long-term survival in clinical trials of new agents, particularly those that are effective in BRCA1/2 mutation carriers.
doi:10.1158/1078-0432.CCR-14-2497
PMCID: PMC4338615  PMID: 25398451
Ovarian cancer; Epithelial ovarian cancer; BRCA1 gene; BRCA2 gene; Survival
10.  Essential role of aldehyde dehydrogenase 1A3 (ALDH1A3) for the maintenance of non-small cell lung cancer stem cells is associated with the STAT3 pathway 
Purpose
Lung cancer stem cells (CSCs) with elevated aldehyde dehydrogenase (ALDH) activity are self-renewing, clonogenic and tumorigenic. The purpose of our study is to elucidate the mechanisms by which lung CSCs are regulated.
Experimental Design
A genome-wide gene expression analysis was performed to identify genes differentially expressed in the ALDH+ vs. ALDH− cells. RT-PCR, western blot and Aldefluor assay were used to validate identified genes. To explore the function in CSCs we manipulated their expression followed by colony and tumor formation assays.
Results
We identified a subset of genes that were differentially expressed in common in ALDH+ cells, among which ALDH1A3 was the most upregulated gene in ALDH+ vs. ALDH− cells. ShRNA-mediated knockdown of ALDH1A3 in NSCLCs resulted in a dramatic reduction in ALDH activity, clonogenicity and tumorigenicity, indicating that ALDH1A3 is required for tumorigenic properties. By contrast, overexpression of ALDH1A3 by itself it was not sufficient to increase tumorigenicity. The ALDH+ cells also expressed more activated Signal Transducers and Activators of Transcription 3 (STAT3) than ALDH− cells. Inhibition of STAT3 or its activator EZH2 genetically or pharmacologically diminished the level of ALDH+ cells and clonogenicity. Unexpectedly, ALDH1A3 was highly expressed in female, never smokers, well differentiated tumors, or adenocarcinoma. ALDH1A3 low expression was associated with poor overall survival.
Conclusion
Our data show that ALDH1A3 is the predominant ALDH isozyme responsible for ALDH activity and tumorigenicity in most NSCLCs, and that inhibiting either ALDH1A3 or the STAT3 pathway are potential therapeutic strategies to eliminate the ALDH+ subpopulation in NSCLCs.
doi:10.1158/1078-0432.CCR-13-3292
PMCID: PMC4438754  PMID: 24907115
Lung cancer; cancer stem cells; ALDH1A3; STAT3; Stattic
11.  CD44 expression denotes a subpopulation of gastric cancer cells in which Hedgehog signaling promotes chemotherapy resistance 
Purpose
Gastric cancers (GC) may harbor a small subset of cells with cancer stem cell (CSC) properties including chemotherapy (CT) resistance. The Hedgehog (HH) pathway is a key developmental pathway that can be subverted by CSCs during tumorigenesis. Here we examine the role of HH signaling in CD44(+) GC cells.
Experimental Design
GC cell lines, tumor xenografts, and patient tumors were examined.
Results
GC cell lines AGS, MKN-45, and NCI-N87 grown as spheroids or sorted for CD44(+) were found to have upregulation of HH pathway proteins. HH inhibition using Smo shRNA or vismodegib (VIS) decreased spheroid formation and colony formation. CD44(+) cells, compared to unselected cells, were also resistant to 5-fluorouracil and cisplatin CT, and this resistance was reversed in vitro and in xenografts with Smo shRNA or VIS. CD44(+) cells also had significantly more migration, invasion, and anchorage-independent growth, and these properties could all be blocked with HH inhibition. Clinical tumor samples from a phase II trial for advanced GC of CT with or without VIS were analyzed for CD44 expression. In the CT alone group, high CD44 expression was associated with decreased survival, while in the CT plus VIS group, high CD44 expression was associated with improved survival.
Conclusions
HH signaling maintains CSC phenotypes and malignant transformation phenotypes in CD44(+) GC cells, and HH inhibition can block CT resistance in CD44(+) cells. GC is a heterogeneous disease, and the strategy of combining CT with HH inhibition may only be effective in the subset with high CD44 levels.
doi:10.1158/1078-0432.CCR-14-0011
PMCID: PMC4135312  PMID: 24947926
12.  Serum CA19-9 is significantly up-regulated up to 2 years prior to diagnosis with pancreatic cancer: implications for early disease detection 
Purpose
Biomarkers for the early detection of pancreatic cancer are urgently needed. The primary objective of this study was to evaluate whether increased levels of serum CA19-9, CA125, CEACAM1 and REG3A are present prior to clinical presentation of pancreatic cancer and to assess the performance of combined markers for early detection and prognosis.
Experimental Design
This nested case control study within UKCTOCS included 118 single- and 143 serial-serum samples from 154 post-menopausal women who were subsequently diagnosed with pancreatic cancer and 304 matched non-cancer controls. Samples were split randomly into independent training and test sets. CA19-9, CA125, CEACAM1 and REG3A were measured using ELISA and/or CLIA. Performance of markers to detect cancers at different times prior to diagnosis and for prognosis was evaluated.
Results
At 95% specificity, CA19-9 (>37 U/mL) had a sensitivity of 68% up to 1 year, and 53% up to 2 yrs before diagnosis. Combining CA19-9 and CA125 improved sensitivity as CA125 was elevated (>30 U/mL) in ~20% of CA19-9-negative cases. CEACAM1 and REG3A were late markers adding little in combined models. Average lead times of 20-23 months were estimated for test-positive cases. Pre-diagnostic levels of CA19-9 and CA125 were associated with poor overall survival (HR 2.69 and 3.15, respectively).
Conclusions
CA19-9 and CA125 have encouraging sensitivity for detecting pre-clinical pancreatic cancer and both markers can be used as prognostic tools. This work challenges the prevailing view that CA19-9 is up-regulated late in the course of pancreatic cancer development.
doi:10.1158/1078-0432.CCR-14-0365
PMCID: PMC4181906  PMID: 24938522
pancreatic cancer; preclinical serum biomarkers; UKCTOCS; CA19-9; CA125; CEACAM1; REG3A
13.  Serum CA19-9 is significantly up-regulated up to 2 years prior to diagnosis with pancreatic cancer: implications for early disease detection 
Purpose
Biomarkers for the early detection of pancreatic cancer are urgently needed. The primary objective of this study was to evaluate whether increased levels of serum CA19-9, CA125, CEACAM1 and REG3A are present prior to clinical presentation of pancreatic cancer and to assess the performance of combined markers for early detection and prognosis.
Experimental Design
This nested case control study within UKCTOCS included 118 single- and 143 serial-serum samples from 154 post-menopausal women who were subsequently diagnosed with pancreatic cancer and 304 matched non-cancer controls. Samples were split randomly into independent training and test sets. CA19-9, CA125, CEACAM1 and REG3A were measured using ELISA and/or CLIA. Performance of markers to detect cancers at different times prior to diagnosis and for prognosis was evaluated.
Results
At 95% specificity, CA19-9 (>37 U/mL) had a sensitivity of 68% up to 1 year, and 53% up to 2 yrs before diagnosis. Combining CA19-9 and CA125 improved sensitivity as CA125 was elevated (>30 U/mL) in ~20% of CA19-9-negative cases. CEACAM1 and REG3A were late markers adding little in combined models. Average lead times of 20–23 months were estimated for test-positive cases. Pre-diagnostic levels of CA19-9 and CA125 were associated with poor overall survival (HR 2.69 and 3.15, respectively).
Conclusions
CA19-9 and CA125 have encouraging sensitivity for detecting pre-clinical pancreatic cancer and both markers can be used as prognostic tools. This work challenges the prevailing view that CA19-9 is up-regulated late in the course of pancreatic cancer development.
doi:10.1158/1078-0432.CCR-14-0365
PMCID: PMC4181906  PMID: 24938522
pancreatic cancer; preclinical serum biomarkers; UKCTOCS; CA19-9; CA125; CEACAM1; REG3A
14.  Phase I Study of Topotecan, Ifosfamide, and Etoposide (TIME) with Autologous Stem Cell Transplant in Refractory Cancer: Pharmacokinetic and Pharmacodynamic Correlates 
Purpose
To determine the maximum tolerated dose (MTD) of topotecan in combination with ifosfamide, mesna, and etoposide (TIME), followed by autologous hematopoietic cell transplant (HCT), in patients with chemotherapy-refractory malignancies.
Experimental Design
Patients were treated with (in mg/m2/d) ifosfamide 3,333, mesna 3,333, and topotecan 3.3 to 28.3 during days-8 through-6 and etoposide 500 (days-5 through-3) followed by HCT on day 0. Once MTD was defined, we expanded this dosing cohort to include patients with high-risk lymphoma due to activity seen during dose escalation. Topotecan pharmacokinetic analyses were carried out, and topoisomerase I levels and activity were measured.
Results
The topotecan MTD in this regimen was 64 mg/m2 (21.3 mg/m2/d). Mucositis was dose limiting and correlated with topotecan dose level and area under the curve (AUC). Dose level was also correlated with length of hospitalization, number of days of parenteral nutrition, and neutrophil and platelet engraftment. Topotecan AUC was significantly correlated with time to platelet recovery. The baseline peripheral blood mononuclear cell topoisomerase I level was found to be a significant positive predictor for overall and progression-free survival. Topotecan AUC was positively correlated with dose level, with a trend toward decreasing clearance with increasing dose.
Conclusion
Topotecan can be a useful drug in the high-dose setting given its activity in some malignancies when given in standard dose. Pharmacokinetic monitoring may be a valuable tool for optimizing the use of topotecan and to avoid toxicity seen with high-systemic exposures. Baseline topoisomerase I levels may have an important role in predicting topotecan efficacy.
doi:10.1158/1078-0432.CCR-11-1540
PMCID: PMC4521635  PMID: 22028494
15.  Consensus Recommendations to Accelerate Clinical Trials for Neurofibromatosis Type 2 
Purpose
Neurofibromatosis type 2 (NF2) is a rare autosomal dominant disorder associated primarily with bilateral schwannomas seen on the superior vestibular branches of the eighth cranial nerves. Significant morbidity can result from surgical treatment of these tumors. Meningiomas, ependymomas, and other benign central nervous system tumors are also common in NF2. The lack of effective treatments for NF2 marks an unmet medical need.
Experimental Design
Here, we provide recommendations from a workshop, cochaired by Drs. D. Gareth Evans and Marco Giovannini, of 36 international researchers, physicians, representatives of the biotechnology industry, and patient advocates on how to accelerate progress toward NF2 clinical trials.
Results
Workshop participants reached a consensus that, based on current knowledge, the time is right to plan and implement NF2 clinical trials. Obstacles impeding NF2 clinical trials and how to address them were discussed, as well as the candidate therapeutic pipeline for NF2.
Conclusions
Both phase 0 and phase II NF2 trials are near-term options for NF2 clinical trials. The number of NF2 patients in the population remains limited, and successful recruitment will require ongoing collaboration efforts between NF2 clinics.
doi:10.1158/1078-0432.CCR-08-3011
PMCID: PMC4513640  PMID: 19671848
16.  Significant Association of oncogene YAP1 with Poor Prognosis and Cetuximab Resistance in Colorectal Cancer Patients 
Purpose
Activation of YAP1, novel oncogene in Hippo pathway, has been observed in many cancers including colorectal cancer (CRC). We investigated if activation of YAP1 is significantly associated with prognosis or treatment outcomes in CRC
Experimental Design
A gene expression signature reflecting YAP1 activation was identified in CRC cells, and CRC patients were stratified into two groups according to this signature: activated YAP1 CRC (AYCC) or inactivated YAP1 CRC (IYCC). Stratified patients in five test cohorts were evaluated to determine the effect of the signature on CRC prognosis and response to cetuximab treatment.
Results
The activated YAP1 signature was associated with poor prognosis for CRC 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 the 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 CRC 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 CRC and may be useful in identifying patients with metastatic CRC resistant to cetuximab.
doi:10.1158/1078-0432.CCR-14-1374
PMCID: PMC4513664  PMID: 25388162
17.  A Targeted RNAi Screen of the Breast Cancer Genome Identifies KIF14 and TLN1 as Genes That Modulate Docetaxel Chemosensitivity in Triple-Negative Breast Cancer 
Purpose
To identify biomarkers within the breast cancer genome that may predict chemosensitivity in breast cancer.
Experimental Design
We conducted an RNA interference (RNAi) screen within the breast cancer genome for genes whose loss-of-function enhanced docetaxel chemosensitivity in an estrogen receptor–negative, progesterone receptor–negative, and Her2-negative (ER−, PR−, and Her2−, respectively) breast cancer cell line, MDA-MB-231. Top candidates were tested for their ability to modulate chemosensitivity in 8 breast cancer cell lines and to show in vivo chemosensitivity in a mouse xenograft model.
Results
From ranking chemosensitivity of 328 short hairpin RNA (shRNA) MDA-MB-231 cell lines (targeting 133 genes with known somatic mutations in breast cancer), we focused on the top two genes, kinesin family member 14 (KIF14) and talin 1 (TLN1). KIF14 and TLN1 loss-of-function significantly enhanced chemosensitivity in four triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, HCC38, HCC1937, and Hs478T) but not in three hormone receptor–positive cell lines (MCF7, T47D, and HCC1428) or normal human mammary epithelial cells (HMEC). Decreased expression of KIF14, but not TLN1, also enhanced docetaxel sensitivity in a Her2-amplified breast cancer cell line, SUM190PT. Higher KIF14 and TLN1 expressions are found in TNBCs compared with the other clinical subtypes. Mammary fat pad xenografts of KIF14- and TLN1-deficient MDA-MB-231 cells revealed reduced tumor mass compared with control MDA-MB-231 cells after chemotherapy. KIF14 expression is also prognostic of relapse-free and overall survival in representative breast cancer expression arrays.
Conclusion
KIF14 and TLN1 are modulators of response to docetaxel and potential therapeutic targets in TNBC.
doi:10.1158/1078-0432.CCR-13-0082
PMCID: PMC4513911  PMID: 23479679
18.  First-In-Human Study of PF-05212384 (PKI-587), a Small-Molecule, Intravenous, Dual Inhibitor of PI3K and mTOR In Patients With Advanced Cancer 
Purpose
To evaluate safety (primary endpoint), tolerability, pharmacokinetics, pharmacodynamic profile, and preliminary activity of the intravenous, pan-class I isoform PI3K/mTOR inhibitor PF-05212384 in patients with advanced solid tumors.
Experimental Design
Part 1 of this open-label phase I study was designed to estimate the maximum tolerated dose (MTD) in patients with non-selected solid tumors, using a modified continual reassessment method to guide dose escalation. Objectives of Part 2 were MTD confirmation and assessment of preliminary activity in patients with selected tumor types and PI3K pathway dysregulation.
Results
Seventy-seven of the 78 enrolled patients received treatment. The MTD for PF-05212384, administered intravenously once weekly, was estimated to be 154 mg. The most common treatment-related adverse events (AEs) were mucosal inflammation/stomatitis (58.4%), nausea (42.9%), hyperglycemia (26%), decreased appetite (24.7%), fatigue (24.7%), and vomiting (24.7%). The majority of patients treated at the MTD experienced only grade 1 treatment-related AEs. Grade 3 treatment-related AEs occurred in 23.8% of patients at the MTD. No treatment-related grade 4–5 AEs were reported at any dose level. Antitumor activity was noted in this heavily pretreated patient population, with two partial responses (PR) and an unconfirmed PR. Eight patients had long-lasting stable disease (>6 months). Pharmacokinetic analyses showed a biphasic concentration-time profile for PF-05212384 (half-life, 30–37 hours after multiple dosing). PF-05212384 inhibited downstream effectors of the PI3K pathway in paired tumor biopsies.
Conclusions
These findings demonstrate the manageable safety profile and antitumor activity of the PI3K/mTOR inhibitor PF-05212384, supporting further clinical development for patients with advanced solid malignancies.
doi:10.1158/1078-0432.CCR-14-1306
PMCID: PMC4508327  PMID: 25652454
PF-05212384; PI3K; mTOR; solid tumors; PI3K/TORC1/2; AKT
19.  Global Promoter Methylation Analysis Reveals Novel Candidate Tumor Suppressor Genes in Natural Killer Cell Lymphoma 
Purpose
To identify tumor suppressor genes epigenetically silenced by promoter hypermethylation in extranodal natural killer cell lymphoma (NKCL).
Experimental Design
Promoter methylation was analyzed with global and locus-specific methylation assays in NKCL cases and NK cell lines. Gene expression profiles were used to identify genes for which aberrant promoter methylation was associated with transcriptional silencing. Selected DNA methylations were validated by RRBS, pyrosequencing, or q-MSP. Decitabine treatment was performed to evaluate reactivation of methylated genes. The tumor suppressor effect of silenced genes was evaluated functionally by reintroducing them into NK cell lines.
Results
We observed significant promoter hypermethylation in most NKCL samples compared with normal NK cells. Correlation of global promoter methylation with gene expression profiles identified 95 genes with strong evidence for being silenced because of promoter methylation, including BCL2L11 (BIM), DAPK1, PTPN6 (SHP1), TET2, SOCS6, and ASNS. Known tumor suppressor genes were significantly overrepresented in this set of genes. Decitabine treatment of NK cell lines was associated with reexpression of all 10 selected methylated and silenced genes. Ectopic expression of frequently silenced BIM in two BIM-nonexpressing NK cell lines led to increased apoptosis and eventual elimination of BIM-transduced cells. It also sensitized these cell lines to chemotherapy-induced apoptosis. Similarly, reintroduction of SOCS6 significantly inhibited growth in SOCS6-nonexpressing NK cell lines. NK cell lines lacking ASNS expression showed increased sensitivity to treatment with l-asparaginase. Reintroduction of ASNS reduced drug sensitivity.
Conclusion
Promoter region hypermethylation is frequent in NKCL, and aberrantly methylated genes are pathologically and clinically significant.
doi:10.1158/1078-0432.CCR-14-1216
PMCID: PMC4504988  PMID: 25614448
20.  Biomarker Analyses from a Placebo-Controlled Phase II Study Evaluating Erlotinib ± Onartuzumab in Advanced Non–Small Cell Lung Cancer: MET Expression Levels Are Predictive of Patient Benefit 
Purpose
In a recent phase II study of onartuzumab (MetMAb), patients whose non–small cell lung cancer (NSCLC) tissue scored as positive for MET protein by immunohistochemistry (IHC) experienced a significant benefit with onartuzumab plus erlotinib (O+E) versus erlotinib. We describe development and validation of a standardized MET IHC assay and, retrospectively, evaluate multiple biomarkers as predictors of patient benefit.
Experimental Design
Biomarkers related to MET and/or EGF receptor (EGFR) signaling were measured by IHC, FISH, quantitative reverse transcription PCR, mutation detection techniques, and ELISA.
Results
A positive correlation between IHC, Western blotting, and MET mRNA expression was observed in NSCLC cell lines/tissues. An IHC scoring system of MET expression taking proportional and intensity-based thresholds into consideration was applied in an analysis of the phase II study and resulted in the best differentiation of outcomes. Further analyses revealed a nonsignificant overall survival (OS) improvement with O+E in patients with high MET copy number (mean ≥5 copies/cell by FISH); however, benefit was maintained in “MET IHC-positive”/MET FISH-negative patients (HR, 0.37; P = 0.01). MET, EGFR, amphiregulin, epiregulin, or HGF mRNA expression did not predict a significant benefit with onartuzumab; a nonsignificant OS improvement was observed in patients with high tumor MET mRNA levels (HR, 0.59; P = 0.23). Patients with low baseline plasma hepatocyte growth factor (HGF) exhibited an HR for OS of 0.519 (P = 0.09) in favor of onartuzumab treatment.
Conclusions
MET IHC remains the most robust predictor of OS and progression-free survival benefit from O+E relative to all examined exploratory markers.
doi:10.1158/1078-0432.CCR-13-1836
PMCID: PMC4504679  PMID: 24687921
21.  A randomized phase II presurgical trial of transdermal 4-Hydroxytamoxifen gel versus oral tamoxifen in women with ductal carcinoma in situ of the breast 
Purpose
Local transdermal therapy to the breast may achieve effective target-organ drug delivery, while diminishing systemic effects. We conducted a randomized, double-blind, placebo-controlled phase II trial comparing transdermal 4-hydroxytamoxifen gel (4-OHT) to oral tamoxifen (oral-T) in women with ductal carcinoma in-situ (DCIS).
Methods
27pre and postmenopausal women were randomized to 4-OHT (4mg/day) or oral-T (20mg/day) for 6-10 weeks before surgery. Plasma, nipple aspirate fluid, and breast adipose tissue concentrations of tamoxifen and its major metabolites were determined by liquid chromatography-tandem mass spectrometry. The primary endpoint was Ki67 labeling in DCIS lesions, measured by immunohistochemistry. In plasma, insulin-like growth factor-1 (IGF-1), sex hormone-binding globulin (SHBG), and coagulation protein concentrations were determined.
Results
Post-therapy Ki-67 decreased by 3.4% in the 4-OHT and 5.1% in the oral-T group (p < 0.03 in both, between-group p=0. 99). Mean plasma 4-OHT was 0.2 and 1.1 ng/mL in 4-OHT and oral groups, respectively (p=0.0003), while mean breast adipose tissue concentrations of 4-OHT were 5.8 ng/g in the 4-OHT group and 5.4 ng/g in the oral group (p=0.88). There were significant increases in plasma SHBG, Factor VIII and von Willebrand factor and a significant decrease in plasma IGF-1 with oral-T, but not with 4-OHT. The incidence of hot flashes was similar in both groups.
Conclusions
The anti-proliferative effect of 4-OHT gel applied to breast skin was similar to that of oral-T, but effects on endocrine and coagulation parameters were reduced. These findings support the further evaluation of local transdermal therapy for DCIS and breast cancer prevention.
doi:10.1158/1078-0432.CCR-13-3045
PMCID: PMC4101910  PMID: 25028506
breast cancer prevention; tamoxifen; 4-OHT; endoxifen; ductal carcinoma in-situ
22.  Squamous cell carcinoma of the oral tongue in young non-smokers is genomically similar to tumors in older smokers 
Purpose
Epidemiological studies have identified an increasing incidence of squamous cell carcinoma of the oral tongue (SCCOT) in younger patients.
Experimental Design
DNA isolated from tongue tumors of young (<45 yrs, non-smokers) and old (>45 yrs) patients at was subjected to whole-exome sequencing and copy number analysis. These data were compared to data from similar patients in the The Cancer Genome Atlas (TCGA) project.
Results
In this study, we found that gene-specific mutation and copy number alteration frequencies were similar between young and old SCCOT patients in two independent cohorts. Likewise, the types of base changes observed in the young cohort were similar to those in the old cohort even though they differed in smoking history. TCGA data also demonstrate that the genomic effects of smoking are tumor-site specific, and we find that smoking has only a minor impact on the types of mutations observed in SCCOT.
Conclusions
Overall, tumors from young SCCOT patients appear genomically similar to those of older SCCOT patients, and the cause for the increasing incidence of young SCCOT remains unknown. These data indicate that the functional impact of smoking on carcinogenesis in SCCOT is still poorly understood.
doi:10.1158/1078-0432.CCR-14-0565
PMCID: PMC4102633  PMID: 24874835
Young tongue cancer; SCCOT; Head and neck/oral cancers; Tobacco
23.  Prognostic B-Cell Signatures using mRNA-Seq in Patients with Subtype-Specific Breast and Ovarian Cancer 
Purpose
Lymphocytic infiltration of tumors predicts improved survival in breast cancer patients. Previous studies have suggested that this survival benefit is confined predominantly to the basal-like subtype. Immune infiltration in ovarian tumors is also associated with improved prognosis. Currently, it is unclear what aspects of the immune response mediate this improved outcome.
Experimental Design
Using The Cancer Genome Atlas (TCGA) mRNA-seq data and a large microarray data set, we evaluated adaptive immune gene expression by genomic subtype in breast and ovarian cancer. To investigate B-cells observed to be prognostic within specific subtypes, we developed methods to analyze B-cell population diversity and degree of somatic hypermutation (SHM) from B-cell receptor (BCR) sequences in mRNA-seq data.
Results
Improved metastasis-free/progression-free survival was correlated with B-cell gene expression signatures, which were restricted mainly to the basal-like and HER2-enriched breast cancer subtypes and the immunoreactive ovarian cancer subtype. Consistent with a restricted epitope-driven response, a subset of basal-like and HER2-enriched breast tumors and immunoreactive ovarian tumors showed high expression of a low-diversity population of BCR gene segments. More BCR segments showed improved prognosis with increased expression in basal-like breast tumors and immunoreactive ovarian tumors compared with other subtypes. Basal-like and HER2-enriched tumors exhibited more BCR sequence variants in regions consistent with somatic hypermutation.
Conclusion
Taken together, these data suggest the presence of a productive and potentially restricted anti-tumor B-cell response in basal-like breast and immunoreactive ovarian cancers. Immunomodulatory therapies that support B-cell responses may be a promising therapeutic approach to targeting these B-cell infiltrated tumors.
doi:10.1158/1078-0432.CCR-13-3368
PMCID: PMC4102637  PMID: 24916698
24.  Real-Time Immune Monitoring to Guide Plasmid DNA Vaccination Schedule Targeting Prostatic Acid Phosphatase (PAP) in Patients with Castration-Resistant Prostate Cancer 
BACKGROUND
We have previously reported that a DNA vaccine encoding prostatic acid phosphatase (PAP) could elicit PAP-specific T cells in patients with early recurrent prostate cancer. In the current pilot trial we sought to evaluate whether prolonged immunization with regular booster immunizations, or “personalized” schedules of immunization determined using real-time immune monitoring, could elicit persistent, antigen-specific T cells, and whether treatment was associated with changes in PSA doubling time (PSA DT).
METHODS
16 patients with castration-resistant, non-metastatic prostate cancer received six immunizations at two-week intervals, and then either quarterly (Arm 1) or as determined by multi-parameter immune monitoring (Arm 2).
RESULTS
Patients were on study a median of 16 months; four received 24 vaccinations. Only one event associated with treatment > grade 2 was observed. 6/16 (38%) remained metastasis-free at 2 years. PAP-specific T cells were elicited in 12/16 (75%), predominantly of a Th1 phenotype, which persisted in frequency and phenotype for at least one year. IFNγ-secreting T-cell responses measured by ELISPOT were detectable in 5/13 individuals at one year, and this was not statistically different between study arms. The overall median fold change in PSA DT from pre-treatment to post-treatment was 1.6 (range 0.6–7.0, p=0.036).
CONCLUSIONS
Repetitive immunization with a plasmid DNA vaccine was safe and elicited Th1-biased antigen-specific T cells that persisted over time. Modifications in the immunization schedule based on real-time immune monitoring did not increase the frequency of patients developing effector and memory T-cell responses with this DNA vaccine.
doi:10.1158/1078-0432.CCR-14-0169
PMCID: PMC4102643  PMID: 24850844
DNA vaccine; prostate cancer; prostatic acid phosphatase; clinical trial; immune monitoring
25.  FANCD2 is a Potential Therapeutic Target and Biomarker in Alveolar Rhabdomyosarcoma Harboring the PAX3/FOXO1 Fusion Gene 
Purpose
Alveolar rhabdomyosarcoma that harbors the PAX3/FOXO1 fusion gene (t-ARMS) is a common and lethal subtype of this childhood malignancy. Improvement in clinical outcomes in this disease is predicated upon the identification of novel therapeutic targets.
Experimental Design
Robust mouse models were used for in vivo analysis, and molecular studies were performed on xenografts treated in parallel. Two independent patient sets (n=101 and 124) of clinically-annotated tumor specimens were used for analysis of FANCD2 levels and its association with clinical and molecular characteristics and outcomes.
Results
Our xenograft studies reveal a selective suppression of FANCD2 by m-TOR kinase inhibition and radiosensitization of the t-ARMS line only. In the initial patient set, we show FANCD2 transcript levels are prognostic in univariate analysis, and are significantly associated with metastatic disease and that the co-presence of the translocation and high expression of FANCD2 is independently prognostic. We also demonstrate a significant and non-random enrichment of mTOR-associated genes that correlate with FANCD2 gene expression within the t-ARMS samples, but not within other cases. In the second patient set, we show that on a protein level, FANCD2 expression correlates with PAX3/FOXO1 fusion gene and is strongly associated with phospho-P70S6K expression in cases with the fusion gene.
Conclusions
Our data demonstrate that FANCD2 may have a significant role in the radiation resistance and virulence of t-ARMS. Indirectly targeting this DNA repair protein, through mTOR inhibition, may represent a novel and selective treatment strategy.
doi:10.1158/1078-0432.CCR-13-0556
PMCID: PMC4102646  PMID: 24787670
rhabdomyosarcoma; PAX3/FOXO1 fusion gene; FANCD2; mTOR; radioresistance

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