Osteocalcin and serum insulin-like growth factor-1 as biochemical skeletal maturity indicators

With change in concepts of growth determination methods, there is a surge in the measurement of biomarkers for appraisal of growth status. Osteocalcin is a bone-specific protein and was observed to parallel th...

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Optical clearing of small intestine for three-dimensional visualization of cellular proliferation within crypts

Abstract

New methods on optical clearing provide a valuable alternative to traditional physical section histology. Optical clearing allows investigation of relatively large tissue samples at histological resolution while maintaining the three-dimensional architecture of the intact system. There is significant potential for applying optical clearing to gastrointestinal tissues. In particular, intestinal crypts contain high concentrations of stem cells, making these structures especially important for research on cellular proliferation in the intestinal epithelium. The objective of our study is to demonstrate an optical clearing method that is easy to implement and is compatible with mitotic fluorescent labeling. The optical clearing method we present utilizes a Triton/DMSO delipidization step followed by refractive index matching, rendering the tissue nearly transparent. We use EdU click chemistry to fluorescently label mitotic cell nuclei. Our results demonstrate successful clearing of jejunal samples with readily visible EdU staining by means of confocal microscopy. Three-dimensional reconstruction of labeled samples reveals preservation of intestinal cytoarchitecture including muscular, submucosal, and mucosal layers. Additionally, the morphology of intestinal crypts and individual EdU-positive mitotic nuclei are visible in sharp detail within their intact three-dimensional organization. In summary, we present an optical clearing method that is easy to implement and has the potential to provide more accurate assessment of cellular proliferation within the gastrointestinal tract in both healthy and disease states.

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The catecholaminergic innervation of the claustrum of the pig

Abstract

Over the past decades, the number of studies employing the pig brain as a model for neurochemical studies has dramatically increased. The key translational features of the pig brain are the similarities with the cortical and subcortical structures of the human brain. In addition, the caudalmost part of the pig claustrum (CL) is characterized by a wide enlargement called posterior puddle, an ideal structure for physiological recordings. Several hypotheses have been proposed for CL function, the key factor being its reciprocal connectivity with most areas of the cerebral cortex and selected subcortical structures. However, afferents from the brainstem could also be involved. The brainstem is the main source of catecholaminergic axons that play an important neuromodulatory action in different brain functions. To study a possible role of the CL in catecholaminergic pathways, we analyzed the presence and the distribution of afferents immunostained with antibodies against tyrosine hydroxylase (TH) and dopamine betahydroxylase (DBH) in the pig CL. Here we show that the CL contains significant TH immunoreactive axons contacting perikarya, whereas projections staining for DBH are very scarce. Our findings hint at the possibility that brainstem catecholaminergic afferents project to the CL, suggesting (i) a possible role of this nucleus in functions controlled by brainstem structures; and, consequently, (ii) its potential involvement in the pathophysiology of neurodegenerative pathologies, including Parkinson's disease (PD).

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In This Issue

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How to write a Critically Appraised Topic: evidence to underpin routine clinical practice

Summary

Critically appraised topics (CATs) are essential tools for busy clinicians who wish to ensure that their daily clinical practice is underpinned by evidence-based medicine. CATs are short summaries of the most up-to-date, high-quality available evidence that is found using thorough structured methods. They can be used to answer specific, patient-orientated questions that arise recurrently in real-life practice. This article provides readers with a detailed guide to performing their own CATs. It is split into four main sections reflecting the four main steps involved in performing a CAT: formulation of a focused question, a search for the most relevant and highest-quality evidence, critical appraisal of the evidence and application of the results back to the patient scenario. As well as helping to improve patient care on an individual basis by answering specific clinical questions that arise, CATs can help spread and share knowledge with colleagues on an international level through publication in the evidence-based dermatology section of the British Journal of Dermatology.

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Bevacizumab Induces Acute Hypoxia and Cancer Progression in Patients with Refractory Breast Cancer: Multimodal Functional Imaging and Multiplex Cytokine Analysis

Purpose: Bevacizumab, an antibody against endothelial growth factor, is a key but controversial drug in the treatment of metastatic breast cancer. We, therefore, aimed to determine the intrinsic resistance to bevacizumab at the physiologic and molecular levels in advanced breast cancer using PET, dynamic contrast-enhanced MRI, diffuse optical spectroscopic imaging (DOSI), and multiplex cytokine assays.

Experimental Design: In total, 28 patients diagnosed with advanced stage III/IV breast cancer receiving single-agent bevacizumab for 1 week followed by paclitaxel combined with bevacizumab underwent ¹⁸F-fluorodeoxyglucose (FDG)-PET, ¹⁸F-fluoromisonidazole (FMISO)-PET, and MRI at both baseline and two courses after treatment initiation. Hemodynamic measurement using DOSI and blood sample collection were performed at baseline and multiple times during the first week after the initiation of single-agent bevacizumab. We distinguished nonresponders from responders by serial FDG-PET based on their glycolytic changes to chemotherapy.

Results: Nonresponders showed significantly higher hypoxic activity on FMISO-PET and less tumor shrinkage than responders. Hemodynamic parameters showed higher tumor blood volume and a remarkable decrease in the tissue oxygen level in nonresponders compared with responders after the infusion of single-agent bevacizumab. Multiplex cytokine assays revealed increased plasma levels of both proangiogenic and hypoxia-related inflammatory cytokines in nonresponders and decreased levels in responders.

Conclusions: Nonresponders exhibited a higher degree of angiogenesis with more severe hypoxia than responders during bevacizumab treatment. These findings demonstrated that the addition of bevacizumab to paclitaxel treatment under hypoxic conditions could be ineffective and may result in acute hypoxia and increased cytokine secretion associated with cancer progression. Clin Cancer Res; 23(19); 5769–78. ©2017 AACR.

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Hypermutated Circulating Tumor DNA: Correlation with Response to Checkpoint Inhibitor-Based Immunotherapy

Purpose: Tumor mutational burden detected by tissue next-generation sequencing (NGS) correlates with checkpoint inhibitor response. However, tissue biopsy may be costly and invasive. We sought to investigate the association between hypermutated blood-derived circulating tumor DNA (ctDNA) and checkpoint inhibitor response.

Experimental Design: We assessed 69 patients with diverse malignancies who received checkpoint inhibitor–based immunotherapy and blood-derived ctDNA NGS testing (54–70 genes). Rates of stable disease (SD) ≥6 months, partial and complete response (PR, CR), progression-free survival (PFS), and overall survival (OS) were assessed based on total and VUS alterations.

Results: Statistically significant improvement in PFS was associated with high versus low alteration number in variants of unknown significance (VUS, >3 alterations versus VUS ≤3 alterations), SD ≥6 months/PR/CR 45% versus 15%, respectively; P = 0.014. Similar results were seen with high versus low total alteration number (characterized plus VUS, ≥6 vs. <6). Statistically significant OS improvement was also associated with high VUS alteration status. Two-month landmark analysis showed that responders versus nonresponders with VUS >3 had a median PFS of 23 versus 2.3 months (P = 0.0004).

Conclusions: Given the association of alteration number on liquid biopsy and checkpoint inhibitor–based immunotherapy outcomes, further investigation of hypermutated ctDNA as a predictive biomarker is warranted. Clin Cancer Res; 23(19); 5729–36. ©2017 AACR.

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FDA Approval Summary: Pembrolizumab for the Treatment of Patients with Unresectable or Metastatic Melanoma

On December 18, 2015, the FDA granted regular approval to pembrolizumab (KEYTRUDA; Merck Sharp & Dohme Corp.) for treatment of patients with unresectable or metastatic melanoma based on results of two randomized, open-label, active-controlled clinical trials. In trial PN006, 834 patients with ipilimumab-naïve metastatic melanoma were randomized (1:1:1) to pembrolizumab 10 mg/kg i.v. every 2 or 3 weeks until disease progression or ipilimumab 3 mg/kg every 3 weeks for up to four doses. In trial PN002, 540 patients with ipilimumab-refractory metastatic melanoma were randomized (1:1:1) to pembrolizumab 2 or 10 mg/kg i.v. every 3 weeks or to investigator's choice of chemotherapy. In trial PN006, patients randomized to pembrolizumab demonstrated a statistically significant improvement in overall survival compared with ipilimumab [every-2-week arm: hazard ratio (HR) = 0.63; 95% confidence interval (CI), 0.47–0.83; P < 0.001; every-3-week arm: HR = 0.69; 95% CI, 0.52–0.90; P = 0.004]. In both trials, patients receiving pembrolizumab demonstrated statistically significant improvements in progression-free survival. The most common (≥2%) immune-mediated adverse reactions in a pooled safety analysis were hypothyroidism, pneumonitis, and hyperthyroidism. Key considerations for approval were determination of pembrolizumab dose and interpretation of tumor response–based endpoints using RECIST or immune-related RECIST. Clin Cancer Res; 23(19); 5661–5. ©2017 AACR.

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Therapy of Small Cell Lung Cancer (SCLC) with a Topoisomerase-I-inhibiting Antibody-Drug Conjugate (ADC) Targeting Trop-2, Sacituzumab Govitecan

Purpose: We evaluated a Trop-2–targeting antibody conjugated with SN-38 in metastatic small cell lung cancer (mSCLC) patients.

Experimental Design: Sacituzumab govitecan was studied in patients with pretreated (median, 2; range, 1–7) mSCLC who received either 8 or 10 mg/kg i.v. on days 1 and 8 of 21-day cycles. The primary endpoints were safety and objective response rate (ORR); duration of response, progression-free survival (PFS), and overall survival (OS) were secondary endpoints.

Results: Sixty percent of patients showed tumor shrinkage from baseline CTs. On an intention-to-treat basis (N = 50), the ORR was 14% (17% for the 10-mg/kg group); the median response duration, 5.7 months; the clinical benefit rate (CBR ≥4 months), 34%; median PFS, 3.7 months; and median OS, 7.5 months. There was a suggested improvement in PR, CBR, and PFS with sacituzumab govitecan in second-line patients who were sensitive to first-line therapy, but no difference between first-line chemosensitive versus chemoresistant patients in the overall population. There was a statistically significant higher OS in those patients who received prior topotecan versus no topotecan therapy in a small subgroup. Grade ≥3 adverse events included neutropenia (34%), fatigue (13%), diarrhea (9%), and anemia (6%). Trop-2 tumor staining was not required for patient selection. No antibodies to the drug conjugate or its components were detected on serial blood collections.

Conclusions: Sacituzumab govitecan appears to have a safe and effective therapeutic profile in heavily pretreated mSCLC patients, including those who are chemosensitive or chemoresistant to first-line chemotherapy. Additional studies as a monotherapy or combination therapy are warranted. Clin Cancer Res; 23(19); 5711–9. ©2017 AACR.

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FDA Approval Summary: Accelerated Approval of Pembrolizumab for Second-Line Treatment of Metastatic Melanoma

On September 4, 2014, the FDA approved pembrolizumab (KEYTRUDA; Merck Sharp & Dohme Corp.) with a recommended dose of 2 mg/kg every 3 weeks by intravenous infusion for the treatment of patients with unresectable or metastatic melanoma who have progressed following treatment with ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor. Approval was based on demonstration of objective tumor responses with prolonged response durations in 89 patients enrolled in a randomized, multicenter, open-label, dose-finding, and activity-estimating phase 1 trial. The overall response rate (ORR) by blinded independent central review per RECIST v1.1 was 24% (95% confidence interval, 15–34); with 6 months of follow-up, 86% of responses were ongoing. The most common (≥20%) adverse reactions were fatigue, cough, nausea, pruritus, rash, decreased appetite, constipation, arthralgia, and diarrhea. Immune-mediated adverse reactions included pneumonitis, colitis, hepatitis, hypophysitis, and thyroid disorders. The benefits of the observed ORR with prolonged duration of responses outweighed the risks of immune-mediated adverse reactions in this life-threatening disease and represented an improvement over available therapy. Important regulatory issues in this application were role of durability of response in the evaluation of ORR for accelerated approval, reliance on data from a first-in-human trial, and strategies for dose selection. Clin Cancer Res; 23(19); 5666–70. ©2017 AACR.

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Diffusion MRI Phenotypes Predict Overall Survival Benefit from Anti-VEGF Monotherapy in Recurrent Glioblastoma: Converging Evidence from Phase II Trials

Purpose: Anti-VEGF therapies remain controversial in the treatment of recurrent glioblastoma (GBM). In the current study, we demonstrate that recurrent GBM patients with a specific diffusion MR imaging signature have an overall survival (OS) advantage when treated with cediranib, bevacizumab, cabozantinib, or aflibercept monotherapy at first or second recurrence. These findings were validated using a separate trial comparing bevacizumab with lomustine.

Experimental Design: Patients with recurrent GBM and diffusion MRI from the monotherapy arms of 5 separate phase II clinical trials were included: (i) cediranib (NCT00035656); (ii) bevacizumab (BRAIN Trial, AVF3708g; NCT00345163); (iii) cabozantinib (XL184-201; NCT00704288); (iv) aflibercept (VEGF Trap; NCT00369590); and (v) bevacizumab or lomustine (BELOB; NTR1929). Apparent diffusion coefficient (ADC) histogram analysis was performed prior to therapy to estimate "ADC_L," the mean of the lower ADC distribution. Pretreatment ADC_L, enhancing volume, and clinical variables were tested as independent prognostic factors for OS.

Results: The coefficient of variance (COV) in double baseline ADC_L measurements was 2.5% and did not significantly differ (P = 0.4537). An ADC_L threshold of 1.24 μm²/ms produced the largest OS differences between patients (HR ~ 0.5), and patients with an ADC_L > 1.24 μm²/ms had close to double the OS in all anti-VEGF therapeutic scenarios tested. Training and validation data confirmed that baseline ADC_L was an independent predictive biomarker for OS in anti-VEGF therapies, but not in lomustine, after accounting for age and baseline enhancing tumor volume.

Conclusions: Pretreatment diffusion MRI is a predictive imaging biomarker for OS in patients with recurrent GBM treated with anti-VEGF monotherapy at first or second relapse. Clin Cancer Res; 23(19); 5745–56. ©2017 AACR.

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Efficacy of Sequential Ipilimumab Monotherapy versus Best Supportive Care for Unresectable Locally Advanced/Metastatic Gastric or Gastroesophageal Junction Cancer

Purpose: Ipilimumab, a monoclonal antibody that blocks cytotoxic T-lymphocyte–associated protein-4 interactions, enhances T-cell activation and promotes tumor immunity. This phase II study evaluated the safety and efficacy of ipilimumab monotherapy versus best supportive care (BSC) among patients with advanced/metastatic gastric or gastroesophageal junction cancer who achieved at least stable disease with first-line chemotherapy.

Experimental Design: Eligible patients were randomized to ipilimumab 10 mg/kg every 3 weeks for four doses, then 10 mg/kg every 12 weeks for up to 3 years, or BSC, which could include continuation of fluoropyrimidine until progression or toxicity. The primary endpoint was immune-related progression-free survival (irPFS); secondary endpoints included PFS by modified World Health Organization criteria and overall survival (OS).

Results: Of 143 patients screened, 57 were randomized to each arm. irPFS with ipilimumab versus BSC was not improved [2.92 months, 95% confidence interval (CI), 1.61–5.16 vs. 4.90 months, 95% CI, 3.45–6.54, HR = 1.44; 80% CI, 1.09–1.91; P = 0.097], resulting in study cessation. At study closeout, which occurred 8 months after the interim analysis, the median OS durations were 12.7 months (95% CI, 10.5–18.9) and 12.1 months (95% CI, 9.3–not estimable), respectively. Grade 3/4 treatment-related adverse events occurred in 23% of ipilimumab-treated patients, in whom diarrhea (9%) and fatigue (5%) were most frequent, and in 9% of active BSC-treated patients.

Conclusions: Although ipilimumab at 10 mg/kg was manageable, it did not improve irPFS versus BSC. However, comparable median OS of approximately 1 year and a favorable safety profile support the investigation of ipilimumab in combination with other therapies for advanced gastric cancer. Clin Cancer Res; 23(19); 5671–8. ©2017 AACR.

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Co-administration of RANKL and CTLA4 Antibodies Enhances Lymphocyte-Mediated Antitumor Immunity in Mice

Purpose: Novel partners for established immune checkpoint inhibitors in the treatment of cancer are needed to address the problems of primary and acquired resistance. The efficacy of combination RANKL and CTLA4 blockade in antitumor immunity has been suggested by recent case reports in melanoma. Here, we provide a rationale for this combination in mouse models of cancer.

Experimental Design: The efficacy and mechanism of a combination of RANKL and CTLA4 blockade was examined by tumor-infiltrating lymphocyte analysis, tumor growth, and metastasis using a variety of neutralizing antibodies and gene-targeted mice.

Results: RANKL blockade improved the efficacy of anti-CTLA4 mAbs against solid tumors and experimental metastases, with regulatory T-cell (Treg)–depleting anti-CTLA4 mAbs of the mouse IgG2a isotype showing greatest combinatorial activity. The optimal combination depended on the presence of activating Fc receptors and lymphocytes (NK cells for metastatic disease and predominantly CD8⁺ T cells for subcutaneous tumor control), whereas anti-RANKL alone did not require FcR. The significantly higher T-cell infiltration into solid tumors post anti-RANKL and anti-CTLA4 was accompanied by increased T-cell effector function (cytokine polyfunctionality), and anti-RANKL activity occurred independently of Treg depletion. The majority of RANKL expression in tumors was on T cells whereas RANK-expressing cells were mostly tumor-associated macrophages (TAM), with some expression also observed on dendritic cells (DC) and myeloid-derived suppressor cells (MDSC).

Conclusions: These results provide a rationale for the further investigation of RANKL–RANK interactions in tumor immunity and a basis for development of translational markers of interest in human clinical trials. Clin Cancer Res; 23(19); 5789–801. ©2017 AACR.

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Local Adjuvant Treatment with Low-Dose CpG-B Offers Durable Protection against Disease Recurrence in Clinical Stage I-II Melanoma: Data from Two Randomized Phase II Trials

Purpose: Although risk of recurrence after surgical removal of clinical stage I–II melanoma is considerable, there is no adjuvant therapy with proven efficacy. Here, we provide clinical evidence that a local conditioning regimen, aimed at immunologic arming of the tumor-draining lymph nodes, may provide durable protection against disease recurrence (median follow-up, 88.8 months).

Experimental Design: In two randomized phase II trials, patients, diagnosed with stage I–II melanoma after excision of the primary tumor, received local injections at the primary tumor excision site within 7 days preceding re-excision and sentinel lymph node (SLN) biopsy of either a saline placebo (n = 22) or low-dose CpG type B (CpG-B) with (n = 9) or without (n = 21) low-dose GM-CSF.

Results: CpG-B treatment was shown to be safe, to boost locoregional and systemic immunity, to be associated with lower rates of tumor-involved SLN (10% vs. 36% in controls, P = 0.04), and, at a median follow-up of 88.8 months, to profoundly improve recurrence-free survival (P = 0.008), even for patients with histologically confirmed (i.e., pathologic) stage I–II disease (P = 0.02).

Conclusions: Potentially offering durable protection, local low-dose CpG-B administration in early-stage melanoma provides an adjuvant treatment option for a large group of patients currently going untreated despite being at considerable risk for disease recurrence. Once validated in a larger randomized phase III trial, this nontoxic immunopotentiating regimen may prove clinically transformative. Clin Cancer Res; 23(19); 5679–86. ©2017 AACR.

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Sorafenib in Patients with Hepatocellular Carcinoma--Results of the Observational INSIGHT Study

Purpose: Sorafenib is the only currently approved systemic therapy for advanced hepatocellular carcinoma (HCC). We aimed to evaluate the safety and efficacy of sorafenib therapy in patients with HCC under real-life conditions regarding patient, tumor characteristics, and any adverse events at study entry and at follow-up visits every 2 to 4 months.

Experimental Design: The current INSIGHT study is a noninterventional, prospective, multicenter, observational study performed in 124 sites across Austria and Germany between 2008 and 2014.

Results: Median overall survival and time to progression (RECIST) were found to be dependent on baseline Barcelona Clinic Liver Cancer (BCLC) tumor stage (A: 29.2, B: 19.6, C: 13.6, D: 3.1 and A: 6.0, B: 5.5, C: 3.9, and D: 1.7 months, respectively), Child–Pugh liver function (A: 17.6, B: 8.1, C: 5.6 and A: 5.3, B: 3.3, C: 2.5 months, respectively), and performance status of the patient; however, age did not affect prognosis. Sorafenib-related adverse events at any grade occurred in 64.9% of patients, with diarrhea (35.4%), hand–foot–skin reaction (16.6%), nausea (10.3%), and fatigue (11.2%) occurring most frequently.

Conclusions: Sorafenib treatment was shown to be effective in a real-life setting, in agreement with previously reported clinical trial data. The therapy was found to have an acceptable safety profile, with predominantly mild to moderate side effects. Clin Cancer Res; 23(19); 5720–8. ©2017 AACR.

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Neratinib Efficacy and Circulating Tumor DNA Detection of HER2 Mutations in HER2 Nonamplified Metastatic Breast Cancer

Purpose: Based on promising preclinical data, we conducted a single-arm phase II trial to assess the clinical benefit rate (CBR) of neratinib, defined as complete/partial response (CR/PR) or stable disease (SD) ≥24 weeks, in HER2^mut nonamplified metastatic breast cancer (MBC). Secondary endpoints included progression-free survival (PFS), toxicity, and circulating tumor DNA (ctDNA) HER2^mut detection.

Experimental Design: Tumor tissue positive for HER2^mut was required for eligibility. Neratinib was administered 240 mg daily with prophylactic loperamide. ctDNA sequencing was performed retrospectively for 54 patients (14 positive and 40 negative for tumor HER2^mut).

Results: Nine of 381 tumors (2.4%) sequenced centrally harbored HER2^mut (lobular 7.8% vs. ductal 1.6%; P = 0.026). Thirteen additional HER2^mut cases were identified locally. Twenty-one of these 22 HER2^mut cases were estrogen receptor positive. Sixteen patients [median age 58 (31–74) years and three (2–10) prior metastatic regimens] received neratinib. The CBR was 31% [90% confidence interval (CI), 13%–55%], including one CR, one PR, and three SD ≥24 weeks. Median PFS was 16 (90% CI, 8–31) weeks. Diarrhea (grade 2, 44%; grade 3, 25%) was the most common adverse event. Baseline ctDNA sequencing identified the same HER2^mut in 11 of 14 tumor-positive cases (sensitivity, 79%; 90% CI, 53%–94%) and correctly assigned 32 of 32 informative negative cases (specificity, 100%; 90% CI, 91%–100%). In addition, ctDNA HER2^mut variant allele frequency decreased in nine of 11 paired samples at week 4, followed by an increase upon progression.

Conclusions: Neratinib is active in HER2^mut, nonamplified MBC. ctDNA sequencing offers a noninvasive strategy to identify patients with HER2^mut cancers for clinical trial participation. Clin Cancer Res; 23(19); 5687–95. ©2017 AACR.

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Tumor Response Dynamics of Advanced Non-small Cell Lung Cancer Patients Treated with PD-1 Inhibitors: Imaging Markers for Treatment Outcome

Purpose: We evaluated tumor burden dynamics in patients with advanced non–small cell lung cancer (NSCLC) treated with commercial PD-1 inhibitors to identify imaging markers associated with improved overall survival (OS).

Experimental Design: The study included 160 patients with advanced NSCLC treated with commercial nivolumab or pembrolizumab monotherapy as a part of clinical care. Tumor burden dynamics were studied for the association with OS.

Results: Tumor burden change at best overall response (BOR) ranged from –100% to +278% (median, +3.5%). Response rate (RR) was 18% (29/160). Current and former smokers had a higher RR than never smokers (P = 0.04). Durable disease control for at least 6 months was noted in 26 patients (16%), which included 10 patients with stable disease as BOR. Using a landmark analysis, patients with <20% tumor burden increase from baseline within 8 weeks of therapy had longer OS than patients with ≥20% increase (median OS, 12.4 vs. 4.6 months, P < 0.001). Patients with <20% tumor burden increase throughout therapy had significantly reduced hazards of death (HR, 0.24; Cox P < 0.0001) after adjusting for smoking (HR, 0.86; P = 0.61) and baseline tumor burden (HR, 1.55; P = 0.062), even though some patients met criteria for RECIST progression while on therapy. One patient (0.6%) had atypical response pattern consistent with pseudoprogression.

Conclusions: Objective response or durable disease control was noted in 24% of patients with advanced NSCLC treated with commercial PD-1 inhibitors. A tumor burden increase of <20% from baseline during therapy was associated with longer OS, proposing a practical marker of treatment benefit. Pseudoprogression is rare in NSCLCs treated with PD-1 inhibitors. Clin Cancer Res; 23(19); 5737–44. ©2017 AACR.

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Phase I Trial of Intravenous Oncolytic Vaccinia Virus (GL-ONC1) with Cisplatin and Radiotherapy in Patients with Locoregionally Advanced Head and Neck Carcinoma

Purpose: Preclinical models have shown that the effectiveness of GL-ONC1, a modified oncolytic vaccinia virus, is enhanced by radiation and chemotherapy. The purpose of this study was to determine the safety of GL-ONC1 when delivered intravenously with chemoradiotherapy to patients with primary, nonmetastatic head and neck cancer.

Experimental Design: Patients with locoregionally advanced unresected, nonmetastatic carcinoma of the head/neck, excluding stage III–IVA p16-positive oropharyngeal cancers, were treated with escalating doses and cycles of intravenous GL-ONC1, along with radiotherapy and chemotherapy. The primary aims were to define the MTD and dose-limiting toxicities, and to recommend a dose for phase II trials.

Results: Between May 2012 and December 2014, 19 patients were enrolled. The most frequent adverse reactions included grade 1–2 rigors, fever, fatigue, and rash. Grade 3 adverse reactions included hypotension, mucositis, nausea, and vomiting. In 2 patients, the rash was confirmed as viral in origin by fluorescence imaging and viral plaque assay. In 4 patients, viral presence in tumor was confirmed on midtreatment biopsy by quantitative PCR. In 1 patient, live virus was confirmed in a tongue tumor 7 days after receiving the first dose of virus. The MTD was not reached. With median follow-up of 30 months, 1-year (2-year) progression-free survival and overall survival were 74.4% (64.1%) and 84.6% (69.2%), respectively.

Conclusions: Delivery of GL-ONC1 is safe and feasible in patients with locoregionally advanced head/neck cancer undergoing standard chemoradiotherapy. A phase II study is warranted to further investigate this novel treatment strategy. Clin Cancer Res; 23(19); 5696–702. ©2017 AACR.

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Clinical and Genome-Wide Analysis of Cisplatin-Induced Peripheral Neuropathy in Survivors of Adult-Onset Cancer

Purpose: Our purpose was to characterize the clinical influences, genetic risk factors, and gene mechanisms contributing to persistent cisplatin-induced peripheral neuropathy (CisIPN) in testicular cancer survivors (TCSs).

Experimental Design: TCS given cisplatin-based therapy completed the validated EORTC QLQ-CIPN20 questionnaire. An ordinal CisIPN phenotype was derived, and associations with age, smoking, excess drinking, hypertension, body mass index, diabetes, hypercholesterolemia, cumulative cisplatin dose, and self-reported health were examined for 680 TCS. Genotyping was performed on the Illumina HumanOmniExpressExome chip. Following quality control and imputation, 5.1 million SNPs in 680 genetically European TCS formed the input set. GWAS and PrediXcan were used to identify genetic variation and genetically determined gene expression traits, respectively, contributing to CisIPN. We evaluated two independent datasets for replication: Vanderbilt's electronic health database (BioVU) and the CALGB 90401 trial.

Results: Eight sensory items formed a subscale with good internal consistency (Cronbach α = 0.88). Variables significantly associated with CisIPN included age at diagnosis (OR per year, 1.06; P = 2 x 10^–9), smoking (OR, 1.54; P = 0.004), excess drinking (OR, 1.83; P = 0.007), and hypertension (OR, 1.61; P = 0.03). CisIPN was correlated with lower self-reported health (OR, 0.56; P = 2.6 x 10^–9) and weight gain adjusted for years since treatment (OR per kg/m², 1.05; P = 0.004). PrediXcan identified lower expressions of MIDN and RPRD1B, and higher THEM5 expression as associated with CisIPN (P value for each < 5 x 10^–6) with replication of RPRD1B meeting significance criteria (Fisher combined P = 0.0089).

Conclusions: CisIPN is associated with age, modifiable risk factors, and genetically determined expression level of RPRD1B. Further study of implicated genes could elucidate the pathophysiologic underpinnings of CisIPN. Clin Cancer Res; 23(19); 5757–68. ©2017 AACR.

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First-in-Human Study of AMG 820, a Monoclonal Anti-Colony-Stimulating Factor 1 Receptor Antibody, in Patients with Advanced Solid Tumors

Purpose: Binding of colony-stimulating factor 1 (CSF1) ligand to the CSF1 receptor (CSF1R) regulates survival of tumor-associated macrophages, which generally promote an immunosuppressive tumor microenvironment. AMG 820 is an investigational, fully human CSF1R antibody that inhibits binding of the ligands CSF1 and IL34 and subsequent ligand-mediated receptor activation. This first-in-human phase I study evaluated the safety, pharmacokinetics, pharmacodynamics, and antitumor activity of AMG 820.

Experimental Design: Adult patients with relapsed or refractory advanced solid tumors received intravenous AMG 820 0.5 mg/kg once weekly or 1.5 to 20 mg/kg every 2 weeks until disease progression, adverse event (AE), or consent withdrawal.

Results: Twenty-five patients received ≥1 dose of AMG 820. AMG 820 was tolerated up to 20 mg/kg; the MTD was not reached. One dose-limiting toxicity was observed (20 mg/kg; nonreversible grade 3 deafness). Most patients (76%) had treatment-related AEs; the most common were periorbital edema (44%), increased aspartate aminotransferase (AST; 28%), fatigue (24%), nausea (16%), increased blood alkaline phosphatase (12%), and blurred vision (12%). No patients had serious or fatal treatment-related AEs; 28% had grade ≥3 treatment-related AEs. Grade 3 AST elevations resolved when treatment was withheld. AMG 820 showed linear pharmacokinetics, with minimal accumulation (<2-fold) after repeated dosing. Pharmacodynamic increases in serum CSF1 concentrations and reduced numbers of skin macrophages were observed. Best response was stable disease in 8 patients (32%).

Conclusions: AMG 820 was tolerated with manageable toxicities up to 20 mg/kg every 2 weeks. Pharmacodynamic response was demonstrated, and limited antitumor activity was observed. Clin Cancer Res; 23(19); 5703–10. ©2017 AACR.

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PD-1+ Polyfunctional T Cells Dominate the Periphery after Tumor-Infiltrating Lymphocyte Therapy for Cancer

Purpose: Infusion of highly heterogeneous populations of autologous tumor-infiltrating lymphocytes (TIL) can result in tumor regression of exceptional duration. Initial tumor regression has been associated with persistence of tumor-specific TILs 1 month after infusion, but mechanisms leading to long-lived memory responses are currently unknown. Here, we studied the dynamics of bulk tumor-reactive CD8⁺ T-cell populations in patients with metastatic melanoma following treatment with TILs.

Experimental Design: We analyzed the function and phenotype of tumor-reactive CD8⁺ T cells contained in serial blood samples of 16 patients treated with TILs.

Results: Polyfunctional tumor-reactive CD8⁺ T cells accumulated over time in the peripheral lymphocyte pool. Combinatorial analysis of multiple surface markers (CD57, CD27, CD45RO, PD-1, and LAG-3) showed a unique differentiation pattern of polyfunctional tumor-reactive CD8⁺ T cells, with highly specific PD-1 upregulation early after infusion. The differentiation and functional status appeared largely stable for up to 1 year after infusion. Despite some degree of clonal diversification occurring in vivo within the bulk tumor-reactive CD8⁺ T cells, further analyses showed that CD8⁺ T cells specific for defined tumor antigens had similar differentiation status.

Conclusions: We demonstrated that tumor-reactive CD8⁺ T-cell subsets that persist after TIL therapy are mostly polyfunctional, display a stable partially differentiated phenotype, and express high levels of PD-1. These partially differentiated PD-1⁺ polyfunctional TILs have a high capacity for persistence and may be susceptible to PD-L1/PD-L2–mediated inhibition. Clin Cancer Res; 23(19); 5779–88. ©2017 AACR.

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Inhibition of CDK4/6 by Palbociclib Significantly Extends Survival in Medulloblastoma Patient-Derived Xenograft Mouse Models

Purpose: Bioinformatics analysis followed by in vivo studies in patient-derived xenograft (PDX) models were used to identify and validate CDK 4/6 inhibition as an effective therapeutic strategy for medulloblastoma, particularly group 3 MYC-amplified tumors that have the worst clinical prognosis.

Experimental Design: A protein interaction network derived from a Sleeping Beauty mutagenesis model of medulloblastoma was used to identify potential novel therapeutic targets. The top hit from this analysis was validated in vivo using PDX models of medulloblastoma implanted subcutaneously in the flank and orthotopically in the cerebellum of mice.

Results: Informatics analysis identified the CDK4/6/CYCLIN D/RB pathway as a novel "druggable" pathway for multiple subgroups of medulloblastoma. Palbociclib, a highly specific inhibitor of CDK4/6, was found to inhibit RB phosphorylation and cause G₁ arrest in PDX models of medulloblastoma. The drug caused rapid regression of Sonic hedgehog (SHH) and MYC-amplified group 3 medulloblastoma subcutaneous tumors and provided a highly significant survival advantage to mice bearing MYC-amplified intracranial tumors.

Conclusions: Inhibition of CDK4/6 is potentially a highly effective strategy for the treatment of SHH and MYC-amplified group 3 medulloblastoma. Clin Cancer Res; 23(19); 5802–13. ©2017 AACR.

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Combination Therapy with NHS-muIL12 and Avelumab (anti-PD-L1) Enhances Antitumor Efficacy in Preclinical Cancer Models

Purpose: To determine whether combination therapy with NHS-muIL12 and the anti-programmed death ligand 1 (PD-L1) antibody avelumab can enhance antitumor efficacy in preclinical models relative to monotherapies.

Experimental Design: BALB/c mice bearing orthotopic EMT-6 mammary tumors and μMt^– mice bearing subcutaneous MC38 tumors were treated with NHS-muIL12, avelumab, or combination therapy; tumor growth and survival were assessed. Tumor recurrence following remission and rechallenge was evaluated in EMT-6 tumor-bearing mice. Immune cell populations within spleen and tumors were evaluated by FACS and IHC. Immune gene expression in tumor tissue was profiled by NanoString® assay and plasma cytokine levels were determined by multiplex cytokine assay. The frequency of tumor antigen–reactive IFN-producing CD8⁺ T cells was evaluated by ELISpot assay.

Results: NHS-muIL12 and avelumab combination therapy enhanced antitumor efficacy relative to either monotherapy in both tumor models. Most EMT-6 tumor–bearing mice treated with combination therapy had complete tumor regression. Combination therapy also induced the generation of tumor-specific immune memory, as demonstrated by protection against tumor rechallenge and induction of effector and memory T cells. Combination therapy enhanced cytotoxic NK and CD8⁺ T-cell proliferation and T-bet expression, whereas NHS-muIL12 monotherapy induced CD8⁺ T-cell infiltration into the tumor. Combination therapy also enhanced plasma cytokine levels and stimulated expression of a greater number of innate and adaptive immune genes compared with either monotherapy.

Conclusions: These data indicate that combination therapy with NHS-muIL12 and avelumab increased antitumor efficacy in preclinical models, and suggest that combining NHS-IL12 and avelumab may be a promising approach to treating patients with solid tumors. Clin Cancer Res; 23(19); 5869–80. ©2017 AACR.

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Shaping the Tumor Stroma and Sparking Immune Activation by CD40 and 4-1BB Signaling Induced by an Armed Oncolytic Virus

Purpose: Pancreatic cancer is a severe indication with short expected survival despite surgery and/or combination chemotherapeutics. Checkpoint blockade antibodies are approved for several cancer indications, but pancreatic cancer has remained refractory. However, there are clinical data suggesting that stimulation of the CD40 pathway may be of interest for these patients. Oncolytic viruses armed with immunostimulatory genes represent an interesting approach. Herein, we present LOAd703, a designed adenovirus armed with trimerized CD40L and 4-1BBL that activates the CD40 and 4-1BB pathways, respectively. As many cells in the tumor stroma, including stellate cells and the infiltrating immune cells, express CD40 and some 4-1BB, we hypothesize that LOAd703 activates immunity and simultaneously modulates the biology of the tumor stroma.

Experimental Design: Tumor, stellate, endothelial, and immune cells were infected by LOAd703 and investigated by flow cytometry, proteomics, and functional analyses.

Results: LOAd703-infected pancreatic cell lines were killed by oncolysis, and the virus was more effective than standard-of-care gemcitabine. In in vivo xenograft models, LOAd703 efficiently reduced established tumors and could be combined with gemcitabine for additional effect. Infected stellate and tumor cells reduced factors that promote tumor growth (Spp-1, Gal-3, HGF, TGFβ and collagen type I), while chemokines were increased. Molecules involved in lymphocyte migration were upregulated on infected endothelial cells. Dendritic cells were robustly stimulated by LOAd703 to produce costimulators, cytokines and chemokines, and such DCs potently expanded both antigen-specific T cells and NK cells.

Conclusions: LOAd703 is a potent immune activator that modulates the stroma to support antitumor responses. Clin Cancer Res; 23(19); 5846–57. ©2017 AACR.

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Combined BTK and PI3K{delta} Inhibition with Acalabrutinib and ACP-319 Improves Survival and Tumor Control in CLL Mouse Model

Purpose: Targeting the B-cell receptor (BCR) pathway with inhibitors of Bruton tyrosine kinase (BTK) and PI3K is highly effective for the treatment of chronic lymphocytic leukemia (CLL). However, deep remissions are uncommon, and drug resistance with single-agent therapy can occur. In vitro studies support the effectiveness of combing PI3K and BTK inhibitors.

Experimental Design: As CLL proliferation and survival depends on the microenvironment, we used murine models to assess the efficacy of the BTK inhibitor acalabrutinib combined with the PI3K inhibitor ACP-319 in vivo. We compared single-agent with combination therapy in TCL1-192 cell–injected mice, a model of aggressive CLL.

Results: We found significantly larger reductions in tumor burden in the peripheral blood and spleen of combination-treated mice. Although single-agent therapy improved survival compared with control mice by a few days, combination therapy extended survival by over 2 weeks compared with either single agent. The combination reduced tumor proliferation, NF-B signaling, and expression of BCL-xL and MCL-1 more potently than single-agent therapy.

Conclusions: The combination of acalabrutinib and ACP-319 was superior to single-agent treatment in a murine CLL model, warranting further investigation of this combination in clinical studies. Clin Cancer Res; 23(19); 5814–23. ©2017 AACR.

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Are You What You Eat or What Your Mother Ate or Both?

A high-fat high-sugar (HF-HS) diet promotes cancer development and progression. However, does the timing of diet matter? This is an important question with profound public health relevance. By exposing mice to a HF-HS diet either through feeding to a pregnant mother or nursing mother or after weaning and then chemically inducing breast cancer, the authors found the most crucial time for breast cancer risk was after weaning, while a HF-HS in utero diet actually slowed tumor development. Understanding early-life events provides valuable insight for later life events and proves it is never too early to start preventing disease. Cancer Prev Res; 10(10); 551–2. ©2017 AACR.

See related article by Lambertz, p. 553–62.

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Early Exposure to a High Fat/High Sugar Diet Increases the Mammary Stem Cell Compartment and Mammary Tumor Risk in Female Mice

Obesity and alterations in metabolic programming from early diet exposures can affect the propensity to disease in later life. Through dietary manipulation, developing mouse pups were exposed to a hyperinsulinemic, hyperglycemic milieu during three developmental phases: gestation, lactation, and postweaning. Analyses showed that a postweaning high fat/high sugar (HF/HS) diet had the main negative effect on adult body weight, glucose tolerance, and insulin resistance. However, dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis revealed that animals born to a mother fed a HF/HS gestation diet, nursed by a mother on a mildly diet-restricted, low fat/low sugar diet (DR) and weaned onto a HF/HS diet (HF/DR/HF) had the highest mammary tumor incidence, while HF/HF/DR had the lowest tumor incidence. Cox proportional hazards analysis showed that a HF/HS postweaning diet doubled mammary cancer risk, and a HF/HS diet during gestation and postweaning increased risk 5.5 times. Exposure to a HF/HS diet during gestation, when combined with a postweaning DR diet, had a protective effect, reducing mammary tumor risk by 86% (HR = 0.142). Serum adipocytokine analysis revealed significant diet-dependent differences in leptin/adiponectin ratio and IGF-1. Flow cytometry analysis of cells isolated from mammary glands from a high tumor incidence group, DR/HF/HF, showed a significant increase in the size of the mammary stem cell compartment compared with a low tumor group, HF/HF/DR. These results indicate that dietary reprogramming induces an expansion of the mammary stem cell compartment during mammary development, increasing likely carcinogen targets and mammary cancer risk. Cancer Prev Res; 10(10); 553–62. ©2017 AACR.

See related editorial by Freedland, p. 551–2.

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Tobacco-Specific Carcinogens Induce Hypermethylation, DNA Adducts, and DNA Damage in Bladder Cancer

Smoking is a major risk factor for the development of bladder cancer; however, the functional consequences of the carcinogens in tobacco smoke and bladder cancer–associated metabolic alterations remain poorly defined. We assessed the metabolic profiles in bladder cancer smokers and non-smokers and identified the key alterations in their metabolism. LC/MS and bioinformatic analysis were performed to determine the metabolome associated with bladder cancer smokers and were further validated in cell line models. Smokers with bladder cancer were found to have elevated levels of methylated metabolites, polycyclic aromatic hydrocarbons, DNA adducts, and DNA damage. DNA methyltransferase 1 (DNMT1) expression was significantly higher in smokers than non-smokers with bladder cancer. An integromics approach, using multiple patient cohorts, revealed strong associations between smokers and high-grade bladder cancer. In vitro exposure to the tobacco smoke carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and benzo[a]pyrene (BaP) led to increase in levels of methylated metabolites, DNA adducts, and extensive DNA damage in bladder cancer cells. Cotreatment of bladder cancer cells with these carcinogens and the methylation inhibitor 5-aza-2'-deoxycytidine rewired the methylated metabolites, DNA adducts, and DNA damage. These findings were confirmed through the isotopic-labeled metabolic flux analysis. Screens using smoke-associated metabolites and DNA adducts could provide robust biomarkers and improve individual risk prediction in bladder cancer smokers. Noninvasive predictive biomarkers that can stratify the risk of developing bladder cancer in smokers could aid in early detection and treatment. Cancer Prev Res; 10(10); 588–97. ©2017 AACR.

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Prospective Evaluation of Multimodal Optical Imaging with Automated Image Analysis to Detect Oral Neoplasia In Vivo

The 5-year survival rate for patients with oral cancer remains low, in part because diagnosis often occurs at a late stage. Early and accurate identification of oral high-grade dysplasia and cancer can help improve patient outcomes. Multimodal optical imaging is an adjunctive diagnostic technique in which autofluorescence imaging is used to identify high-risk regions within the oral cavity, followed by high-resolution microendoscopy to confirm or rule out the presence of neoplasia. Multimodal optical images were obtained from 206 sites in 100 patients. Histologic diagnosis, either from a punch biopsy or an excised surgical specimen, was used as the gold standard for all sites. Histopathologic diagnoses of moderate dysplasia or worse were considered neoplastic. Images from 92 sites in the first 30 patients were used as a training set to develop automated image analysis methods for identification of neoplasia. Diagnostic performance was evaluated prospectively using images from 114 sites in the remaining 70 patients as a test set. In the training set, multimodal optical imaging with automated image analysis correctly classified 95% of nonneoplastic sites and 94% of neoplastic sites. Among the 56 sites in the test set that were biopsied, multimodal optical imaging correctly classified 100% of nonneoplastic sites and 85% of neoplastic sites. Among the 58 sites in the test set that corresponded to a surgical specimen, multimodal imaging correctly classified 100% of nonneoplastic sites and 61% of neoplastic sites. These findings support the potential of multimodal optical imaging to aid in the early detection of oral cancer. Cancer Prev Res; 10(10); 563–70. ©2017 AACR.

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In Silico Systems Biology Analysis of Variants of Uncertain Significance in Lynch Syndrome Supports the Prioritization of Functional Molecular Validation

Lynch syndrome (LS) is a genetic condition secondary to germline alterations in the DNA mismatch repair (MMR) genes with 30% of changes being variants of uncertain significance (VUS). Our aim was to perform an in silico reclassification of VUS from a large single institutional cohort that will help prioritizing functional validation. A total of 54 VUS were detected with 33 (61%) novel variants. We integrated family history, pathology, and genetic information along with supporting evidence from eight different in silico tools at the RNA and protein level. Our assessment allowed us to reclassify 54% (29/54) of the VUS as probably damaging, 13% (7/54) as possibly damaging, and 28% (15/54) as probably neutral. There are more than 1,000 VUS reported in MMR genes and our approach facilitates the prioritization of further functional efforts to assess the pathogenicity to those classified as probably damaging. Cancer Prev Res; 10(10); 580–7. ©2017 AACR.

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Effect of Green Tea Supplements on Liver Enzyme Elevation: Results from a Randomized Intervention Study in the United States

Liver injury effects of green tea–based products have been reported in sporadic case reports. However, no study has examined systematically such adverse effects in an unbiased manner. We examined the potential effects of a high, sustained oral dose of green tea extract (GTE) on liver injury measures in a randomized, placebo-controlled, double-blinded phase II clinical trial, which enrolled 1,075 women with the original aim to assess the effect of daily GTE consumption for 12 months on biomarkers of breast cancer risk. The current analysis examined the effect of GTE consumption on liver injury in 1,021 participants (513 in GTE and 508 in placebo arm) with normal baseline levels of liver enzymes. Among women in the GTE arm, alanine aminotransferase (ALT) increased by 5.4 U/L [95% confidence interval (CI), 3.6–7.1] and aspartate aminotransferase increased by 3.8 U/L (95% CI, 2.5–5.1), which were significantly higher than those among women in the placebo arm (both P < 0.001). Overall, 26 (5.1%) women in GTE developed moderate or more severe abnormalities in any liver function measure during the intervention period, yielding an OR of 7.0 (95% CI, 2.4–20.3) for developing liver function abnormalities as compared with those in the placebo arm. ALT returned to normal after dechallenge and increased again after one or more rechallenges with GTE. The rise–fall pattern of liver enzyme values following the challenge–dechallenge cycles of GTE consumption strongly implicates the effect of high-dose GTE on liver enzyme elevations. Cancer Prev Res; 10(10); 571–9. ©2017 AACR.

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Topically Applied Carvedilol Attenuates Solar Ultraviolet Radiation Induced Skin Carcinogenesis

In previous studies, the β-blocker carvedilol inhibited EGF-induced epidermal cell transformation and chemical carcinogen-induced mouse skin hyperplasia. As exposure to ultraviolet (UV) radiation leads to skin cancer, the present study examined whether carvedilol can prevent UV-induced carcinogenesis. Carvedilol absorbs UV like a sunscreen; thus, to separate pharmacological from sunscreen effects, 4-hydroxycarbazole (4-OHC), which absorbs UV to the same degree as carvedilol, served as control. JB6 P⁺ cells, an established epidermal model for studying tumor promotion, were used for evaluating the effect of carvedilol on UV-induced neoplastic transformation. Both carvedilol and 4-OHC (1 μmol/L) blocked transformation induced by chronic UV (15 mJ/cm²) exposure for 8 weeks. However, EGF-mediated transformation was inhibited by only carvedilol but not by 4-OHC. Carvedilol (1 and 5 μmol/L), but not 4-OHC, attenuated UV-induced AP-1 and NF-B luciferase reporter activity, suggesting a potential anti-inflammatory activity. In a single-dose UV (200 mJ/cm²)-induced skin inflammation mouse model, carvedilol (10 μmol/L), applied topically after UV exposure, reduced skin hyperplasia and the levels of cyclobutane pyrimidine dimers, IL1β, IL6, and COX-2 in skin. In SKH-1 mice exposed to gradually increasing levels of UV (50–150 mJ/cm²) three times a week for 25 weeks, topical administration of carvedilol (10 μmol/L) after UV exposure increased tumor latency compared with control (week 18 vs. 15), decreased incidence and multiplicity of squamous cell carcinomas, while 4-OHC had no effect. These data suggest that carvedilol has a novel chemopreventive activity and topical carvedilol following UV exposure may be repurposed for preventing skin inflammation and cancer. Cancer Prev Res; 10(10); 598–606. ©2017 AACR.

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Gene expression signatures and immunohistochemical subtypes add prognostic value to each other in breast cancer cohorts

Purpose: Gene signatures and Ki67 stratify the same breast tumour into opposing good/poor prognosis groups in approximately 20% of patients. Given this discrepancy, we hypothesized that the combination of a clinically relevant signature and immunohistochemical (IHC) markers may provide more prognostic information than either classifier alone. Experimental design: We assessed Ki67 alone or combined with ER, PR and HER2 (forming IHC subtypes), and the research versions of the Genomic Grade Index (GGI), 70-gene, cell-cycle score (CCS), Recurrence Score (RS) and PAM50 signatures on matching TMA/whole tumour sections and microarray data in two Swedish breast cancer cohorts of 379 and 209 patients, with median follow-up of 12.4 and 12.5 years, respectively. Firstly, we fit cox proportional hazard models and used the change in likelihood-ratio ( LR) to determine the additional prognostic information provided by signatures beyond that of 1) Ki67 and 2) IHC subtypes. Secondly and uniquely, we then assessed whether signatures could compete well with pathology-based immunohistochemical classifiers by calculating the additional prognostic information of Ki67/IHC subtypes beyond signatures. Results: In cohort 1 only RS and PAM50 provided additional prognostic information beyond Ki67 and IHC subtypes ( LR-2 Ki67: RS= 12.8, PAM50 = 20.7, IHC subtypes: RS= 12.9, PAM50 = 11.7). Conversely, IHC subtypes added prognostic information beyond all signatures except PAM50. Similar results were observed in cohort 2. Conclusion(s): RS and PAM50 provided more prognostic information than the IHC subtypes in all breast cancer patients, however the IHC subtypes did not add any prognostic information to PAM50.

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Transmembrane and coiled-coil domain 1 impairs the AKT signaling pathway in urinary bladder urothelial carcinoma: a characterization of a tumor suppressor

Purpose: Urinary bladder urothelial carcinoma (UBUC) is a common malignant disease in developed countries. Cell cycle dysregulation resulting in uncontrolled cell proliferation has been associated with UBUC development. This study aimed to explore the roles of TMCO1 in UBUCs. Experimental Design: Data mining, branched DNA assay, immunohistochemistry, xenograft, cell culture, quantitative RT-PCR, immunoblotting, stable and transient transfection, lentivirus production and stable knockdown, cell cycle, cell viability and proliferation, soft agar, wound healing, transwell migration and invasion, co-immunoprecipitation, immunocytochemistry, AKT serine/threonine kinase (AKT) activity assays and site-directed mutagenesis were used to study TMCO1 involvement in vivo and in vitro. Results: Data mining identified that the TMCO1 transcript was downregulated during the progression of UBUCs. In distinct UBUC-derived cell lines, changes in TMCO1 levels altered the cell-cycle distribution, cell viability, cell proliferation, colony formation and modulated the AKT pathway. TMCO1 recruited the PH domain and leucine rich repeat protein phosphatase 2 (PHLPP2) to dephosphorylate pAKT1(serine 473) (S473). Mutagenesis at S60 of the TMCO1 protein released TMCO1-induced cell cycle arrest and restored the AKT pathway in BFTC905 cells. Stable TMCO1 (wild-type) overexpression suppressed, while T33A and S60A mutants recovered, tumor size in xenograft mice. Conclusions: Clinical associations, xenograft mice and in vitro indications provide solid evidence that the TMCO1 gene is a novel tumor suppressor in UBUCs. TMCO1 dysregulates cell cycle progression via suppression of the AKT pathway, and S60 of the TMCO1 protein is crucial for its tumor suppressor roles.

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PAM50 provides prognostic information when applied to the lymph node metastases of advanced breast cancer patients

Purpose: Transcriptional pathway activity and the molecular subtypes of breast cancer metastases have been shown to significantly influence patient post-relapse survival. Here we further determine the relevance of clinically employed gene signatures in the advanced breast cancer setting. Experimental Design: Sufficient RNA for expression profiling was obtained from distant metastatic or inoperable loco-regional relapse tissue by fine needle aspiration from 109 patients of the Swedish TEX clinical trial. Gene signatures (GGI, 70 gene, Recurrence score, Cell Cycle Score, Risk of Recurrence score and PAM50) were applied to all metastases and their relationship to long- (5 year) and short-term (1.5 year) post-relapse survival at all and loco-regional lymph nodes (n= 40) versus other metastatic sites (n=69) combined was assessed using Kaplan-Meier and/or multivariate Cox regression analyses. Results: The majority of metastases were classified into intermediate or high-risk groups by all signatures and a significant association was found between metastatic signature subgroups and primary tumor estrogen receptor status and histological grade (P < 0.05). When considering all sites of metastasis only PAM50 was statistically significant in Kaplan-Meier analysis (Logrank P = 0.008 and 0.008 for long and short term post-relapse BCSS, respectively). This significance remained in both uni and multi-variate models when restricting analyses to lymph node metastases only and a similar trend was observed in other metastatic sites combined, but did not reach formal significance. Conclusions: Our findings are the first to demonstrate that the PAM50 signature can provide prognostic information from the lymph node metastases of advanced breast cancer patients.

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Downregulation of DNMT3A by miR-708-5p inhibits lung cancer stem cell-like phenotypes through repressing Wnt/{beta}-catenin signaling

Purpose: Lung cancer is the leading cause of cancer death in the world, and emerging evidences suggest that lung cancer stem cells (CSCs) are associated with its poor prognosis, tumor recurrence and therapy resistance. Here we reveal a novel role for miR-708-5p in inhibiting lung cancer stem cell-like features. Experimental Design: Phenotypic effects of miR-708-5p on the lung CSC-like properties were examined by in vitro sphere formation assay and in xenografted animal models. Immunoblotting, dual luciferase reporter, and immunocytochemistry were performed to determine the target of miR-708-5p. DNA methylation of CDH1 promoter region was tested using bisulfate sequencing. Genome-wide miRNA sequencing data of 990 patients from the cancer genome atlas (TCGA) dataset and 148 patients from China cohort were analyzed to excavate the pathogenic implications of miR-708-5p. Results: Expression of miR-708-5p inhibits the CSC traits of NSCLC cells in vitro while antagonizing miR-708-5p promotes tumorigenesis in vivo. MiR-708-5p directly suppresses the translation of DNMT3A, which results in a substantial reduction of global DNA methylation and the up-regulated expression of tumor suppressor CDH1. The up-regulation of CDH1 decreased the activity of Wnt/β-catenin signaling and then impaired the stemness charactertics of NSCLC cells. Clinically, patients with high miR-708-5p expression show significantly better survival and lower recurrence. Furthermore, miR-708-5p has a promising potential to apply to differentiating histological subtypes in NSCLC. Conclusion: Our findings support that miR-708-5p suppresses NSCLC initiation, development and stemness through interfering DNMT3A-dependent DNA methylation. MiR-708-5p may function as a novel diagnostic and prognostic biomarker in NSCLC.

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A Randomized Phase II Trial of Adjuvant Galinpepimut-S, WT-1 Analog Peptide Vaccine, after Multimodality Therapy for Patients with Malignant Pleural Mesothelioma

Purpose: Determine the 1-year progression-free survival (PFS) among patients with malignant pleural mesothelioma (MPM) receiving the WT1 peptide vaccine galinpepimut-S after multimodality therapy vs those receiving control adjuvants. Patients and Methods: This double-blind, controlled, two center phase II trial randomized MPM patients after surgery and another treatment modality to galinpepimut-S with GM-CSF and Montanide or GM-CSF and Montanide alone. An improvement in 1-year PFS from 50% to 70% was the predefined efficacy threshold, and 78 patients total were planned. The study was not powered for comparison between the two arms. Results: 41 patients were randomized. Treatment related adverse events were mild, self-limited, and not clinically significant. Based on a stringent prespecified futility analysis (futility = > 10 of 20 patients on one arm experiencing progression < 1 year), the control arm closed early. The treatment arm was subsequently closed because of the resultant unblinding. The PFS rate at 1 year from beginning study treatment was 33% and 45% in the control and vaccine arms, respectively. Median PFS was 7.4 months vs 10.1 months and median OS was 18.3 months vs 22.8 months in the control and vaccine arms, respectively. Conclusion: The favorable safety profile was confirmed. PFS and OS were greater in those who received vaccine but the trial was neither designed nor powered for comparison between the arms. Based on these promising results, the investigators are planning a larger randomized trial with greater statistical power to define the optimal use and benefit of galinpepimut-S in the treatment of MPM.

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Multiregional sequencing reveals genomic alterations and clonal dynamics in primary malignant melanoma of the esophagus

Primary malignant melanoma of the esophagus (PMME) is a rare and aggressive disease with high tendency of metastasis. To characterize the genetic basis and intra-tumor heterogeneity of PMME, we performed multiregion exome sequencing and whole genome SNP array genotyping of 12 samples obtained from a PMME patient. High intra-tumor heterogeneity was observed in both somatic mutation and copy number alteration levels. Nine geographically separate samples including two normal samples were clonally related and followed a branched evolution model. Most putative oncogenic drivers such as BRAF and KRAS mutations as well as CDKN2A biallelic inactivation were observed in trunk clones, whereas clinically actionable mutations such as PIK3CA and JAK1 mutations were detected in branch clones. Ancestor tumor clones evolved into three subclonal clades: clade1 fostered metastatic subclones that carried metastatic features of PIK3CA and ARHGAP26 point mutations as well as chr13 arm-level deletion, clade2 owned branch-specific JAK1 mutations and PTEN deletion, and clade3 was found in two vertical distribution samples below the primary tumor area, highlighting the fact that it is possible for PMME to disseminate by the submucosal longitudinal lymphatic route at an early stage of metastasis. These findings facilitate interpretation of the genetic essence of this rare melanoma subtype as well as the pattern of cancer evolution, thus reinforcing the therapeutic challenges associated with PMME.

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Secretory autophagy in cancer-associated fibroblasts promotes head and neck cancer progression and offers a novel therapeutic target

Despite therapeutic advancements, there has been little change in the survival of patients with head and neck squamous cell carcinoma (HNSCC). Recent results suggest that cancer-associated fibroblasts (CAF) drive progression of this disease. Here, we report that autophagy is upregulated in HNSCC-associated CAFs where it is responsible for key pathogenic contributions in this disease. Autophagy is fundamentally involved in cell degradation, but there is emerging evidence that suggests it is also important for cellular secretion. Thus, we hypothesized that autophagy-dependent secretion of tumor-promoting factors by HNSCC-associated CAFs may explain their role in malignant development. In support of this hypothesis, we observed a reduction in CAF-facilitated HNSCC progression after blocking CAF autophagy. Studies of cell growth media conditioned after autophagy blockade revealed levels of secreted IL-6, IL-8 and other cytokines were modulated by autophagy. Notably, when HNSCC cells were co-cultured with normal fibroblasts they upregulated autophagy through IL-6, IL-8 and bFGF. In a mouse xenograft model of HNSCC, pharmacological inhibition of Vps34, a key mediator of autophagy, enhanced the antitumor efficacy of cisplatin. Our results establish an oncogenic function for secretory autophagy in HNSCC stromal cells that promotes malignant progression.

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SKP2 Activation by Thyroid Hormone Receptor {beta}2 bypasses Rb-Dependent Proliferation in Rb-Deficient Cells

Germline RB1 mutations strongly predispose humans to cone-precursor-derived retinoblastomas and strongly predispose mice to pituitary tumors, yet shared cell type-specific circuitry that sensitizes these different cell types to the loss of RB1 has not been defined. Here we show that the cell type-restricted thyroid hormone receptor isoform TRβ2 sensitizes to RB1 loss in both settings by antagonizing the widely expressed and tumor suppressive TRβ1.TRβ2 promoted expression of the E3 ubiquitin ligase SKP2, a critical factor for RB1-mutant tumors, by enabling EMI1/FBXO5-dependent inhibition of SKP2 degradation. In RB1-wild type neuroblastoma cells, endogenous Rb or ectopic TRβ2 was required to sustain SKP2 expression as well as cell viability and proliferation. These results suggest that in certain contexts, Rb loss enables TRβ1-dependent suppression of SKP2 as a safeguard against RB1-deficient tumorigenesis. TRβ2 counteracts TRβ1, thus disrupting this safeguard and promoting development of RB1-deficient malignancies.

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Mechano-signal transduction in mesenchymal stem cells induces prosaposin secretion to drive the proliferation of breast cancer cells

In response to chemical stimuli from cancer cells, mesenchymal stem cells (MSC) can differentiate into cancer-associated fibroblasts (CAF) and promote tumor progression. How mechanical stimuli such as stiffness of the extracellular matrix (ECM) contribute to MSC phenotype in cancer remains poorly understood. Here we show that ECM stiffness leads to mechano-signal transduction in MSC which promotes mammary tumor growth in part through secretion of the signaling protein prosaposin. On a stiff matrix, MSC cultured with conditioned media from mammary cancer cells expressed increased levels of alpha-smooth muscle actin, a marker of CAF, compared to MSC cultured on a soft matrix. By contrast, MSC cultured on a stiff matrix secreted prosaposin which promoted proliferation and survival of mammary carcinoma cells but inhibited metastasis. Our findings suggest that in addition to chemical stimuli, increased stiffness of the ECM in the tumor microenvironment induces differentiation of MSC to CAF, triggering enhanced proliferation and survival of mammary cancer cells.

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JAM-C identifies Src family kinase-activated leukemia-initiating cells and predicts poor prognosis in acute myeloid leukemia

Acute Myeloid Leukemia (AML) originates from hematopoietic stem and progenitor cells that acquire somatic mutations, leading to disease and clonogenic evolution. AML is characterized by accumulation of immature myeloid cells in the bone marrow and phenotypic cellular heterogeneity reflective of normal hematopoietic differentiation. Here we show that JAM-C expression defines a subset of leukemic cells endowed with leukemia-initiating cell activity (LIC). Stratification of de novo AML patients at diagnosis based on JAM-C-expressing cells frequencies in the blood served as an independent prognostic marker for disease outcome. Using publicly available leukemic stem cell (LSC) gene expression profiles and gene expression data generated from JAM-C-expressing leukemic cells, we defined a single cell core gene expression signature correlated to JAM-C expression that reveals LSC heterogeneity. Finally, we demonstrated that JAM-C controls Src family kinase (SFK) activation in LSC and that LIC with exacerbated SFK activation were uniquely found within the JAM-C-expressing LSC compartment.

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Clonality, heterogeneity and evolution of synchronous bilateral ovarian cancer

Synchronous bilateral ovarian cancer (SBOC) represents a relatively frequent occurrence and clinically relevant diagnostic dilemma. Delineation of its clonal architecture, genetic heterogeneity and evolutionary trajectories may have important implications for prognosis and management of patients with SBOC. Here we describe the results of next-generation whole-exome or whole-genome sequencing of specimens from 12 SBOC cases and report that bilateral tumors from each individual display a comparable number of genomic abnormalities and similar mutational signatures of single nucleotide variations. Clonality indices based on tumor-specific alterations supported monoclonal origins of SBOC. Each of the ovarian lesions was nevertheless oligoclonal, with inferred metastatic tumors harboring more subclones than their primary counterparts. The phylogenetic structure of SBOC indicated that most cancer cell dissemination occurred early, when the primary carcinoma was still relatively small (<100 million cells). Accordingly, the mutation spectra and mutational signatures of somatic variants exhibited pronounced spatiotemporal differences in each patient. Overall, these findings suggest that SBOC are clonally related and form through pelvic spread rather than independent multifocal oncogenesis. Metastatic dissemination is often an early event, with dynamic mutational processes leading to divergent evolution and intratumor and intertumor heterogeneity, ultimately contributing substantially to phenotypic plasticity and diverse clinical course in SBOC.

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S100A4 Is a Biomarker and Regulator of Glioma Stem Cells That Is Critical for Mesenchymal Transition in Glioblastoma

Glioma stem cells (GSC) and epithelial–mesenchymal transition (EMT) are strongly associated with therapy resistance and tumor recurrence, but the underlying mechanisms are incompletely understood. Here, we show that S100A4 is a novel biomarker of GSCs. S100A4+ cells in gliomas are enriched with cancer cells that have tumor-initiating and sphere-forming abilities, with the majority located in perivascular niches where GSCs are found. Selective ablation of S100A4-expressing cells was sufficient to block tumor growth in vitro and in vivo. We also identified S100A4 as a critical regulator of GSC self-renewal in mouse and patient-derived glioma tumorspheres. In contrast with previous reports of S100A4 as a reporter of EMT, we discovered that S100A4 is an upstream regulator of the master EMT regulators SNAIL2 and ZEB along with other mesenchymal transition regulators in glioblastoma. Overall, our results establish S100A4 as a central node in a molecular network that controls stemness and EMT in glioblastoma, suggesting S100A4 as a candidate therapeutic target. Cancer Res; 77(19); 5360–73. ©2017 AACR.

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Highlights from Recent Cancer Literature

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Roles for Innate Immunity in Combination Immunotherapies

Immunity to infectious agents involves a coordinated response of innate and adaptive immune cells working in concert, with many feed-forward and regulatory interactions between both arms of the immune system. In contrast, many therapeutic strategies to augment immunity against tumors have focused predominantly on stimulation of adaptive immunity. However, a growing appreciation of the potential contributions of innate immune effectors to antitumor immunity, especially in the context of combination immunotherapy, is leading to novel strategies to elicit a more integrated immune response against cancer. Here we review antitumor activities of innate immune cells, mechanisms of their synergy with adaptive immune responses against tumors, and discuss recent studies highlighting the potential of combination therapies recruiting both innate and adaptive immune effectors to eradicate established tumors. Cancer Res; 77(19); 5215–21. ©2017 AACR.

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Integrating Models to Quantify Environment-Mediated Drug Resistance

Drug resistance is the single most important driver of cancer treatment failure for modern targeted therapies, and the dialog between tumor and stroma has been shown to modulate the response to molecularly targeted therapies through proliferative and survival signaling. In this work, we investigate interactions between a growing tumor and its surrounding stroma and their role in facilitating the emergence of drug resistance. We used mathematical modeling as a theoretical framework to bridge between experimental models and scales, with the aim of separating intrinsic and extrinsic components of resistance in BRAF-mutated melanoma; the model describes tumor–stroma dynamics both with and without treatment. Integration of experimental data into our model revealed significant variation in either the intensity of stromal promotion or intrinsic tissue carrying capacity across animal replicates. Cancer Res; 77(19); 5409–18. ©2017 AACR.

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New Advances and Challenges of Targeting Cancer Stem Cells

The second International Cancer Stem Cell Conference in Cleveland, Ohio, on September 20–23, 2016, convened 330 attendees from academic, industrial, and clinical organizations. It featured a debate on the concepts and challenges of the cancer stem cells (CSC) as well as CSC-centered scientific sessions on clinical trials, genetics and epigenetics, tumor microenvironment, immune suppression, metastasis, therapeutic resistance, and emerging novel concepts. The conference hosted 35 renowned speakers, 100 posters, 20 short talks, and a preconference workshop. The reported advances of CSC research and therapies fostered new collaborations across national and international borders, and inspired the next generation's young scientists. Cancer Res; 77(19); 5222–7. ©2017 AACR.

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Chromatin-Associated Protein SIN3B Prevents Prostate Cancer Progression by Inducing Senescence

Distinguishing between indolent and aggressive prostate adenocarcinoma remains a priority to accurately identify patients who need therapeutic intervention. SIN3B has been implicated in the initiation of senescence in vitro. Here we show that in a mouse model of prostate cancer, SIN3B provides a barrier to malignant progression. SIN3B was required for PTEN-induced cellular senescence and prevented progression to invasive prostate adenocarcinoma. Furthermore, SIN3B was downregulated in human prostate adenocarcinoma correlating with upregulation of its target genes. Our results suggest a tumor suppressor function for SIN3B that limits prostate adenocarcinoma progression, with potential implications for the use of SIN3B and its target genes as candidate diagnostic markers to distinguish indolent from aggressive disease. Cancer Res; 77(19); 5339–48. ©2017 AACR.

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Targeting a Single Alternative Polyadenylation Site Coordinately Blocks Expression of Androgen Receptor mRNA Splice Variants in Prostate Cancer

Prostate cancer is the second leading cause of male cancer deaths due to disease progression to castration-resistant prostate cancer (CRPC). Androgen receptor (AR) splice variants including AR-V7 function as constitutively active transcription factors in CRPC cells, thereby promoting resistance to AR-targeted therapies. To date, there are no AR variant–specific treatments for CRPC. Here we report that the splicing of AR variants AR-V7 as well as AR-V1 and AR-V9 is regulated coordinately by a single polyadenylation signal in AR intron 3. Blocking this signal with morpholino technology or silencing of the polyadenylation factor CPSF1 caused a splice switch that inhibited expression of AR variants and blocked androgen-independent growth of CRPC cells. Our findings support the development of new therapies targeting the polyadenylation signal in AR intron 3 as a strategy to prevent expression of a broad array of AR variants in CRPC. Cancer Res; 77(19); 5228–35. ©2017 AACR.

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Combination Therapy with Bispecific Antibodies and PD-1 Blockade Enhances the Antitumor Potency of T Cells

The DOCK-AND-LOCK (DNL) method is a platform technology that combines recombinant engineering and site-specific conjugation to create multispecific, multivalent antibodies of defined composition with retained bioactivity. We have applied DNL to generate a novel class of trivalent bispecific antibodies (bsAb), each comprising an anti-CD3 scFv covalently conjugated to a stabilized dimer of different antitumor Fabs. Here, we report the further characterization of two such constructs, (E1)-3s and (14)-3s, which activate T cells and target Trop-2– and CEACAM5-expressing cancer cells, respectively. (E1)-3s and (14)-3s, in the presence of human T cells, killed target cells grown as monolayers at subnanomolar concentrations, with a similar potency observed for drug-resistant cells. Antitumor efficacy was demonstrated for (E1)-3s coadministered with human peripheral blood mononuclear cells (PBMC) in NOD/SCID mice harboring xenografts of MDA-MB-231, a triple-negative breast cancer line constitutively expressing Trop-2 and PD-L1. Growth inhibition was observed following treatment with (E1)-3s or (14)-3s combined with human PBMC in 3D spheroids generated from target cell lines to mimic the in vivo behavior and microenvironment of these tumors. Moreover, addition of an antagonistic anti–PD-1 antibody increased cell death in 3D spheroids and extended survival of MDA-MB-231-bearing mice. These preclinical results emphasize the potential of combining T-cell–redirecting bsAbs with antagonists or agonists that mitigate T-cell inhibition within the tumor microenvironment to improve immunotherapy of solid cancers in patients. They also support the use of 3D spheroids as a predictive alternative to in vivo models for evaluating T-cell functions. Cancer Res; 77(19); 5384–94. ©2017 AACR.

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Loss of Igf2 Gene Imprinting in Murine Prostate Promotes Widespread Neoplastic Growth

Loss of imprinting (LOI) is an epigenetic event that relaxes an allele-specific restriction on gene expression. One gene that experiences LOI is the paracrine insulin-like growth factor IGF2, which occurs commonly in human prostate tissues during aging and tumorigenesis. However, the relationship between IGF2 LOI and prostate tumorigenesis has not been established functionally. In this study, we created a mouse model with CTCF-binding site mutations at the Igf2-H19 imprint control region that abolishes CTCF insulator activity, resulting in biallelic Igf2 expression that mimics increased levels seen with aging-induced LOI. We found that Igf2 LOI increased the prevalence and severity of prostatic intraepithelial neoplasia (PIN), a premalignant lesion. Engineering Nkx3.1 deficiency into our model increased the frequency of PIN lesions in an additive fashion. Prostates harboring LOI displayed increased MAPK signaling and epithelial proliferation. In human prostate tissue arrays, we documented a positive correlation in benign tissues of IGF2 levels with phospho-ERK and phospho-AKT levels. Overall, our results establish that Igf2 LOI is sufficient on its own to increase rates of neoplastic development in the prostate by upregulating critical cancer-associated signaling pathways. Cancer Res; 77(19); 5236–47. ©2017 AACR.

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Identification of Novel Breast Cancer Risk Loci

It has been estimated that >1,000 genetic loci have yet to be identified for breast cancer risk. Here we report the first study utilizing targeted next-generation sequencing to identify single-nucleotide polymorphisms (SNP) associated with breast cancer risk. Targeted sequencing of 283 genes was performed in 240 women with early-onset breast cancer (≤40 years) or a family history of breast and/or ovarian cancer. Common coding variants with minor allele frequencies (MAF) >1% that were identified were presumed initially to be SNPs, but further database inspections revealed variants had MAF of ≤1% in the general population. Through prioritization and stringent selection criteria, we selected 24 SNPs for further genotyping in 1,516 breast cancer cases and 1,189 noncancer controls. Overall, we identified the JAK2 SNP rs56118985 to be significantly associated with overall breast cancer risk. Subtype analysis performed for patient subgroups defined by ER, PR, and HER2 status suggested additional associations of the NOTCH3 SNP rs200504060 and the HIF1A SNP rs142179458 with breast cancer risk. In silico analysis indicated that coding amino acids encoded at these three SNP sites were conserved evolutionarily and associated with decreased protein stability, suggesting a likely impact on protein function. Our results offer proof of concept for identifying novel cancer risk loci from next-generation sequencing data, with iterative data analysis from targeted, whole-exome, or whole-genome sequencing a wellspring to identify new SNPs associated with cancer risk. Cancer Res; 77(19); 5428–37. ©2017 AACR.

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A Novel Functional Splice Variant of AKT3 Defined by Analysis of Alternative Splice Expression in HPV-Positive Oropharyngeal Cancers

The incidence of HPV-related oropharyngeal squamous cell carcinoma (OPSCC) has increased more than 200% in the past 20 years. Recent genetic sequencing efforts have elucidated relevant genes in head and neck cancer, but HPV-related tumors have consistently shown few DNA mutations. In this study, we sought to analyze alternative splicing events (ASE) that could alter gene function independent of mutations. To identify ASE unique to HPV-related tumors, RNA sequencing was performed on 46 HPV-positive OPSCC and 25 normal tissue samples. A novel algorithm using outlier statistics on RNA-sequencing junction expression identified 109 splicing events, which were confirmed in a validation set from The Cancer Genome Atlas. Because the most common type of splicing event identified was an alternative start site (39%), MBD-seq genome-wide CpG methylation data were analyzed for methylation alterations at promoter regions. ASE in six genes showed significant negative correlation between promoter methylation and expression of an alternative transcriptional start site, including AKT3. The novel AKT3 transcriptional variant and methylation changes were confirmed using qRT-PCR and qMSP methods. In vitro silencing of the novel AKT3 variant resulted in significant growth inhibition of multiple head and neck cell lines, an effect not observed with wild-type AKT3 knockdown. Analysis of ASE in HPV-related OPSCC identified multiple alterations likely involved in carcinogenesis, including a novel, functionally active transcriptional variant of AKT3. Our data indicate that ASEs represent a significant mechanism of oncogenesis with untapped potential for understanding complex genetic changes that result in the development of cancer. Cancer Res; 77(19); 5248–58. ©2017 AACR.

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MRE11 Promotes Tumorigenesis by Facilitating Resistance to Oncogene-Induced Replication Stress

Hypomorphic mutations in the genes encoding the MRE11/RAD50/NBS1 (MRN) DNA repair complex lead to cancer-prone syndromes. MRN binds DNA double-strand breaks, where it functions in repair and triggers cell-cycle checkpoints via activation of the ataxia-telangiectasia mutated kinase. To gain understanding of MRN in cancer, we engineered mice with B lymphocytes lacking MRN, or harboring MRN in which MRE11 lacks nuclease activities. Both forms of MRN deficiency led to hallmarks of cancer, including oncogenic translocations involving c-Myc and the immunoglobulin locus. These preneoplastic B lymphocytes did not progress to detectable B lineage lymphoma, even in the absence of p53. Moreover, Mre11 deficiencies prevented tumorigenesis in a mouse model strongly predisposed to spontaneous B-cell lymphomas. Our findings indicate that MRN cannot be considered a standard tumor suppressor and instead imply that nuclease activities of MRE11 are required for oncogenesis. Inhibition of MRE11 nuclease activity increased DNA damage and selectively induced apoptosis in cells overexpressing oncogenes, suggesting MRE11 serves an important role in countering oncogene-induced replication stress. Thus, MRE11 may offer a target for cancer therapeutic development. More broadly, our work supports the idea that subtle enhancements of endogenous genome instability can exceed the tolerance of cancer cells and be exploited for therapeutic ends. Cancer Res; 77(19); 5327–38. ©2017 AACR.

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PRC2-Mediated Transcriptomic Alterations at the Embryonic Stage Govern Tumorigenesis and Clinical Outcome in MYCN-Driven Neuroblastoma

Pediatric cancers such as neuroblastoma are thought to involve a dysregulation of embryonic development. However, it has been difficult to identify the critical events that trigger tumorigenesis and differentiate them from normal development. In this study, we report the establishment of a spheroid culture method that enriches early-stage tumor cells from TH-MYCN mice, a preclinical model of neuroblastoma. Using this method, we found that tumorigenic cells were evident as early as day E13.5 during embryo development, when the MYC and PRC2 transcriptomes were significantly altered. Ezh2, an essential component of PRC2, was expressed in embryonic and postnatal tumor lesions and physically associated with N-MYC and we observed that H3K27me3 was increased at PRC2 target genes. PRC2 inhibition suppressed in vitro sphere formation, derepressed its target genes, and suppressed in situ tumor growth. In clinical specimens, expression of MYC and PRC2 target genes correlated strongly and predicted survival outcomes. Together, our findings highlighted PRC2-mediated transcriptional control during embryogenesis as a critical step in the development and clinical outcome of neuroblastoma. Cancer Res; 77(19); 5259–71. ©2017 AACR.

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Oncogenic KRAS and p53 Loss Drive Gastric Tumorigenesis in Mice That Can Be Attenuated by E-Cadherin Expression

Gastric adenocarcinoma is the third leading cause of cancer-related death worldwide, but no models exist to readily investigate distant metastases that are mainly responsible for mortality in this disease. Here we report the development of a genetically engineered mouse model of gastric adenocarcinoma tumorigenesis based on KrasG12D expression plus inactivation of E-cadherin (Cdh1) and p53 in the gastric parietal cell lineage. Intestinal and diffuse gastric tumors arise rapidly in this model that displays a median survival of 76 days. Tumors occur throughout the stomach, with metastases documented in lymph nodes, lung, and liver. Mice otherwise identical but retaining one wild-type Cdh1 allele exhibited longer survival with only 20% penetrance of invasive tumors and no apparent lung or liver metastases. Notably, increased RAS activity and downstream MAPK signaling was observed in stomachs only when E-cadherin was absent. This model offers a valuable tool to investigate gastric adenocarcinoma subtypes where RAS/MAPK pathway activation and E-cadherin attenuation are common. Cancer Res; 77(19); 5349–59. ©2017 AACR.

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Aneuploid Cell Survival Relies upon Sphingolipid Homeostasis

Aneuploidy, a hallmark of cancer cells, poses an appealing opportunity for cancer treatment and prevention strategies. Using a cell-based screen to identify small molecules that could selectively kill aneuploid cells, we identified the compound N-[2-hydroxy-1-(4-morpholinylmethyl)-2-phenylethyl]-decanamide monohydrochloride (DL-PDMP), an antagonist of UDP-glucose ceramide glucosyltransferase. DL-PDMP selectively inhibited proliferation of aneuploid primary mouse embryonic fibroblasts and aneuploid colorectal cancer cells. Its selective cytotoxic effects were based on further accentuating the elevated levels of ceramide, which characterize aneuploid cells, leading to increased apoptosis. We observed that DL-PDMP could also enhance the cytotoxic effects of paclitaxel, a standard-of-care chemotherapeutic agent that causes aneuploidy, in human colon cancer and mouse lymphoma cells. Our results offer pharmacologic evidence that the aneuploid state in cancer cells can be targeted selectively for therapeutic purposes, or for reducing the toxicity of taxane-based drug regimens. Cancer Res; 77(19); 5272–86. ©2017 AACR.

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Trastuzumab Increases HER2 Uptake and Cross-Presentation by Dendritic Cells

Early-phase clinical trials evaluating CD8+ T cell–eliciting, HER2-derived peptide vaccines administered to HER2+ breast cancer patients in the adjuvant setting suggest synergy between the vaccines and trastuzumab, the mAb targeting the HER2 protein. Among 60 patients enrolled in clinical trials evaluating the E75 + GM-CSF and GP2 + GM-CSF vaccines, there have been no recurrences in patients vaccinated after receiving trastuzumab as part of standard therapy in the per treatment analyses conducted after a median follow-up of greater than 34 months. Here, we describe a mechanism by which this synergy may occur. Flow cytometry showed that trastuzumab facilitated uptake of HER2 by dendritic cells (DC), which was mediated by the Fc receptor and was specific to trastuzumab. In vitro, increased HER2 uptake by DC increased cross-presentation of E75, the immunodominant epitope derived from the HER2 protein, an observation confirmed in two in vivo mouse models. This increased E75 cross-presentation, mediated by trastuzumab treatment, enabled more efficient expansion of E75-specific cytotoxic T cells (E75-CTL). These results demonstrate a mechanism by which trastuzumab links innate and adaptive immunity by facilitating activation of antigen-specific T cells. On the basis of these data, we conclude that HER2-positive breast cancer patients that have been treated with trastuzumab may experience a more robust antitumor immune response by restimulation of T cells with the E75 peptide vaccine, thereby accounting for the improved disease-free survival observed with combination therapy. Cancer Res; 77(19); 5374–83. ©2017 AACR.

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JCAD Promotes Progression of Nonalcoholic Steatohepatitis to Liver Cancer by Inhibiting LATS2 Kinase Activity

Nonalcoholic steatohepatitis-associated hepatocellular carcinoma (NASH-HCC) is a malignancy whose incidents are rapidly increasing. However, the mechanisms that drive development of HCC in a steatotic microenvironment remain unknown. Here we report that the obesity-associated protein JCAD is expressed at significantly higher levels in human NASH-HCC specimens compared with pericarcinoma specimens. High JCAD expression was verified in multiple hepatoma cell lines. Forced overexpression of JCAD in hepatoma cells promoted tumor growth and proliferation, whereas JCAD silencing yielded opposite effects. JCAD interacted with the kinase domain of the tumor suppressor kinase LATS2, a core component of the Hippo signaling pathway. JCAD overexpression inhibited the ability of LATS2 to phosphorylate YAP in this pathway, in turn upregulating CCND1 and GLI2 to promote hepatoma cell proliferation. JCAD was induced by fatty acid overload in hepatic cells and was highly expressed in a mouse model of NASH-precarcinoma lesions, where the ratio of phospho-YAP to YAP was decreased. In human NASH-HCC specimens, JCAD expression and YAP phosphorylation patterns paralleled with the mouse model. Our findings illuminate a new role for JCAD and its critical interplay in the Hippo signaling cascade during the transition of NASH to HCC, with potential implications for therapeutic development in this setting. Cancer Res; 77(19); 5287–300. ©2017 AACR.

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Mitotic Vulnerability in Triple-Negative Breast Cancer Associated with LIN9 Is Targetable with BET Inhibitors

Triple-negative breast cancers (TNBC) are highly aggressive, lack FDA-approved targeted therapies, and frequently recur, making the discovery of novel therapeutic targets for this disease imperative. Our previous analysis of the molecular mechanisms of action of bromodomain and extraterminal protein inhibitors (BETi) in TNBC revealed these drugs cause multinucleation, indicating BET proteins are essential for efficient mitosis and cytokinesis. Here, using live cell imaging, we show that BET inhibition prolonged mitotic progression and induced mitotic cell death, both of which are indicative of mitotic catastrophe. Mechanistically, the mitosis regulator LIN9 was a direct target of BET proteins that mediated the effects of BET proteins on mitosis in TNBC. Although BETi have been proposed to function by dismantling super-enhancers (SE), the LIN9 gene lacks an SE but was amplified or overexpressed in the majority of TNBCs. In addition, its mRNA expression predicted poor outcome across breast cancer subtypes. Together, these results provide a mechanism for cancer selectivity of BETi that extends beyond modulation of SE-associated genes and suggest that cancers dependent upon LIN9 overexpression may be particularly vulnerable to BETi. Cancer Res; 77(19); 5395–408. ©2017 AACR.

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Nuclear FAK and Runx1 Cooperate to Regulate IGFBP3, Cell-Cycle Progression, and Tumor Growth

Nuclear focal adhesion kinase (FAK) is a potentially important regulator of gene expression in cancer, impacting both cellular function and the composition of the surrounding tumor microenvironment. Here, we report in a murine model of skin squamous cell carcinoma (SCC) that nuclear FAK regulates Runx1-dependent transcription of insulin-like growth factor binding protein 3 (IGFBP3), and that this regulates SCC cell-cycle progression and tumor growth in vivo. Furthermore, we identified a novel molecular complex between FAK and Runx1 in the nucleus of SCC cells and showed that FAK interacted with a number of Runx1-regulatory proteins, including Sin3a and other epigenetic modifiers known to alter Runx1 transcriptional function through posttranslational modification. These findings provide important new insights into the role of FAK as a scaffolding protein in molecular complexes that regulate gene transcription. Cancer Res; 77(19); 5301–12. ©2017 AACR.

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Genomic Alterations in Circulating Tumor DNA from Diverse Cancer Patients Identified by Next-Generation Sequencing

Noninvasive genomic profiling of tumors may be possible with next-generation sequencing (NGS) of blood-derived circulating tumor DNA (ctDNA), but proof of concept in a large cohort of patients with diverse cancers has yet to be reported. Here we report the results of an analysis of plasma-derived ctDNA from 670 patients with diverse cancers. The tumors represented in the patient cohort were mainly gastrointestinal (31.8%), brain (22.7%), or lung (20.7%). ctDNA obtained from most patients [N = 423 (63%)] displayed at least one alteration. The most frequent alterations seen, as characterized mutations or variants of unknown significance, occurred in TP53 (32.5% of patients), EGFR (13%), KRAS (12.5%), and PIK3CA (9.1%); for characterized alterations, 30.7% (TP53), 7.6% (EGFR), 12.2% (KRAS), and 7.7% (PIK3CA). We found that 32% of brain tumors had at least one ctDNA alteration. Head and neck tumors were independently associated with a higher number of alterations in a multivariable analysis (P = 0.019). Notably, 320/670 (48%) of patients displayed potentially actionable alterations, with 241 patients possible candidates for on-label or off-label treatment with an FDA-approved drug. Several illustrations of the clinical utility of the information obtained for improving treatment of specific patients is provided. Our findings demonstrate the feasibility and impact of genomic profiling of tumors by ctDNA NGS, greatly encouraging broader investigations of the application of this technology for precision medicine in cancer management. Cancer Res; 77(19); 5419–27. ©2017 AACR.

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The meta‐analysis

Fernando de Andrade Quintanilha Ribeiro
Braz J Otorhinolaryngol 2017;83:497

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First Clinical Consensus and National Recommendations on Tracheostomized Children of the Brazilian Academy of Pediatric Otorhinolaryngology (ABOPe) and Brazilian Society of Pediatrics (SBP)

Melissa A.G. Avelino, Rebecca Maunsell, Fabiana Cardoso Pereira Valera, José Faibes Lubianca Neto, Cláudia Schweiger, Carolina Sponchiado Miura, Vitor Guo Chen, Dayse Manrique, Raquel Oliveira, Fabiano Gavazzoni, Isabela Furtado de Mendonça Picinin, Paulo Bittencourt, Paulo Camargos, Fernanda Peixoto, Marcelo Barciela Brandão, Tania Maria Sih, Wilma Terezinha Anselmo‐Lima
Braz J Otorhinolaryngol 2017;83:498-506

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Impact of cartilage graft size on success of tympanoplasty

Waleed Abdelhameed, Ibrahim Rezk, Alhussein Awad
Braz J Otorhinolaryngol 2017;83:507-11

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Encoding of speech sounds at auditory brainstem level in good and poor hearing aid performers

Hemanth Narayan Shetty, Manjula Puttabasappa
Braz J Otorhinolaryngol 2017;83:512-22

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Profile and prevalence of hearing complaints in the elderly

Magda Aline Bauer, Ângela Kemel Zanella, Irênio Gomes Filho, Geraldo de Carli, Adriane Ribeiro Teixeira, Ângelo José Gonçalves Bós
Braz J Otorhinolaryngol 2017;83:523-9

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