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Κυριακή 1 Οκτωβρίου 2017

Feeling Sated Can Become a Cue to Eat More

When hunger pangs strike, we usually interpret them as a cue to reach for a snack; when we start to feel full, we take it as a sign that we should stop eating. But new research shows that these associations can be learned the other way around, such that satiety becomes a cue to eat more, not less.

The findings, published in Psychological Science, a journal of the Association for Psychological Science, suggest that internal, physical states themselves can serve as contexts that cue specific learned behaviors.

"We already know that extreme diets are susceptible to fail. One reason might be that the inhibition of eating learned while dieters are hungry doesn't transfer well to a non-hungry state," says psychological scientist Mark E. Bouton of the University of Vermont, one of the authors on the study. "If so, dieters might 'relapse' to eating, or perhaps overeating, when they feel full again."

To test this hypothesis, Bouton and co-author Scott T. Schepers conducted a behavioral conditioning study using 32 female Wistar rats as their participants.

Every day for 12 days, the rats – who were already satiated – participated in a 30-minute conditioning session. They were placed in a box that contained a lever and learned that they would receive tasty treats if they pressed that lever. Then, over the next 4 days, the rats were placed in the same box while they were hungry, and they discovered that lever presses no longer produced treats.

Through these two phases, the rats were conditioned to associate satiety with receiving tasty food and hunger with receiving no food. But what would the rats do if they were placed in the box again?

The results were clear: When the rats were tested again, they pressed the lever far more often if they were full than if they were hungry. In other words, they relapsed back to seeking treats.

"Rats that learned to respond for highly palatable foods while they were full and then inhibited their behavior while hungry, tended to relapse when they were full again," Bouton explains.

This relapse pattern emerged even when food was removed from the cage before both the learning and unlearning sessions, indicating that the rats' internal physical states, and not the presence or absence of food, cued their learned behavior.

Findings from three different studies supported the researchers' hypothesis that hunger and satiety could be learned as contextual cues in a classic ABA (sated-hungry-sated) renewal design. But the researchers found no evidence that an AAB design — in which the rats learned and subsequently inhibited the lever-treat association in a hungry state and were tested in a sated state – had any effect on the rats' lever pressing.

Together, these results show that seeking food and not seeking food are behaviors that are specific to the context in which they are learned. Although our body may drive food seeking behavior according to physiological needs, this research suggests that food-related behaviors can become associated with internal physical cues in ways that are divorced from our physiological needs.

"A wide variety of stimuli can come to guide and promote specific behaviors through learning. For example, the sights, sounds, and the smell of your favorite restaurant might signal the availability of your favorite food, causing your mouth to water and ultimately guiding you to eat," say Schepers and Bouton. "Like sights, sounds, and smells, internal sensations can also come to guide behavior, usually in adaptive and useful ways: We learn to eat when we feel hunger, and learn to drink when we feel thirst. However, internal stimuli such as hunger or satiety may also promote behavior in ways that are not so adaptive."

All data have been made publicly available via the Open Science Framework. The complete Open Practices Disclosure for this article is available online. This article has received the badge for Open Data.

This research was supported by National Institute on Drug Abuse Grant R01-DA033123 (to M.E. Bouton)



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Issue Information

Thumbnail image of graphical abstract

Cover of this issue. Radiation-resistant X60 cells contained increased amounts of mitochondria. See also Sato et al. (pages 2004-2010 of this issue).



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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 "ADCL," the mean of the lower ADC distribution. Pretreatment ADCL, enhancing volume, and clinical variables were tested as independent prognostic factors for OS.

Results: The coefficient of variance (COV) in double baseline ADCL measurements was 2.5% and did not significantly differ (P = 0.4537). An ADCL threshold of 1.24 μm2/ms produced the largest OS differences between patients (HR ~ 0.5), and patients with an ADCL > 1.24 μm2/ms had close to double the OS in all anti-VEGF therapeutic scenarios tested. Training and validation data confirmed that baseline ADCL 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 HER2mut nonamplified metastatic breast cancer (MBC). Secondary endpoints included progression-free survival (PFS), toxicity, and circulating tumor DNA (ctDNA) HER2mut detection.

Experimental Design: Tumor tissue positive for HER2mut 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 HER2mut).

Results: Nine of 381 tumors (2.4%) sequenced centrally harbored HER2mut (lobular 7.8% vs. ductal 1.6%; P = 0.026). Thirteen additional HER2mut cases were identified locally. Twenty-one of these 22 HER2mut 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 HER2mut 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 HER2mut variant allele frequency decreased in nine of 11 paired samples at week 4, followed by an increase upon progression.

Conclusions: Neratinib is active in HER2mut, nonamplified MBC. ctDNA sequencing offers a noninvasive strategy to identify patients with HER2mut 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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Explaining the Obesity Paradox--Response



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A Prospective Study of Chronic Inflammation in Benign Prostate Tissue and Risk of Prostate Cancer: Linked PCPT and SELECT Cohorts

Background: We leveraged two trials to test the hypothesis of an inflammation–prostate cancer link prospectively in men without indication for biopsy.

Methods: Prostate Cancer Prevention Trial (PCPT) participants who had an end-of-study biopsy performed per protocol that was negative for cancer and who subsequently enrolled in the Selenium and Vitamin E Cancer Prevention Trial (SELECT) were eligible. We selected all 100 cases and sampled 200 frequency-matched controls and used PCPT end-of-study biopsies as "baseline." Five men with PSA > 4 ng/mL at end-of-study biopsy were excluded. Tissue was located for 92 cases and 193 controls. We visually assessed inflammation in benign tissue. We estimated ORs and 95% confidence intervals (CI) using logistic regression adjusting for age and race.

Results: Mean time between biopsy and diagnosis was 5.9 years. In men previously in the PCPT placebo arm, 78.1% of cases (N = 41) and 68.2% of controls (N = 85) had at least one baseline biopsy core (~5 evaluated per man) with inflammation. The odds of prostate cancer (N = 41 cases) appeared to increase with increasing mean percentage of tissue area with inflammation, a trend that was statistically significant for Gleason sum <4+3 disease (N = 31 cases; vs. 0%, >0–<1.8% OR = 1.70, 1.8–<5.0% OR = 2.39, ≥5% OR = 3.31, Ptrend = 0.047). In men previously in the finasteride arm, prevalence of inflammation did not differ between cases (76.5%; N = 51) and controls (75.0%; N = 108).

Conclusions: Benign tissue inflammation was positively associated with prostate cancer.

Impact: This first prospective study of men without biopsy indication supports the hypothesis that inflammation influences prostate cancer development. Cancer Epidemiol Biomarkers Prev; 26(10); 1549–57. ©2017 AACR.



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Associations of Coffee Drinking and Cancer Mortality in the Cancer Prevention Study-II

Background: Associations of coffee consumption with cancer mortality are inconsistent for many types of cancer, and confounding by smoking is an important concern.

Methods: Cox proportional hazards regression was used to estimate multivariable-adjusted HRs for coffee consumption associated with death from all cancers combined and from specific cancer types among 922,896 Cancer Prevention Study-II participants ages 28–94 years who completed a four-page questionnaire and were cancer free at baseline in 1982.

Results: During follow-up through 2012, there were 118,738 cancer-related deaths. There was a nonlinear association between coffee consumption and all-cancer death among current smokers and former smokers and no association among never smokers. Among nonsmokers, a 2 cup/day increase in coffee consumption was inversely associated with death from colorectal [HR = 0.97; 95% confidence interval (CI) 0.95–0.99], liver [HR = 0.92; 95% CI, 0.88–0.96], and female breast (HR = 0.97; 95% CI, 0.94–0.99) cancers, and positively associated with esophageal cancer–related death (HR = 1.07; 95% CI, 1.02–1.12). For head and neck cancer, a nonlinear inverse association was observed starting at 2–3 cups per day (HR = 0.72; 95% CI, 0.55–0.95), with similar associations observed at higher levels of consumption.

Conclusions: These findings are consistent with many other studies that suggest coffee drinking is associated with a lower risk of colorectal, liver, female breast, and head and neck cancer. The association of coffee consumption with higher risk of esophageal cancer among nonsmokers in our study should be confirmed.

Impact: These results underscore the importance of assessing associations between coffee consumption and cancer mortality by smoking status. Cancer Epidemiol Biomarkers Prev; 26(10); 1477–86. ©2017 AACR.



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Novel Gene and Network Associations Found for Acute Lymphoblastic Leukemia Using Case-Control and Family-Based Studies in Multiethnic Populations

Background: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, suggesting that germline variants influence ALL risk. Although multiple genome-wide association (GWA) studies have identified variants predisposing children to ALL, it remains unclear whether genetic heterogeneity affects ALL susceptibility and how interactions within and among genes containing ALL-associated variants influence ALL risk.

Methods: Here, we jointly analyzed two published datasets of case–control GWA summary statistics along with germline data from ALL case–parent trios. We used the gene-level association method PEGASUS to identify genes with multiple variants associated with ALL. We then used PEGASUS gene scores as input to the network analysis algorithm HotNet2 to characterize the genomic architecture of ALL.

Results: Using PEGASUS, we confirmed associations previously observed at genes such as ARID5B, IKZF1, CDKN2A/2B, and PIP4K2A, and we identified novel candidate gene associations. Using HotNet2, we uncovered significant gene subnetworks that may underlie inherited ALL risk: a subnetwork involved in B-cell differentiation containing the ALL-associated gene CEBPE, and a subnetwork of homeobox genes, including MEIS1.

Conclusions: Gene and network analysis uncovered loci associated with ALL that are missed by GWA studies, such as MEIS1. Furthermore, ALL-associated loci do not appear to interact directly with each other to influence ALL risk, and instead appear to influence leukemogenesis through multiple, complex pathways.

Impact: We present a new pipeline for post hoc analysis of association studies that yields new insight into the etiology of ALL and can be applied in future studies to shed light on the genomic underpinnings of cancer. Cancer Epidemiol Biomarkers Prev; 26(10); 1531–9. ©2017 AACR.



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The Association of Mammographic Density and Molecular Breast Cancer Subtype

Background: Mammographic density (MD) is associated with increased breast cancer risk, yet limited data exist on an association between MD and breast cancer molecular subtypes.

Methods: Women ages 18 years and older with breast cancer and available mammograms between 2003 and 2012 were enrolled in a larger study on MD. MD was classified by the Breast Imaging Reporting and Data System (BI-RADS) classification and by volumetric breast percent density (Volpara Solutions). Subtype was assigned by hormone receptor status, tumor grade, and mitotic score (MS). Subtypes included: Luminal-A (ER/PR+ and grade = 1; ER/PR+ and grade = 2 and MS = 1; ER+/PR and grade = 1; n = 233); Luminal-B (ER+ and grade = 3 or MS = 3; ER+/PR and grade = 2; ER/PR+ and grade = 2 and MS = 2; n = 79); Her-2-neu+ (H2P; n = 59); triple-negative (ER/PR, Her-2; n = 86). Precancer factors including age, race, body mass index (kg/m2), family history of breast cancer, and history of lobular carcinoma in situ were analyzed.

Results: A total of 604 patients had invasive cancer; 457 had sufficient information for analysis. Women with H2P tumors were younger (P = 0.011) and had the highest volumetric percent density (P = 0.002) among subgroups. Multinomial logistic regression (LA = reference) demonstrated that although quantitative MD does not significantly differentiate between all subtypes (P = 0.123), the association between MD and H2P tumors is significant (OR = 1.06; confidence interval, 1.01–1.12). This association was not seen using BI-RADS classification in bivariable analysis but was statistically significant (P = 0.047) when controlling for other precancer factors.

Conclusions: Increased MD is more strongly associated with H2P tumors when compared with LA.

Impact: Delineating risk factors specific to breast cancer subtype may promote development of individualized risk prediction models and screening strategies. Cancer Epidemiol Biomarkers Prev; 26(10); 1487–92. ©2017 AACR.



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The Prevalence of Cancer-Associated Autoantibodies in Patients with Gastric Cancer and Progressive Grades of Premalignant Lesions

Background: Serum autoantibodies against tumor-associated antigens (TAAs) are detectable in early-stage gastric cancer patients; however, the time point during cancerogenesis when they appear in circulation is still obscure.

Methods: In this study, we developed a recombinant antigen microarray and analyzed the prevalence of autoantibodies against 102 TAAs in 829 gastric cancer patients and 929 healthy controls from Caucasian and Asian populations, as well as 100 patients with chronic atrophic gastritis and 775 individuals staged according to different grades of intestinal metaplasia.

Results: Six antigens, including CTAG1B/CTAG2, DDX53, IGF2BP2, TP53, and MAGEA3, were predominantly reacting with sera from gastric cancer patients when compared with healthy controls, and the seroreactivity was associated with intestinal-type gastric cancer, but not with patients' Helicobacter pylori status, grade, age, gender, or stage of gastric cancer. We detected gastric cancer–associated seroreactivity in 13% of patients with advanced/severe intestinal metaplasia, which was increased in comparison with mild/moderate intestinal metaplasia (5.3%) and was comparable with that seen in early-stage gastric cancer patients (12%). Moreover, by testing serum samples taken 1 to 9 years before the clinical diagnosis of 18 incident gastric cancer cases, we detected autoantibody responses against several TAAs—SOX2, MYC, BIRC5, IGF2BP1, and MUC1.

Conclusions: Our results suggest that humoral immune response against TAAs is generated already during premalignant stages.

Impact: Based on the obtained results, cancer-associated autoantibodies might make a valuable contribution to the stratification of high-risk patients with premalignant lesions in the stomach through enhancing the positive predictive power of existing risk models. Cancer Epidemiol Biomarkers Prev; 26(10); 1564–74. ©2017 AACR.



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Cervical Cancer Screening among Women from Muslim-Majority Countries in Ontario, Canada

Background: Immigrant women are less likely to be screened for cervical cancer in Ontario. Religion may play a role for some women. In this population-based retrospective cohort study, we used country of birth as a proxy for religious affiliation and examined screening uptake among foreign-born women from Muslim-majority versus other countries, stratified by region of origin.

Methods: We linked provincial databases and identified all women eligible for cervical cancer screening between April 1, 2012, and March 31, 2015. Women were classified into regions based on country of birth. Countries were classified as Muslim-majority or not.

Results: Being born in a Muslim-majority country was significantly associated with lower likelihood of being up-to-date on Pap testing, after adjustment for region of origin, neighborhood income, and primary care–related factors [adjusted relative risk (ARR), 0.93; 95% (confidence interval) CI, 0.92–0.93]. Sub-Saharan African women from Muslim-majority countries had the highest prevalence of being overdue (59.6%), and the lowest ARR for screening when compared with women from non–Muslim-majority Sub-Saharan African countries (ARR, 0.77; 95% CI, 0.76–0.79). ARRs were lowest for women with no primary care versus those in a capitation-based model (ARR, 0.28; 95% CI, 0.27–0.29 overall).

Conclusions: We have shown that being born in a Muslim-majority country is associated with a decreased likelihood of being up-to-date on cervical screening in Ontario and that access to primary care has a sizeable impact on screening uptake.

Impact: Screening efforts need to take into account the background characteristics of population subgroups and to focus on increasing primary care access for all. Cancer Epidemiol Biomarkers Prev; 26(10); 1493–9. ©2017 AACR.



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Highlights of This Issue



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The Cost-Effectiveness of Visual Triage of Human Papillomavirus-Positive Women in Three Low- and Middle-Income Countries

Background: World Health Organization guidelines support human papillomavirus (HPV) testing alone (followed by treatment with cryotherapy) or in conjunction with visual inspection with acetic acid (VIA) triage testing. Our objective was to determine the cost-effectiveness of VIA triage for HPV-positive women in low-resource settings.

Methods: We calibrated mathematical simulation models of HPV infection and cervical cancer to epidemiologic data from India, Nicaragua, and Uganda. Using cost and test performance data from the START-UP demonstration projects, we assumed screening took place either once or three times in a lifetime between ages 30 and 40 years. Strategies included (i) HPV alone, followed by cryotherapy for all eligible HPV-positive women; and (ii) HPV testing with VIA triage for HPV-positive women, followed by cryotherapy for eligible women who were also VIA-positive (HPV-VIA). Model outcomes included lifetime risk of cervical cancer and incremental cost-effectiveness ratios (ICERs; international dollars/year of life saved).

Results: In all three countries, HPV alone was more effective than HPV-VIA. In Nicaragua and Uganda, HPV alone was also less costly than HPV-VIA; ICERs associated with screening three times in a lifetime (HPV alone) were below per capita GDP. In India, both HPV alone and HPV-VIA had ICERs below per capita GDP.

Conclusions: VIA triage of HPV-positive women is not likely to be cost-effective in settings with high cervical cancer burden. HPV alone followed by treatment may achieve greater health benefits and value for public health dollars.

Impact: This study provides early evidence on the cost-effectiveness of HPV testing followed by VIA triage versus an HPV screen-and-treat strategy. Cancer Epidemiol Biomarkers Prev; 26(10); 1500–10. ©2017 AACR.



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Impact of Sixteen Established Pancreatic Cancer Susceptibility Loci in American Jews

Background: The higher risk of pancreatic cancer in Ashkenazi Jews compared with non-Jews is only partially explained by the increased frequency of BRCA1 and BRCA2 mutations in Ashkenazi Jews.

Methods: We evaluated the impact of 16 established pancreatic cancer susceptibility loci in a case–control sample of American Jews, largely Ashkenazi, including 406 full-Jewish pancreatic cancer patients and 2,332 full-Jewish controls, genotyped as part of the Pancreatic Cancer Cohort and Case–Control Consortium I/II (PanScan I/II), Pancreatic Cancer Case-Control Consortium (PanC4), and Resource for Genetic Epidemiology Research on Adult Health and Aging (GERA) datasets. We compared risk in full-Jewish subjects with risk in part-Jewish; non-Jewish Southern European; and in the combined non-Jewish Eastern, Northern, Southern, and Western European (non-Jewish white European) subjects from the same datasets. Jewish ancestries were genetically identified using seeded Fast principal component analysis. Data were analyzed by unconditional logistic regression, and adjusted for age, sex, and principal components.

Results: One SNP on chromosome 13q22.1 (rs9543325; OR, 1.36; 95% confidence interval, 1.16–1.58; P = 10–4.1) was significant in full-Jews. Individual ORs and minor allele frequencies were similar between Jewish and non-Jewish white European subjects. The average ORs across the 16 pancreatic cancer susceptibility loci for full-Jewish, full- plus part-Jewish, non-Jewish Southern European, and non-Jewish white European subjects were 1.25, 1.30, 1.31, and 1.26, respectively.

Conclusions: The 16 pancreatic cancer susceptibility loci similarly impact Jewish and non-Jewish white European subjects, both individually and as summary odds.

Impact: These 16 pancreatic cancer susceptibility loci likely do not explain the higher risk seen in Ashkenazi Jews. Cancer Epidemiol Biomarkers Prev; 26(10); 1540–8. ©2017 AACR.



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Recent Trends in Ovarian Cancer Incidence and Relative Survival in the United States by Race/Ethnicity and Histologic Subtypes

Background: Incidence and survival rates of nonserous epithelial ovarian cancer in racial/ethnic minorities remain relatively unknown in the United States. We examined the trends in incidence and survival rates for epithelial ovarian cancer by histologic subtypes and race/ethnicity.

Methods: Ovarian cancer incidence and mortality data from 2000 to 2013 were obtained from the Surveillance, Epidemiology, and End Results database. Age-adjusted incidence rate, incidence rate ratio, and annual percentage changes (APC) were calculated by histology and race/ethnicity subgroups and stratified by age at diagnosis. Five-year relative survival rates were calculated by stage and race/ethnicity.

Results: A small but significant decrease in incidence rates was seen in non-Hispanic white (NHW), non-Hispanic black (NHB), and Hispanic women (APC –1.58, –0.84, and –1.31, respectively), while incidence rates remained relatively stable in Asian women (APC –0.37). With exception of significant increase in the incidence rate of clear cell carcinoma among Asian woman (APC 1.85), an overall trend toward decreasing incidence rates was seen across histologic subtypes and age-strata, although not all results were statistically significant. Compared with NHW women, NHB women experienced poorer 5-year survival at every stage across histologic subtypes, while Hispanic and Asian women had equivalent or better survival.

Conclusions: Over the last decade, incidence rates of epithelial ovarian cancer in the United States have decreased or remained stable across race/ethnic and histologic subgroups, except for clear cell carcinoma. Survival remains poorest among NHB women.

Impact: Comparative histologic subtype distribution and incidence trends do not explain the ovarian cancer survival disparity disproportionately affecting NHB women. Cancer Epidemiol Biomarkers Prev; 26(10); 1511–8. ©2017 AACR.



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Mediating Effect of Postsurgical Chemotherapy on Presence of Dementia and Survival among Patients 65 and Older with Stage III Colon Cancer

Introduction: Both colon cancer and dementia are prevalent among the elderly and have a high risk of cooccurrence. Previous studies found that patients with dementia were treated less aggressively. In this study, we hypothesized that presence of preexisting dementia was associated with worse survival for stage III colon cancer patients, and that postoperative chemotherapy was on the causal pathway.

Methods: We defined preexisting dementia in Surveillance Epidemiology and End Results Medicare data through either a formal diagnosis or a prescription for dementia drugs or both before the diagnosis of cancer. We applied multivariable Cox regression to estimate the effect of preexisting dementia on survival, adjusting for demographic factors, tumor characteristics, and receipt of chemotherapy. We assessed mediating effects in the context of the counterfactual framework using the accelerated failure time model.

Results: There were 4,573 patients diagnosed with stage III colon cancer between 2007 and 2009 identified. A preexisting diagnosis of dementia significantly increased the risk of death by 45% (HR = 1.45, 95% CI: 1.29–1.63). Patients with either a formal diagnosis of dementia or a related prescription had significantly lower cause-specific survival than their cognitively healthy counterparts. Receipt of chemotherapy was a significant mediator on the causal pathway. The effect of presence of dementia was mediated by receipt of chemotherapy by 13% for preexisting dementia.

Conclusions: Preexisting dementia is significantly associated with worse survival for stage III colon cancer patients, and its deleterious effect is partially explained by decreased likelihood of postoperative chemotherapy receipt.

Impact: This is the first study that provides estimate of the mediating effect of diminished chemotherapy in patients with stage III colon cancer and dementia, simultaneously demonstrating the cancer-specific survival benefit of chemotherapy in the presence of dementia. Cancer Epidemiol Biomarkers Prev; 26(10); 1558–63. ©2017 AACR.



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Serum Insulin, Glucose, Indices of Insulin Resistance, and Risk of Lung Cancer

Background: Although insulin may increase the risk of some cancers, few studies have examined fasting serum insulin and lung cancer risk.

Methods: We examined serum insulin, glucose, and indices of insulin resistance [insulin:glucose molar ratio and homeostasis model assessment of insulin resistance (HOMA-IR)] and lung cancer risk using a case-cohort study within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study of Finnish men. A total of 196 cases and 395 subcohort members were included. Insulin and glucose were measured in fasting serum collected 5 to 12 years before diagnosis. Cox proportional hazards models were utilized to estimate the relative risk of lung cancer.

Results: The average time between blood collection and lung cancer was 9.6 years. Fasting serum insulin levels were 8.7% higher in subcohort members than cases. After multivariable adjustment, men in the fourth quartile of insulin had a significantly higher risk of lung cancer than those in the first quartile [HR = 2.10; 95% confidence interval (CI), 1.12–3.94]. A similar relationship was seen with HOMA-IR (HR = 1.83; 95% CI, 0.99–3.38). Risk was not strongly associated with glucose or the insulin:glucose molar ratio (Ptrend = 0.55 and Ptrend = 0.27, respectively).

Conclusions: Higher fasting serum insulin concentrations, as well as the presence of insulin resistance, appear to be associated with an elevated risk of lung cancer development.

Impact: Although insulin is hypothesized to increase risk of some cancers, insulin and lung cancer remain understudied. Higher insulin levels and insulin resistance were associated with increased lung cancer risk. Although smoking cessation is the best method of lung cancer prevention, other lifestyle changes that affect insulin concentrations and sensitivity may reduce lung cancer risk. Cancer Epidemiol Biomarkers Prev; 26(10); 1519–24. ©2017 AACR.



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Explaining the Obesity Paradox--Letter



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Tobacco Product Use Patterns, and Nicotine and Tobacco-Specific Nitrosamine Exposure: NHANES 1999-2012

Background: Few studies have examined differences in product consumption patterns and nicotine and tobacco-specific nitrosamines (TSNA) exposure between single versus dual- and poly-tobacco users. We applied the Tobacco Product Use Patterns (T-PUPs) model to fill this gap in the literature.

Methods: Data from adults (age ≥18 years) who used any tobacco products during the 5 days prior to participating in the 1999–2012 National Health and Nutrition Examination Survey (NHANES) were analyzed. Participants were classified into seven T-PUPs: (1) cigarettes only, (2) noncigarette combustibles only, (3) noncombustibles only, (4) dual noncigarette combustibles and noncombustibles, (5) dual cigarettes and noncombustibles, (6) dual cigarettes and noncigarette combustibles, and (7) poly-tobacco use. Weighted regression models were used to compare product consumption, serum cotinine, and urinary total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (i.e., NNAL) levels between single-, dual-, and poly-tobacco T-PUPs.

Results: Dual- and poly-tobacco T-PUPs were associated with lower product consumption compared with single-product T-PUPs only in some cases (e.g., dual cigarette and noncombustible users smoked cigarettes on 0.6 fewer days in the past 5 days compared with cigarette-only users; P < 0.05). Dual- and poly-tobacco T-PUPs had either nondistinguishable or higher levels of serum cotinine and urinary total NNAL than corresponding single-product T-PUPs.

Conclusions: Product consumption, and nicotine and TSNAs exposure of dual- and poly-tobacco product category users somewhat differ from those of single-product category users as defined by the T-TUPs model.

Impact: Higher levels of cotinine and NNAL among dual- and poly-tobacco T-TUPs users compared with the single-product T-TUPs users may indicate health concerns. Cancer Epidemiol Biomarkers Prev; 26(10); 1525–30. ©2017 AACR.



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Cigarette Prices and Smoking Cessation--Letter



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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.



http://ift.tt/2yCVugy

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.

http://ift.tt/2kdlmNP

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.

http://ift.tt/2yDuayN

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.

http://ift.tt/2ki3T74

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.

http://ift.tt/2yDjjF6

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.

http://ift.tt/2kgr8hx

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.

http://ift.tt/2yCVq0i

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



http://ift.tt/2xR96qR

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.

http://ift.tt/2g1uGzM

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.

http://ift.tt/2xR2PM1

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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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.

http://ift.tt/2g3nO51

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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