Superenhancers Drive Neuroblastoma Differentiation States [Neuroblastoma]

Superenhancer-associated transcriptional networks promote neuroblastoma heterogeneity.

http://ift.tt/2eu6zwb

Transcription Elongation Factors Are Potential Targets in Glioblastoma [Glioblastoma]

JMJD6 promotes transcription pause-release and elongation and glioblastoma cell survival in vivo.

http://ift.tt/2gnEF1I

Epacadostat Shows Value in Two SCCHN Trials [News in Brief]

In the ECHO-202/KEYNOTE-037 and ECHO-204 trials reported at the 2017 Annual Meeting of the American Society of Clinical Oncology, patients with squamous cell carcinoma of the head and neck responded well to the combinations of epacadostat plus pembrolizumab and epacadostat plus nivolumab. An IDO1 inhibitor, epacadostat also demonstrated promising activity in combination with the PD-1 checkpoint inhibitors in other solid tumors, including melanoma, urothelial carcinoma, renal cell carcinoma, and non–small cell lung cancer.

http://ift.tt/2eJEgqs

GPX4 Blocks Ferroptosis to Drive the Survival of Chemoresistant Cells [Drug Resistance]

The lipid peroxidase GPX4 is critical for the survival of therapy-resistant ZEB1⁺ cancer cells.

http://ift.tt/2guvC2D

New Biomarker Identified for PDAC [News in Brief]

A new study suggests that the protein THBS2 could lead to early detection of pancreatic ductal adenocarcinoma. By testing for THBS2 and another marker, CA19-9, researchers identified blood samples from patients with the disease with 98% specificity and 87% sensitivity.

http://ift.tt/2eJJRNk

Integrative Genomics Characterize Medulloblastoma Subtypes [Medulloblastoma]

Integrative genomic analysis characterized the genomic landscapes of medulloblastoma subtypes.

http://ift.tt/2gtPoeR

Savolitinib Heads for Phase III Trial in PRCC [News in Brief]

A phase II trial of savolitinib, a MET inhibitor, found that the drug induced partial responses in some patients with papillary renal cell carcinoma and was well tolerated, prompting drug makers Chi-Med and AstraZeneca to launch a phase III study.

http://ift.tt/2eJMTBo

Disrupting the APC-Asef Interaction Suppresses Cancer Cell Migration [Drug Design]

Structure-based design yields peptidomimetic APC–ARM pocket inhibitors that block Asef binding.

http://ift.tt/2guzsZG

New SNPs from Testicular Cancer GWAS [News in Brief]

Two new genome-wide association studies identify 30 new risk variants for testicular cancer. One study suggests that genes involved in transcription regulation during development and microtubule assembly promote development of the disease. The second study implicates genes involved in mitochondrial metabolism, germ cell maturation, and DNA damage repair.

http://ift.tt/2eJMSxk

Biallelic Germline Mutations in RFWD3 May Induce Fanconi Anemia [Fanconi Anemia]

The E3 ubiquitin ligase RFWD3 is mutated in a patient with Fanconi anemia lacking known Fanconi mutations.

http://ift.tt/2wnlLRK

Genomic Analysis Detects Recurrent Promoter Mutations in Breast Cancer [Breast Cancer]

Deep sequencing of 360 primary breast tumors identified 9 genes with recurrently mutated promoters.

http://ift.tt/2vPsq3K

CAMKII{gamma} Stabilizes MYC to Promote T-cell Lymphomagenesis [Lymphoma]

Inhibition of CAMKII reduces MYC protein levels, T-cell lymphomagenesis, and tumor burden in vivo.

http://ift.tt/2wn4HeC

Nivolumab Plus Ipilimumab Has Antitumor Activity in Metastatic RCC [Clinical Trials]

Nivolumab plus ipilimumab achieves durable responses and has manageable safety in metastatic RCC.

http://ift.tt/2vOs4uj

FOXA1-Dependent Enhancer Reprogramming Promotes Metastasis [Metastasis]

Recurrent enhancer changes accompany the metastatic transition in pancreatic cancer models.

http://ift.tt/2wnq9An

Discovery and Optimization of HKT288, a Cadherin-6-Targeting ADC for the Treatment of Ovarian and Renal Cancers [Research Articles]

Despite an improving therapeutic landscape, significant challenges remain in treating the majority of patients with advanced ovarian or renal cancer. We identified the cell–cell adhesion molecule cadherin-6 (CDH6) as a lineage gene having significant differential expression in ovarian and kidney cancers. HKT288 is an optimized CDH6-targeting DM4-based antibody–drug conjugate (ADC) developed for the treatment of these diseases. Our study provides mechanistic evidence supporting the importance of linker choice for optimal antitumor activity and highlights CDH6 as an antigen for biotherapeutic development. To more robustly predict patient benefit of targeting CDH6, we incorporate a population-based patient-derived xenograft (PDX) clinical trial (PCT) to capture the heterogeneity of response across an unselected cohort of 30 models—a novel preclinical approach in ADC development. HKT288 induces durable tumor regressions of ovarian and renal cancer models in vivo, including 40% of models on the PCT, and features a preclinical safety profile supportive of progression toward clinical evaluation.

Significance: We identify CDH6 as a target for biotherapeutics development and demonstrate how an integrated pharmacology strategy that incorporates mechanistic pharmacodynamics and toxicology studies provides a rich dataset for optimizing the therapeutic format. We highlight how a population-based PDX clinical trial and retrospective biomarker analysis can provide correlates of activity and response to guide initial patient selection for first-in-human trials of HKT288. Cancer Discov; 7(9); 1030–45. ©2017 AACR.

This article is highlighted in the In This Issue feature, p. 920

http://ift.tt/2vOKI5b

Analysis of Circulating Cell-Free DNA Identifies Multiclonal Heterogeneity of BRCA2 Reversion Mutations Associated with Resistance to PARP Inhibitors [Research Briefs]

Approximately 20% of metastatic prostate cancers harbor mutations in genes required for DNA repair by homologous recombination repair (HRR) such as BRCA2. HRR defects confer synthetic lethality to PARP inhibitors (PARPi) such as olaparib and talazoparib. In ovarian or breast cancers, olaparib resistance has been associated with HRR restoration, including by BRCA2 mutation reversion. Whether similar mechanisms operate in prostate cancer, and could be detected in liquid biopsies, is unclear. Here, we identify BRCA2 reversion mutations associated with olaparib and talazoparib resistance in patients with prostate cancer. Analysis of circulating cell-free DNA (cfDNA) reveals reversion mutation heterogeneity not discernable from a single solid-tumor biopsy and potentially allows monitoring for the emergence of PARPi resistance.

Significance: The mechanisms of clinical resistance to PARPi in DNA repair–deficient prostate cancer have not been described. Here, we show BRCA2 reversion mutations in patients with prostate cancer with metastatic disease who developed resistance to talazoparib and olaparib. Furthermore, we show that PARPi resistance is highly multiclonal and that cfDNA allows monitoring for PARPi resistance. Cancer Discov; 7(9); 999–1005. ©2017 AACR.

See related commentary by Domchek, p. 937.

See related article by Kondrashova et al., p. 984.

See related article by Goodall et al., p. 1006.

This article is highlighted in the In This Issue feature, p. 920

http://ift.tt/2wnyxQe

Secondary Somatic Mutations Restoring RAD51C and RAD51D Associated with Acquired Resistance to the PARP Inhibitor Rucaparib in High-Grade Ovarian Carcinoma [Research Briefs]

High-grade epithelial ovarian carcinomas containing mutated BRCA1 or BRCA2 (BRCA1/2) homologous recombination (HR) genes are sensitive to platinum-based chemotherapy and PARP inhibitors (PARPi), while restoration of HR function due to secondary mutations in BRCA1/2 has been recognized as an important resistance mechanism. We sequenced core HR pathway genes in 12 pairs of pretreatment and postprogression tumor biopsy samples collected from patients in ARIEL2 Part 1, a phase II study of the PARPi rucaparib as treatment for platinum-sensitive, relapsed ovarian carcinoma. In 6 of 12 pretreatment biopsies, a truncation mutation in BRCA1, RAD51C, or RAD51D was identified. In five of six paired postprogression biopsies, one or more secondary mutations restored the open reading frame. Four distinct secondary mutations and spatial heterogeneity were observed for RAD51C. In vitro complementation assays and a patient-derived xenograft, as well as predictive molecular modeling, confirmed that resistance to rucaparib was associated with secondary mutations.

Significance: Analyses of primary and secondary mutations in RAD51C and RAD51D provide evidence for these primary mutations in conferring PARPi sensitivity and secondary mutations as a mechanism of acquired PARPi resistance. PARPi resistance due to secondary mutations underpins the need for early delivery of PARPi therapy and for combination strategies. Cancer Discov; 7(9); 984–98. ©2017 AACR.

See related commentary by Domchek, p. 937.

See related article by Quigley et al., p. 999.

See related article by Goodall et al., p. 1006.

This article is highlighted in the In This Issue feature, p. 920

http://ift.tt/2vOJCX5

Circulating Cell-Free DNA to Guide Prostate Cancer Treatment with PARP Inhibition [Research Articles]

Biomarkers for more precise patient care are needed in metastatic prostate cancer. We have reported a phase II trial (TOPARP-A) of the PARP inhibitor olaparib in metastatic prostate cancer, demonstrating antitumor activity associating with homologous recombination DNA repair defects. We now report targeted and whole-exome sequencing of serial circulating cell-free DNA (cfDNA) samples collected during this trial. Decreases in cfDNA concentration independently associated with outcome in multivariable analyses (HR for overall survival at week 8: 0.19; 95% CI, 0.06–0.56; P = 0.003). All tumor tissue somatic DNA repair mutations were detectable in cfDNA; allele frequency of somatic mutations decreased selectively in responding patients (²P < 0.001). At disease progression, following response to olaparib, multiple subclonal aberrations reverting germline and somatic DNA repair mutations (BRCA2, PALB2) back in frame emerged as mechanisms of resistance. These data support the role of liquid biopsies as a predictive, prognostic, response, and resistance biomarker in metastatic prostate cancer.

Significance: We report prospectively planned, serial, cfDNA analyses from patients with metastatic prostate cancer treated on an investigator-initiated phase II trial of olaparib. These analyses provide predictive, prognostic, response, and resistance data with "second hit" mutations first detectable at disease progression, suggesting clonal evolution from treatment-selective pressure and platinum resistance. Cancer Discov; 7(9); 1006–17. ©2017 AACR.

See related commentary by Domchek, p. 937.

See related article by Kondrashova et al., p. 984.

See related article by Quigley et al., p. 999.

This article is highlighted in the In This Issue feature, p. 920

http://ift.tt/2wnyu70

A Next-Generation TRK Kinase Inhibitor Overcomes Acquired Resistance to Prior TRK Kinase Inhibition in Patients with TRK Fusion-Positive Solid Tumors [Research Briefs]

Larotrectinib, a selective TRK tyrosine kinase inhibitor (TKI), has demonstrated histology-agnostic efficacy in patients with TRK fusion–positive cancers. Although responses to TRK inhibition can be dramatic and durable, duration of response may eventually be limited by acquired resistance. LOXO-195 is a selective TRK TKI designed to overcome acquired resistance mediated by recurrent kinase domain (solvent front and xDFG) mutations identified in multiple patients who have developed resistance to TRK TKIs. Activity against these acquired mutations was confirmed in enzyme and cell-based assays and in vivo tumor models. As clinical proof of concept, the first 2 patients with TRK fusion–positive cancers who developed acquired resistance mutations on larotrectinib were treated with LOXO-195 on a first-in-human basis, utilizing rapid dose titration guided by pharmacokinetic assessments. This approach led to rapid tumor responses and extended the overall duration of disease control achieved with TRK inhibition in both patients.

Significance: LOXO-195 abrogated resistance in TRK fusion–positive cancers that acquired kinase domain mutations, a shared liability with all existing TRK TKIs. This establishes a role for sequential treatment by demonstrating continued TRK dependence and validates a paradigm for the accelerated development of next-generation inhibitors against validated oncogenic targets. Cancer Discov; 7(9); 963–72. ©2017 AACR.

See related commentary by Parikh and Corcoran, p. 934.

This article is highlighted in the In This Issue feature, p. 920

http://ift.tt/2vON3gf

New Horizons for Precision Medicine in Biliary Tract Cancers [Review]

Biliary tract cancers (BTC), including cholangiocarcinoma and gallbladder cancer, are poor-prognosis and low-incidence cancers, although the incidence of intrahepatic cholangiocarcinoma is rising. A minority of patients present with resectable disease but relapse rates are high; benefit from adjuvant capecitabine chemotherapy has been demonstrated. Cisplatin/gemcitabine combination chemotherapy has emerged as the reference first-line treatment regimen; there is no standard second-line therapy. Selected patients may be suitable for liver-directed therapy (e.g., radioembolization or external beam radiation), pending confirmation of benefit in randomized studies. Initial trials targeting the epithelial growth factor receptor and angiogenesis pathways have failed to deliver new treatments. Emerging data from next-generation sequencing analyses have identified actionable mutations (e.g., FGFR fusion rearrangements and IDH1 and IDH2 mutations), with several targeted drugs entering clinical development with encouraging results. The role of systemic therapies, including targeted therapies and immunotherapy for BTC, is rapidly evolving and is the subject of this review.

Significance: The authors address genetic drivers and molecular biology from a translational perspective, in an intent to offer a clear view of the recent past, present, and future of BTC. The review describes a state-of-the-art update of the current status and future directions of research and therapy in advanced BTC. Cancer Discov; 7(9); 943–62. ©2017 AACR.

http://ift.tt/2wnyrIm

HER2 Reactivation through Acquisition of the HER2 L755S Mutation as a Mechanism of Acquired Resistance to HER2-targeted Therapy in HER2+ Breast Cancer

Purpose: Resistance to anti-HER2 therapies in HER2⁺ breast cancer can occur through activation of alternative survival pathways or reactivation of the HER signaling network. Here we employed BT474 parental and treatment-resistant cell line models to investigate a mechanism by which HER2⁺ breast cancer can reactivate the HER network under potent HER2-targeted therapies.

Experimental Design: Resistant derivatives to lapatinib (L), trastuzumab (T), or the combination (LR/TR/LTR) were developed independently from two independent estrogen receptor ER⁺/HER2⁺ BT474 cell lines (AZ/ATCC). Two derivatives resistant to the lapatinib-containing regimens (BT474/AZ-LR and BT474/ATCC-LTR lines) that showed HER2 reactivation at the time of resistance were subjected to massive parallel sequencing and compared with parental lines. Ectopic expression and mutant-specific siRNA interference were applied to analyze the mutation functionally. In vitro and in vivo experiments were performed to test alternative therapies for mutant HER2 inhibition.

Results: Genomic analyses revealed that the HER2L755S mutation was the only common somatic mutation gained in the BT474/AZ-LR and BT474/ATCC-LTR lines. Ectopic expression of HER2L755S induced acquired lapatinib resistance in the BT474/AZ, SK-BR-3, and AU565 parental cell lines. HER2L755S-specific siRNA knockdown reversed the resistance in BT474/AZ-LR and BT474/ATCC-LTR lines. The HER1/2–irreversible inhibitors afatinib and neratinib substantially inhibited both resistant cell growth and the HER2 and downstream AKT/MAPK signaling driven by HER2L755S in vitro and in vivo.

Conclusions: HER2 reactivation through acquisition of the HER2L755S mutation was identified as a mechanism of acquired resistance to lapatinib-containing HER2-targeted therapy in preclinical HER2-amplified breast cancer models, which can be overcome by irreversible HER1/2 inhibitors. Clin Cancer Res; 23(17); 5123–34. ©2017 AACR.

http://ift.tt/2vOFgzj

The Novel Association of Circulating Tumor Cells and Circulating Megakaryocytes with Prostate Cancer Prognosis

Purpose: To develop an approach for the investigation of different subtypes of circulating tumor cells (CTC) and other cells to evaluate their potential prognostic value of prostate cancer.

Experimental Design: Malignancy of CTCs undergoing epithelial-to-mesenchymal transition (EMT) was confirmed by repeated FISH. Subgroups of CTCs in 81 patients with prostate cancer (43 castration resistant and 38 untreated localized) were correlated to disease aggressiveness parameters. AUC analysis was applied to compare the performance for metastasis prediction between serum PSA level alone and a combined risk score using both PSA and EMTing CTC count. Circulating megakaryocytes and cancer patient survival association was performed using Cox model.

Results: The majority of vimentin (VIM)⁺/CD45^– cells were malignant, with genomic alterations in several genomic regions. The number of cytokeratin (CK)^–/VIM⁺/CD45^– CTCs correlated with disease burden, tumor aggressiveness, and poorer survival. Meanwhile, CK⁺/VIM⁺/CD45^– CTCs were associated with metastases better than other subtypes of CTCs in these limited samples. Combination of PSA level and the number of CK⁺/VIM⁺/CD45^– CTCs enhanced the prediction of cancer metastases [AUC, 0.921; 95% confidence interval (CI), 0.858–0.985]. The number of circulating megakaryocytes was potentially associated with good patient survival in advanced prostate cancer (HR, 0.849; 95% CI, 0.628–1.146, per cell increase), and the difference between the number of mesenchymal CTCs and megakaryocytes strongly correlated to poor survival (HR, 10.17; 95% CI, 2.164–47.789, if score ≥2.0).

Conclusions: This CTC analysis approach and the potential association of megakaryocytes with cancer prognosis may greatly enhance our ability to investigate the cancer metastasis process and to predict/monitor cancer progression. Clin Cancer Res; 23(17); 5112–22. ©2017 AACR.

http://ift.tt/2wrIfPX

Retraction: Polymorphisms of the CYP1B1 Gene as Risk Factors for Human Renal Cell Cancer

http://ift.tt/2vwF8sL

mTOR Pathway Mutations and Response to Rapalogs in RCC--Letter

http://ift.tt/2wrHXIR

Evaluation of Tumor-Derived Exosomal miRNA as Potential Diagnostic Biomarkers for Early-Stage Non-Small Cell Lung Cancer Using Next-Generation Sequencing

Purpose: To identify tumor-derived exosomal biomarkers that are able to discriminate between adenocarcinoma and squamous cell carcinoma (SCC) as a noninvasive method in the early diagnosis of non–small cell lung cancer (NSCLC).

Experimental Design: Tumor-derived exosomes from the plasma of early-stage NSCLC patients were isolated. Exosomal miRNA profiling of 46 stage I NSCLC patients and 42 healthy individuals was performed using miRNA-seq to identify and validate adenocarcinoma- and SCC-specific miRNAs. The diagnostic accuracy of select miRNAs was tested further with an additional 60 individuals.

Results: There were 11 and 6 miRNAs expressed at remarkably higher levels, 13 and 8 miRNAs expressed at lower levels in adenocarcinoma and SCC patients, respectively, compared with healthy volunteers. Distinct adenocarcinoma- and SCC-specific exosomal miRNAs were validated. The reliability of miRNA-seq data was verified with several demonstrated diagnostic potential miRNAs for NSCLC and other carcinomas, as reported in previous studies, such as let-7, miR-21, miR-24, and miR-486. The results indicated that miR-181-5p, miR-30a-3p, miR-30e-3p, and miR-361-5p were adenocarcinoma-specific, and miR-10b-5p, miR-15b-5p, and miR-320b were SCC-specific. The diagnostic accuracy of three combination miRNA panels was evaluated using an AUC value of 0.899, 0.936, and 0.911 for detecting NSCLC, adenocarcinoma, and SCC, respectively.

Conclusions: Tumor-derived exosomal miRNAs, adenocarcinoma-specific miR-181-5p, miR-30a-3p, miR-30e-3p and miR-361-5p, and SCC-specific miR-10b-5p, miR-15b-5p, and miR-320b were observed by next-generation sequencing, and their diagnostic accuracy were verified. These miRNAs may be promising and effective candidates in the development of highly sensitive, noninvasive biomarkers for early NSCLC diagnosis. Clin Cancer Res; 23(17); 5311–9. ©2017 AACR.

http://ift.tt/2vwvzKt

Unique and Novel Urinary Metabolomic Features in Malignant versus Benign Adrenal Neoplasms

Purpose: Adrenal incidentalomas must be differentiated from adrenocortical cancer (ACC). Currently, size, growth, and imaging characteristics determine the potential for malignancy but are imperfect. The aim was to evaluate whether urinary small molecules (<800 Da) are associated with ACC.

Experimental Design: Preoperative fasting urine specimens from patients with ACC (n = 19) and benign adrenal tumors (n = 46) were analyzed by unbiased ultraperformance liquid chromatography/mass spectrometry. Creatinine-normalized features were analyzed by Progenesis, SIMCA, and unpaired t test adjusted by FDR. Features with an AUC >0.8 were identified through fragmentation patterns and database searches. All lead features were assessed in an independent set from patients with ACC (n = 11) and benign adrenal tumors (n = 46) and in a subset of tissue samples from patients with ACC (n = 15) and benign adrenal tumors (n = 15) in the training set.

Results: Sixty-nine features were discovered and four known metabolites identified. Urinary creatine riboside was elevated 2.1-fold (P = 0.0001) in patients with ACC. L-tryptophan, N,N,N-trimethyl-L-lysine, and 3-methylhistidine were lower 0.33-fold (P < 0.0001), 0.56-fold (P < 0.0001), and 0.33-fold (P = 0.0003) in patients with ACC, respectively. Combined multivariate analysis of the four biomarkers showed an AUC of 0.89 [sensitivity 94.7% (confidence interval {CI}, 73.9%–99.1%), specificity 82.6% (CI, 68.6%–92.2%), PPV 69.2% (CI, 48.2%–85.6%), and NPV 97.4% (CI, 86.5%–99.6%)] for distinguishing ACC from benign tumors. Of the four, creatine riboside and four unknown features were validated. Creatine riboside, N,N,N-trimethyl-L-lysine, and two unknown features were elevated in ACC tumors.

Conclusions: There are unique urinary metabolic features in patients with ACC with some metabolites present in patient tumor samples. Urinary creatine riboside can differentiate benign adrenal neoplasms from ACC. Clin Cancer Res; 23(17); 5302–10. ©2017 AACR.

http://ift.tt/2wrZcty

Chronic Lymphocytic Leukemia with Mutated IGHV4-34 Receptors: Shared and Distinct Immunogenetic Features and Clinical Outcomes

Purpose: We sought to investigate whether B cell receptor immunoglobulin (BcR IG) stereotypy is associated with particular clinicobiological features among chronic lymphocytic leukemia (CLL) patients expressing mutated BcR IG (M-CLL) encoded by the IGHV4-34 gene, and also ascertain whether these associations could refine prognostication.

Experimental Design: In a series of 19,907 CLL cases with available immunogenetic information, we identified 339 IGHV4-34–expressing cases assigned to one of the four largest stereotyped M-CLL subsets, namely subsets #4, #16, #29 and #201, and investigated in detail their clinicobiological characteristics and disease outcomes.

Results: We identified shared and subset-specific patterns of somatic hypermutation (SHM) among patients assigned to these subsets. The greatest similarity was observed between subsets #4 and #16, both including IgG-switched cases (IgG-CLL). In contrast, the least similarity was detected between subsets #16 and #201, the latter concerning IgM/D-expressing CLL. Significant differences between subsets also involved disease stage at diagnosis and the presence of specific genomic aberrations. IgG subsets #4 and #16 emerged as particularly indolent with a significantly (P < 0.05) longer time-to-first-treatment (TTFT; median TTFT: not yet reached) compared with the IgM/D subsets #29 and #201 (median TTFT: 11 and 12 years, respectively).

Conclusions: Our findings support the notion that BcR IG stereotypy further refines prognostication in CLL, superseding the immunogenetic distinction based solely on SHM load. In addition, the observed distinct genetic aberration landscapes and clinical heterogeneity suggest that not all M-CLL cases are equal, prompting further research into the underlying biological background with the ultimate aim of tailored patient management. Clin Cancer Res; 23(17); 5292–301. ©2017 AACR.

http://ift.tt/2vwThX1

miR-193a-3p is a Key Tumor Suppressor in Ulcerative Colitis-Associated Colon Cancer and Promotes Carcinogenesis through Upregulation of IL17RD

Purpose: Patients with ulcerative colitis are at increased risk for colorectal cancer, although mechanisms underlying neoplastic transformation are poorly understood. We sought to evaluate the role of microRNAs in neoplasia development in this high-risk population.

Experimental Design: Tissue from 12 controls, 9 ulcerative colitis patients without neoplasia, and 11 ulcerative colitis patients with neoplasia was analyzed. miRNA array analysis was performed and select miRNAs assayed by real-time PCR on the discovery cohort and a validation cohort. DNA methylation of miR-193a was assessed. Following transfection of miR-193a-3p, proliferation, IL17RD expression, and luciferase activity of the 3'UTR of IL17RD were measured. Tumor growth in xenografts as well as EGFR signaling were assessed in HCT116 cells expressing IL17RD with either a mutant 3' untranslated region (UTR) or wild-type (WT) 3'UTR.

Results: miR-31, miR-34a, miR-106b, and miR-193a-3p were significantly dysregulated in ulcerative colitis-neoplasia and adjacent tissue. Significant down-regulation of miR-193a-3p was also seen in an independent cohort of ulcerative colitis cancers. Changes in methylation of miR-193a or expression of pri-miR-193a were not observed in ulcerative colitis cancer. Transfection of miR-193a-3p resulted in decreased proliferation, and identified IL17RD as a direct target of miR-193a-3p. IL17RD expression was increased in ulcerative colitis cancers, and miR-193a-3p treatment decreased growth and EGFR signaling of HCT116 cells in xenografts expressing both IL17RD with WT 3'UTR compared with cells expressing IL17RD with mutant 3'UTR.

Conclusions: miR-193a-3p is downregulated in ulcerative colitis neoplasia, and its loss promotes carcinogenesis through upregulation of IL17RD. These findings provide novel insight into inflammation-driven colorectal cancer and could suggest new therapeutic targets in this high-risk population. Clin Cancer Res; 23(17); 5281–91. ©2017 AACR.

http://ift.tt/2wrYSek

Targeting the Wnt Pathway and Cancer Stem Cells with Anti-progastrin Humanized Antibodies as a Potential Treatment for K-RAS-Mutated Colorectal Cancer

Purpose: Patients with metastatic colorectal cancer suffer from disease relapse mainly due to cancer stem cells (CSC). Interestingly, they have an increased level of blood progastrin, a tumor-promoting peptide essential for the self-renewal of colon CSCs, which is also a direct β-catenin/TCF4 target gene. In this study, we aimed to develop a novel targeted therapy to neutralize secreted progastrin to inhibit Wnt signaling, CSCs, and reduce relapses.

Experimental Design: Antibodies (monoclonal and humanized) directed against progastrin were produced and selected for target specificity and affinity. After validation of their effectiveness on survival of colorectal cancer cell lines harboring B-RAF or K-RAS mutations, their efficacy was assessed in vitro and in vivo, alone or concomitantly with chemotherapy, on CSC self-renewal capacity, tumor recurrence, and Wnt signaling.

Results: We show that anti-progastrin antibodies decrease self-renewal of CSCs both in vitro and in vivo, either alone or in combination with chemotherapy. Furthermore, migration and invasion of colorectal cancer cells are diminished; chemosensitivity is prolonged in SW620 and HT29 cells and posttreatment relapse is significantly delayed in T84 cells, xenografted nude mice. Finally, we show that the Wnt signaling activity in vitro is decreased, and, in transgenic mice developing Wnt-driven intestinal neoplasia, the tumor burden is alleviated, with an amplification of cell differentiation in the remaining tumors.

Conclusions: Altogether, these data show that humanized anti-progastrin antibodies might represent a potential new treatment for K-RAS–mutated colorectal patients, for which there is a crucial unmet medical need. Clin Cancer Res; 23(17); 5267–80. ©2017 AACR.

http://ift.tt/2vwzTtc

Expression and Therapeutic Potential of SOX9 in Chordoma

Purpose: Conventional chemotherapeutic agents are ineffective in the treatment of chordoma. We investigated the functional roles and therapeutic relevance of the sex-determining region Y (SRY)-box 9 (SOX9) in chordoma.

Experimental Design: SOX9 expression was examined by immunohistochemistry (IHC) using 50 chordoma tissue samples. SOX9 expression in chordoma cell lines was examined by Western blot and immunofluorescent assays. We used synthetic human SOX9 siRNA to inhibit the expression of SOX9. Cell proliferation ability and cytotoxicity of inhibiting SOX9 were assessed by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) and clonogenic assays. The effect of SOX9 knockdown on chordoma cell motility was evaluated by a wound-healing assay and a Transwell invasion chamber assay. Knockdown of SOX9 induced apoptosis, cell-cycle arrest, as well as decreased expression of cancer stem cell markers were determined by Western blot and flow cytometric assays. The effect of the combination of SOX9 siRNA and the chemotherapeutic drug doxorubicin/cisplatin on chordoma cells was assessed by an MTT assay.

Results: Tissue microarray and IHC analysis showed that SOX9 is broadly expressed in chordomas and that higher expression levels of SOX9 correlated with a poor prognosis. RNA interference (RNAi)-mediated knockdown of SOX9 inhibited chordoma cell growth, decreased cell motility, and induced apoptosis as well as cell-cycle arrest. Moreover, the combination of SOX9 inhibition and chemotherapeutic drugs had an enhanced anti-cancer effect on chordoma cells.

Conclusions: Our results demonstrate that SOX9 plays a crucial role in chordoma. Targeting SOX9 provides a new rationale for treatment of chordoma. Clin Cancer Res; 23(17); 5176–86. ©2017 AACR.

http://ift.tt/2wrSzHR

Can Microsatellite Status of Colorectal Cancer Be Reliably Assessed after Neoadjuvant Therapy?

Purpose: Determination of microsatellite instability (MSI) by PCR is the gold standard; however, IHC of mismatch repair (MMR) proteins is frequently performed instead. The reliability of these methods on postneoadjuvant therapy specimens is unknown. We examined the effect of neoadjuvant therapy on MSI results by PCR and IHC.

Experimental design: A total of 239 colorectal cancers resected after neoadjuvant therapy were assessed for MSI with PCR and IHC. PCR and IHC results for matched paired pre- and posttreatment specimens were compared. In parallel, 2 isogenic cell lines conditioned for MMR functioning and 2 different patient-derived xenografts (PDXs) were exposed to chemotherapy, radiation, or both. We also examined whether establishment of PDXs induced MSI changes in 5 tumors. IHC and MSI were tested after treatment to assess for changes.

Results: We identified paired pre- and posttreatment specimens for 37 patients: 2 with PCR only, 34 with IHC only, and 1 with both. All 3 patients with PCR had microsatellite stable pre- and posttreatment specimens. Of the 35 patients with IHC, 30 had intact MMR proteins in pre- and posttreatment specimens, 1 had equivocal MLH1 staining in the pretreatment and loss in the posttreatment specimen, and 4 had intact pretreatment MSH6 but variable posttreatment staining. In the experimental setting, no changes in MSI status were detected after treatment or tumor implantation in animals.

Conclusions: Our findings show that the expression of MMR proteins, commonly MSH6, can change after neoadjuvant therapy and confirm PCR as the gold-standard test for MSI after neoadjuvant therapy. Clin Cancer Res; 23(17); 5246–54. ©2017 AACR.

http://ift.tt/2vwzAyy

Immune Correlates of GM-CSF and Melanoma Peptide Vaccination in a Randomized Trial for the Adjuvant Therapy of Resected High-Risk Melanoma (E4697)

Purpose: E4697 was a multicenter intergroup randomized placebo-controlled phase III trial of adjuvant GM-CSF and/or a multiepitope melanoma peptide vaccine for patients with completely resected, high-risk stage III/IV melanoma.

Experimental Design: A total of 815 patients were enrolled from December 1999 to October 2006 into this six-arm study. GM-CSF was chosen to promote the numbers and functions of dendritic cells (DC). The melanoma antigen peptide vaccine (Tyrosinase_{368-376 (370D)}, gp100_{209-217 (210M)}, MART-1_27-35) in montanide was designed to promote melanoma-specific CD8⁺ T-cell responses.

Results: Although the overall RFS and OS were not significantly improved with the vaccine or GM-CSF when compared with placebo, immunomodulatory effects were observed in peripheral blood and served as important correlates to this therapeutic study. Peripheral blood was examined to evaluate the impact of GM-CSF and/or the peptide vaccine on peripheral blood immunity and to investigate potential predictive or prognostic biomarkers. A total of 11.3% of unvaccinated patients and 27.1% of vaccinated patients developed peptide-specific CD8⁺ T-cell responses. HLA-A2⁺ patients who had any peptide-specific CD8⁺ T-cell response at day +43 tended to have poorer OS in univariate analysis. Patients receiving GM-CSF had significant reduction in percentages of circulating myeloid dendritic cells (mDC) and plasmacytoid DC (pDC) at day +43. In a subset of patients who received GM-CSF, circulating myeloid-derived suppressor cells (MDSC), and anti-GM-CSF–neutralizing antibodies (Nabs) were also modulated. The majority of patients developed anti-GM-CSF Nabs, which correlated with improved RFS and OS.

Conclusions: The assessment of cellular and humoral responses identified counterintuitive immune system changes correlating with clinical outcome. Clin Cancer Res; 23(17); 5034–43. ©2017 AACR.

http://ift.tt/2wrVNLl

MONARCH 1, A Phase II Study of Abemaciclib, a CDK4 and CDK6 Inhibitor, as a Single Agent, in Patients with Refractory HR+/HER2- Metastatic Breast Cancer

Purpose: The phase II MONARCH 1 study was designed to evaluate the single-agent activity and adverse event (AE) profile of abemaciclib, a selective inhibitor of CDK4 and CDK6, in women with refractory hormone receptor–positive (HR⁺), HER2^– metastatic breast cancer (MBC).

Experimental Design: MONARCH 1 was a phase II single-arm open-label study. Women with HR⁺/HER2^– MBC who had progressed on or after prior endocrine therapy and had 1 or 2 chemotherapy regimens in the metastatic setting were eligible. Abemaciclib 200 mg was administered orally on a continuous schedule every 12 hours until disease progression or unacceptable toxicity. The primary objective of MONARCH 1 was investigator-assessed objective response rate (ORR). Other endpoints included clinical benefit rate, progression-free survival (PFS), and overall survival (OS).

Results: Patients (n = 132) had a median of 3 (range, 1–8) lines of prior systemic therapy in the metastatic setting, 90.2% had visceral disease, and 50.8% had ≥3 metastatic sites. At the 12-month final analysis, the primary objective of confirmed objective response rate was 19.7% (95% CI, 13.3–27.5; 15% not excluded); clinical benefit rate (CR+PR+SD≥6 months) was 42.4%, median progression-free survival was 6.0 months, and median overall survival was 17.7 months. The most common treatment-emergent AEs of any grade were diarrhea, fatigue, and nausea; discontinuations due to AEs were infrequent (7.6%).

Conclusions: In this poor-prognosis, heavily pretreated population with refractory HR⁺/HER2^– metastatic breast cancer, continuous dosing of single-agent abemaciciclib was well tolerated and exhibited promising clinical activity. Clin Cancer Res; 23(17); 5218–24. ©2017 AACR.

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Dual Inhibition of HDAC and Tyrosine Kinase Signaling Pathways with CUDC-907 Inhibits Thyroid Cancer Growth and Metastases

Purpose: There is currently no standard therapy for anaplastic thyroid cancer (ATC) and poorly differentiated thyroid cancer (PDTC), which account for two-thirds of thyroid cancer–related deaths. Driver mutations in the PI3K/AKT and RAF/RAS/MEK/ERK pathways are common in ATC and PDTC. Histone deacetylases (HDAC) regulate cancer initiation and progression. Our aim was to determine the therapeutic efficacy of simultaneously targeting these pathways in thyroid cancer with a single agent and to evaluate biomarkers of treatment response.

Experimental Design: CUDC-907 is a first-in-class compound, functioning as a dual inhibitor of HDACs and the PI3K/AKT pathway. We investigated its antiproliferative effect in vitro and in vivo.

Results: CUDC-907 significantly inhibited cellular proliferation in thyroid cancer cell lines, induced G₂–M arrest with decreased levels of the checkpoint regulators cyclin B1, AURKA, AURKB, PLK1, and increased p21 and p27. Treatment induced apoptosis with increased caspase-3/7 activity and decreased survivin levels and decreased cellular migration and invasion. CUDC-907 treatment caused H3 hyperacetylation and decreased HDAC2 expression. HDAC2 was upregulated in ATC and other thyroid cancer histologic subtypes. CUDC-907 treatment reduced both p-AKT and p-ERK1/2 levels. Finally, CUDC-907 treatment, in a metastatic mouse model of thyroid cancer, showed significant inhibition of growth and metastases, and tumors from treated mice had decreased HDAC2 expression, suggesting that this may be a useful biomarker of response.

Conclusions: Dual inhibition of HDAC and the tyrosine kinase signaling pathways with CUDC-907 is a promising treatment strategy for advanced, metastatic thyroid cancer. Clin Cancer Res; 23(17); 5044–54. ©2017 AACR.

http://ift.tt/2wscYwu

Trabectedin Overrides Osteosarcoma Differentiative Block and Reprograms the Tumor Immune Environment Enabling Effective Combination with Immune Checkpoint Inhibitors

Purpose: Osteosarcoma, the most common primary bone tumor, is characterized by an aggressive behavior with high tendency to develop lung metastases as well as by multiple genetic aberrations that have hindered the development of targeted therapies. New therapeutic approaches are urgently needed; however, novel combinations with immunotherapies and checkpoint inhibitors require suitable preclinical models with intact immune systems to be properly tested.

Experimental Design: We have developed immunocompetent osteosarcoma models that grow orthotopically in the bone and spontaneously metastasize to the lungs, mimicking human osteosarcoma. These models have been used to test the efficacy of trabectedin, a chemotherapeutic drug utilized clinically for sarcomas and ovarian cancer.

Results: Trabectedin, as monotherapy, significantly inhibited osteosarcoma primary tumor growth and lung metastases by both targeting neoplastic cells and reprogramming the tumor immune microenvironment. Specifically, trabectedin induced a striking differentiation of tumor cells by favoring the recruitment of Runx2, the master genetic regulator of osteoblastogenesis, on the promoter of genes involved in the physiologic process of terminal osteoblast differentiation. Differentiated neoplastic cells, as expected, showed reduced proliferation rate. Concomitantly, trabectedin enhanced the number of tumor-infiltrating T lymphocytes, with local CD8 T cells, however, likely post-activated or exhausted, as suggested by their high expression of the inhibitory checkpoint molecule PD-1. Accordingly, the combination with a PD-1–blocking antibody significantly increased trabectedin efficacy in controlling osteosarcoma progression.

Conclusions: These results demonstrate the therapeutic efficacy of trabectedin in osteosarcoma treatment, unveiling its multiple activities and providing a solid rationale for its combination with immune checkpoint inhibitors. Clin Cancer Res; 23(17); 5149–61. ©2017 AACR.

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A Pilot Study of Stereotactic Body Radiation Therapy Combined with Cytoreductive Nephrectomy for Metastatic Renal Cell Carcinoma

Purpose: While stereotactic body radiotherapy (SBRT) can reduce tumor volumes in patients with metastatic renal cell carcinoma (mRCC), little is known regarding the immunomodulatory effects of high-dose radiation in the tumor microenvironment. The main objectives of this pilot study were to assess the safety and feasibility of nephrectomy following SBRT treatment of patients with mRCC and analyze the immunological impact of high-dose radiation.

Experimental Design: Human RCC cell lines were irradiated and evaluated for immunomodulation. In a single-arm feasibility study, patients with mRCC were treated with 15 Gray SBRT at the primary lesion in a single fraction followed 4 weeks later by cytoreductive nephrectomy. RCC specimens were analyzed for tumor-associated antigen (TAA) expression and T-cell infiltration. The trial has reached accrual (ClinicalTrials.gov identifier: NCT01892930).

Results: RCC cells treated in vitro with radiation had increased TAA expression compared with untreated tumor cells. Fourteen patients received SBRT followed by surgery, and treatment was well-tolerated. SBRT-treated tumors had increased expression of the immunomodulatory molecule calreticulin and TAA (CA9, 5T4, NY-ESO-1, and MUC-1). Ki67⁺ -proliferating CD8⁺ T cells and FOXP3⁺ cells were increased in SBRT-treated patient specimens in tumors and at the tumor–stromal interface compared with archived patient specimens.

Conclusions: It is feasible to perform nephrectomy following SBRT with acceptable toxicity. Following SBRT, patient RCC tumors have increased expression of calreticulin, TAA, as well as a higher percentage of proliferating T cells compared with archived RCC tumors. Collectively, these studies provide evidence of immunomodulation following SBRT in mRCC. Clin Cancer Res; 23(17); 5055–65. ©2017 AACR.

http://ift.tt/2wrTND3

B7-H3 Expression in NSCLC and Its Association with B7-H4, PD-L1 and Tumor-Infiltrating Lymphocytes

Purpose: The immune checkpoint PD-1 and its receptor B7-H1 (PD-L1) are successful therapeutic targets in cancer but less is known about other B7 family members. Here, we determined the expression level of B7-H3 protein in non–small cell lung cancer (NSCLC) and evaluated its association with tumor-infiltrating lymphocytes (TIL), PD-L1, B7-H4, and major clinicopathologic characteristics is in 3 NSCLC cohorts.

Experimental design: We used multiplexed automated quantitative immunofluorescence (QIF) to assess the levels of B7-H3, PD-L1, B7-H4, and TILs in 634 NSCLC cases with validated antibodies. Associations between the marker levels, major clinicopathologic variables and survival were analyzed.

Results: Expression of B7-H3 protein was found in 80.4% (510/634) of the cases. High B7-H3 protein level (top 10 percentile) was associated with poor overall survival (P < 0.05). Elevated B7-H3 was consistently associated with smoking history across the 3 cohorts, but not with sex, age, clinical stage, and histology. Coexpression of B7-H3 and PD-L1 was found in 17.6% of the cases (112/634) and with B7-H4 in 10% (63/634). B7-H4 and PD-L1 were simultaneously detected only in 1.8% of NSCLCs (12/634). The expression of B7-H3 was not associated with the levels of CD3-, CD8-, and CD20-positive TILs.

Conclusions: B7-H3 protein is expressed in the majority of NSCLCs and is associated with smoking history. High levels of B7-H3 protein have a negative prognostic impact in lung carcinomas. Coexpression of B7-H3 with PD-L1 and B7-H4 is relatively low, suggesting a nonredundant biological role of these targets. Clin Cancer Res; 23(17); 5202–9. ©2017 AACR.

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Dendritic Cell/Cytokine-Induced Killer Cell Immunotherapy Combined with S-1 in Patients with Advanced Pancreatic Cancer: A Prospective Study

Purpose: Advanced pancreatic cancer has remained challenging to treat effectively. This study aimed to investigate the clinical effects and safety of immunotherapy with dendritic cells and cytokine-induced killer cells (DC-CIK) administered with the chemotherapy (CT) S-1 in this malignancy.

Experimental Design: Consecutive patients (n = 47) with advanced pancreatic cancer were treated with either DC-CIK + S-1, DC-CIK alone, S-1 alone, or best supportive care.

Results: DC-CIK plus S-1 produced significantly longer median OS and PFS (212 and 136 days) compared with DC-CIK (128 and 85 days), CT (141 and 92 days), or supportive care only (52 and 43 days; P < 0.001). After adjusting for competing risk factors, DC-CIK combined with S-1 and receipt of 2 or more cycles of DC-CIK treatment remained independent predictors of disease-free and overall survival (P < 0.05). Phenotypic analysis of PBMCs demonstrated that the CD3⁺, CD3⁺/CD4⁺, and CD8⁺/CD28⁺ T-cell subsets were elevated (P < 0.05), while the CD3⁺/CD8⁺, CD3⁺/CD16⁺/CD56⁺ and CD4⁺/CD25⁺ cell subsets were significantly decreased after DC-CIK cell therapy (P < 0.05). There were no grade 3 or 4 toxicities. In addition, the mutational frequency in cell-free tumor DNA (cfDNA) declined in 4 of 14 patients who received DC-CIK, and was associated with a more favorable survival.

Conclusions: Treatment of advanced pancreatic cancer with combined DC-CIK infusions and S-1 was safe, resulted in favorable PFS and OS, and modulated the peripheral blood immune repertoire. Clin Cancer Res; 23(17); 5066–73. ©2017 AACR.

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Gene Expression Profiling in BRAF-Mutated Melanoma Reveals Patient Subgroups with Poor Outcomes to Vemurafenib That May Be Overcome by Cobimetinib Plus Vemurafenib

Purpose: The association of tumor gene expression profiles with progression-free survival (PFS) outcomes in patients with BRAF^V600-mutated melanoma treated with vemurafenib or cobimetinib combined with vemurafenib was evaluated.

Experimental Design: Gene expression of archival tumor samples from patients in four trials (BRIM-2, BRIM-3, BRIM-7, and coBRIM) was evaluated. Genes significantly associated with PFS (P < 0.05) were identified by univariate Cox proportional hazards modeling, then subjected to unsupervised hierarchical clustering, principal component analysis, and recursive partitioning to develop optimized gene signatures.

Results: Forty-six genes were identified as significantly associated with PFS in both BRIM-2 (n = 63) and the vemurafenib arm of BRIM-3 (n = 160). Two distinct signatures were identified: cell cycle and immune. Among vemurafenib-treated patients, the cell-cycle signature was associated with shortened PFS compared with the immune signature in the BRIM-2/BRIM-3 training set [hazard ratio (HR) 1.8; 95% confidence interval (CI), 1.3–2.6, P = 0.0001] and in the coBRIM validation set (n = 101; HR, 1.6; 95% CI, 1.0–2.5; P = 0.08). The adverse impact of the cell-cycle signature on PFS was not observed in patients treated with cobimetinib combined with vemurafenib (n = 99; HR, 1.1; 95% CI, 0.7–1.8; P = 0.66).

Conclusions: In vemurafenib-treated patients, the cell-cycle gene signature was associated with shorter PFS. However, in cobimetinib combined with vemurafenib-treated patients, both cell cycle and immune signature subgroups had comparable PFS. Cobimetinib combined with vemurafenib may abrogate the adverse impact of the cell-cycle signature. Clin Cancer Res; 23(17); 5238–45. ©2017 AACR.

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Tumor Cell-Free DNA Copy Number Instability Predicts Therapeutic Response to Immunotherapy

Purpose: Chromosomal instability is a fundamental property of cancer, which can be quantified by next-generation sequencing (NGS) from plasma/serum–derived cell-free DNA (cfDNA). We hypothesized that cfDNA could be used as a real-time surrogate for imaging analysis of disease status as a function of response to immunotherapy and as a more reliable tool than tumor biomarkers.

Experimental Design: Plasma cfDNA sequences from 56 patients with diverse advanced cancers were prospectively collected and analyzed in a single-blind study for copy number variations, expressed as a quantitative chromosomal number instability (CNI) score versus 126 noncancer controls in a training set of 23 and a blinded validation set of 33. Tumor biomarker concentrations and a surrogate marker for T regulatory cells (Tregs) were comparatively analyzed.

Results: Elevated CNI scores were observed in 51 of 56 patients prior to therapy. The blinded validation cohort provided an overall prediction accuracy of 83% (25/30) and a positive predictive value of CNI score for progression of 92% (11/12). The combination of CNI score before cycle (Cy) 2 and 3 yielded a correct prediction for progression in all 13 patients. The CNI score also correctly identified cases of pseudo-tumor progression from hyperprogression. Before Cy2 and Cy3, there was no significant correlation for protein tumor markers, total cfDNA, or surrogate Tregs.

Conclusions: Chromosomal instability quantification in plasma cfDNA can serve as an early indicator of response to immunotherapy. The method has the potential to reduce health care costs and disease burden for cancer patients following further validation. Clin Cancer Res; 23(17); 5074–81. ©2017 AACR.

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Inclusion of a Genetic Risk Score into a Validated Risk Prediction Model for Colorectal Cancer in Japanese Men Improves Performance

We previously developed and validated a risk prediction model for colorectal cancer in Japanese men using modifiable risk factors. To further improve risk prediction, we evaluated the degree of improvement obtained by adding a genetic risk score (GRS) using genome-wide association study (GWAS)-identified risk variants to our validated model. We examined the association between 36 risk variants identified by GWAS and colorectal cancer risk using a weighted Cox proportional hazards model in a nested case–control study within the Japan Public Health Center-based Prospective Study. GRS was constructed using six variants associated with risk in this study of the 36 tested. We assessed three models: a nongenetic model that included the same variables used in our previously validated model; a genetic model that used GRS; and an inclusive model, which included both. The c-statistic, integrated discrimination improvement (IDI), and net reclassification improvement (NRI) were calculated by the 5-fold cross-validation method. We estimated 10-year absolute risks for developing colorectal cancer. A statistically significant association was observed between the weighted GRS and colorectal cancer risk. The mean c-statistic for the inclusive model (0.66) was slightly greater than that for the nongenetic model (0.60). Similarly, the mean IDI and NRI showed improvement when comparing the nongenetic and inclusive models. These models for colorectal cancer were well calibrated. The addition of GRS using GWAS-identified risk variants to our validated model for Japanese men improved the prediction of colorectal cancer risk. Cancer Prev Res; 10(9); 535–41. ©2017 AACR.

http://ift.tt/2enO09j

Identification of a Human Airway Epithelial Cell Subpopulation with Altered Biophysical, Molecular, and Metastatic Properties

Lung cancers are documented to have remarkable intratumoral genetic heterogeneity. However, little is known about the heterogeneity of biophysical properties, such as cell motility, and its relationship to early disease pathogenesis and micrometastatic dissemination. In this study, we identified and selected a subpopulation of highly migratory premalignant airway epithelial cells that were observed to migrate through microscale constrictions at up to 100-fold the rate of the unselected immortalized epithelial cell lines. This enhanced migratory capacity was found to be Rac1-dependent and heritable, as evidenced by maintenance of the phenotype through multiple cell divisions continuing more than 8 weeks after selection. The morphology of this lung epithelial subpopulation was characterized by increased cell protrusion intensity. In a murine model of micrometastatic seeding and pulmonary colonization, the motility-selected premalignant cells exhibit both enhanced survival in short-term assays and enhanced outgrowth of premalignant lesions in longer-term assays, thus overcoming important aspects of "metastatic inefficiency." Overall, our findings indicate that among immortalized premalignant airway epithelial cell lines, subpopulations with heritable motility-related biophysical properties exist, and these may explain micrometastatic seeding occurring early in the pathogenesis of lung cancer. Understanding, targeting, and preventing these critical biophysical traits and their underlying molecular mechanisms may provide a new approach to prevent metastatic behavior. Cancer Prev Res; 10(9); 514–24. ©2017 AACR.

See related editorial by Hynds and Janes, p. 491

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Airway Basal Cell Heterogeneity and Lung Squamous Cell Carcinoma

Basal cells are stem/progenitor cells that maintain airway homeostasis, enact repair following epithelial injury, and are a candidate cell-of-origin for lung squamous cell carcinoma. Heterogeneity of basal cells is recognized in terms of gene expression and differentiation capacity. In this Issue, Pagano and colleagues isolate a subset of immortalized basal cells that are characterized by high motility, suggesting that they might also be heterogeneous in their biophysical properties. Motility-selected cells displayed an increased ability to colonize the lung in vivo. The possible implications of these findings are discussed in terms of basal cell heterogeneity, epithelial cell migration, and modeling of metastasis that occurs early in cancer evolution. Cancer Prev Res; 10(9); 491–3. ©2017 AACR.

See related article by Pagano et al., p. 514

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When the Damage Is Done: Selecting Patients for Head and Neck Cancer Chemoprevention Trials

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Signals from the Adipose Microenvironment and the Obesity-Cancer Link--A Systematic Review

Obesity and its associated metabolic dysregulation are established risk factors for many cancers. However, the biologic mechanisms underlying this relationship remain incompletely understood. Given the rising rates of both obesity and cancer worldwide, and the challenges for many people to lose excess adipose tissue, a systematic approach to identify potential molecular and metabolic targets is needed to develop effective mechanism-based strategies for the prevention and control of obesity-driven cancer. Epidemiologic, clinical, and preclinical data suggest that within the growth-promoting, proinflammatory microenvironment accompanying obesity, crosstalk between adipose tissue (comprised of adipocytes, macrophages and other cells) and cancer-prone cells may occur via obesity-associated hormones, cytokines, and other mediators that have been linked to increased cancer risk and/or progression. We report here a systematic review on the direct "crosstalk" between adipose tissue and carcinomas in humans. We identified 4,641 articles with n = 20 human clinical studies, which are summarized as: (i) breast (n = 7); (ii) colorectal (n = 4); (iii) esophageal (n = 2); (iv) esophageal/colorectal (n = 1); (v) endometrial (n = 1); (vi) prostate (n = 4); and (vii) ear-nose-throat (ENT) cancer (n = 1). Findings from these clinical studies reinforce preclinical data and suggest organ-dependent crosstalk between adipose tissue and carcinomas via VEGF, IL6, TNFα, and other mechanisms. Moreover, visceral white adipose tissue plays a more central role, as it is more bioenergetically active and is associated with a more procancer secretome than subcutaneous adipose tissue. Efforts to eavesdrop and ultimately interfere with this cancer-enhancing crosstalk may lead to new targets and strategies for decreasing the burden of obesity-related cancers. Cancer Prev Res; 10(9); 494–506. ©2017 AACR.

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Circulating RANKL and RANKL/OPG and Breast Cancer Risk by ER and PR Subtype: Results from the EPIC Cohort

Receptor activator of nuclear factor-kappa B (RANK)-RANK ligand (RANKL) signaling promotes mammary tumor development in experimental models. Circulating concentrations of soluble RANKL (sRANKL) may influence breast cancer risk via activation of RANK signaling; this may be modulated by osteoprotegerin (OPG), the decoy receptor for RANKL. sRANKL and breast cancer risk by hormone receptor subtype has not previously been investigated. A case–control study was nested in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. This study included 1,976 incident invasive breast cancer cases [estrogen receptor positive (ER+), n = 1,598], matched 1:1 to controls. Women were pre- or postmenopausal at blood collection. Serum sRANKL was quantified using an ELISA, serum OPG using an electrochemiluminescent assay. Risk ratios (RR) and 95% confidence intervals (95% CI) were calculated using conditional logistic regression. Associations between sRANKL and breast cancer risk differed by tumor hormone receptor status (P_het = 0.05). Higher concentrations of sRANKL were positively associated with risk of ER+ breast cancer [5th vs. 1st quintile RR 1.28 (95% CI, 1.01–1.63); P_trend = 0.20], but not ER– disease. For both ER+ and estrogen and progesterone receptor positive (ER+PR+) breast cancer, results considering the sRANKL/OPG ratio were similar to those for sRANKL; we observed a suggestive inverse association between the ratio and ER–PR– disease [5th vs. 1st quintile RR = 0.60 (0.31–1.14); P_trend = 0.03]. This study provides the first large-scale prospective data on circulating sRANKL and breast cancer. We observed limited evidence for an association between sRANKL and breast cancer risk. Cancer Prev Res; 10(9); 525–34. ©2017 AACR.

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High Level of Tobacco Carcinogen-Derived DNA Damage in Oral Cells Is an Independent Predictor of Oral/Head and Neck Cancer Risk in Smokers

Exposure to tobacco-specific nitrosamines (TSNA) and polycyclic aromatic hydrocarbons (PAH) is recognized to play an important role in the development of oral/head and neck squamous cell cancer (HNSCC). We recently reported higher levels of TSNA-associated DNA adducts in the oral cells of smokers with HNSCC as compared with cancer-free smokers. In this study, we further investigated the tobacco constituent exposures in the same smokers to better understand the potential causes for the elevated oral DNA damage in smokers with HNSCC. Subjects included cigarette smokers with HNSCC (cases, n = 30) and cancer-free smokers (controls, n = 35). At recruitment, tobacco/alcohol use questionnaires were completed, and urine and oral cell samples were obtained. Analysis of urinary 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and N'-Nitrosonornicotine (NNN; TSNA biomarkers), 1-hydroxypyrene (1-HOP, a PAH), cotinine, 3'-hydroxycotinine, and the nicotine metabolite ratio (NMR) were performed. Cases and controls differed in mean age, male preponderance, and frequency of alcohol consumption (but not total alcoholic drinks). Univariate analysis revealed similar levels of NNN, 1-HOP, and cotinine between groups but, as reported previously, significantly higher DNA adduct formation in the cases. Multiple regression adjusting for potential confounders showed persistent significant difference in DNA adduct levels between cases and controls [ratio of geometric means, 20.0; 95% CI, 2.7–148.6). Our cohort of smokers with HNSCC demonstrates higher levels of TSNA-derived oral DNA damage in the setting of similar exposure to nicotine and tobacco carcinogens. Among smokers, DNA adduct formation may act as a predictor of eventual development of HNSCC that is independent of carcinogen exposure indicators. Cancer Prev Res; 10(9); 507–13. ©2017 AACR.

See related editorial by Johnson and Bauman, p. 489

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Multiparametric Detection of Antibodies against Different EBV Antigens to Predict Risk for Nasopharyngeal Carcinoma in a High-Risk Population of China

In this study, we aimed to use the combined detection of multiple antibodies against Epstein–Barr virus (EBV) antigens to develop a model for screening and diagnosis of nasopharyngeal carcinoma (NPC). Samples of 300 nasopharyngeal carcinoma patients and 494 controls, including 294 healthy subjects (HC), 99 non-nasopharyngeal carcinoma cancer patients (NNPC), and 101 patients with benign nasopharyngeal lesions (BNL), were incubated with the EUROLINE Anti-EBV Profile 2, and band intensities were used to establish a risk prediction model. The nasopharyngeal carcinoma risk probability analysis based on the panel of VCAgp125 IgA, EBNA-1 IgA, EA-D IgA, EBNA-1 IgG, EAD IgG, and VCAp19 IgG displayed the best performance. When using 26.1% as the cutoff point in ROC analysis, the AUC value and sensitivity/specificity were 0.951 and 90.7%/86.2%, respectively, in nasopharyngeal carcinoma and all controls. In nasopharyngeal carcinoma and controls without the non-nasopharyngeal carcinoma and BNL groups, the AUC value and sensitivity/specificity were 0.957 and 90.7%/88.1%, respectively. The diagnostic specificity and sensitivity of the EUROLINE Anti-EBV Profile 2 assay for both nasopharyngeal carcinoma and early-stage nasopharyngeal carcinoma were higher than that of mono-antibody detection by immune-enzymatic assay and real-time PCR (EBV DNA). In the VCA-IgA–negative group, 82.6% of nasopharyngeal carcinoma patients showed high probability for nasopharyngeal carcinoma, and the negative predictive value was 97.1%. In the VCA-IgA–positive group, 73.3% of healthy subjects showed low probability. The positive predictive value reached 98.2% in this group. The nasopharyngeal carcinoma risk probability value determined by the EUROLINE Anti-EBV Profile 2 might be a suitable tool for nasopharyngeal carcinoma screening. Cancer Prev Res; 10(9); 542–50. ©2017 AACR.

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MELK and EZH2 Cooperate to Regulate Medulloblastoma Cancer Stem-like Cell Proliferation and Differentiation

Medulloblastoma is the most common malignant brain tumor in children. Although accumulated research has suggested that cancer stem-like cells play a key role in medulloblastoma tumorigenesis, the specific molecular mechanism regarding proliferation remains elusive. Here, we reported more abundant expression of maternal embryonic leucine-zipper kinase (MELK) and enhancer of zeste homolog 2 (EZH2) in medulloblastoma stem-like cells than in neural stem cells and the interaction between the two proteins could mediate the self-renewal of sonic hedgehog subtype medulloblastoma. In human medulloblastoma, extensive nodularity and large-cell/anaplastic subgroups differed according to the staining levels of MELK and EZH2 from the other two subgroups. The proportion of MELK- or EZH2-positive staining status could be considered as a potential indicator for survival. Mechanistically, MELK bound to and phosphorylated EZH2, and its methylation was induced by EZH2 in medulloblastoma, which could regulate the proliferation of cancer stem-like cells. In xenografts, loss of MELK or EZH2 attenuated medulloblastoma stem-like cell-derived tumor growth and promoted differentiation. These findings indicate that MELK-induced phosphorylation and EZH2-mediated methylation in MELK/EZH2 pathway are essential for medulloblastoma stem-like cell-derived tumor proliferation, thereby identifying a potential therapeutic strategy for these patients.

Implications: This study demonstrates that the interaction occurring between MELK and EZH2 promotes self-proliferation and stemness, thus representing an attractive therapeutic target and potential candidate for diagnosis of medulloblastoma. Mol Cancer Res; 15(9); 1275–86. ©2017 AACR.

http://ift.tt/2enGuel

PPAR{delta} Reprograms Glutamine Metabolism in Sorafenib-Resistant HCC

The tyrosine kinase inhibitor sorafenib is the only therapeutic agent approved for the treatment of advanced hepatocellular carcinoma (HCC), but acquired resistance to sorafenib is high. Here, we report metabolic reprogramming in sorafenib-resistant HCC and identify a regulatory molecule, peroxisome proliferator–activated receptor- (PPAR), as a potential therapeutic target. Sorafenib-resistant HCC cells showed markedly higher glutamine metabolism and reductive glutamine carboxylation, which was accompanied by increased glucose-derived pentose phosphate pathway and glutamine-derived lipid biosynthetic pathways and resistance to oxidative stress. These glutamine-dependent metabolic alterations were attributed to PPAR, which was upregulated in sorafenib-resistant HCC cells and human HCC tissues. Furthermore, PPAR contributed to increased proliferative capacity and redox homeostasis in sorafenib-resistant HCC cells. Accordingly, inhibiting PPAR activity reversed compensatory metabolic reprogramming in sorafenib-resistant HCC cells and sensitized them to sorafenib. Therefore, targeting compensatory metabolic reprogramming of glutamine metabolism in sorafenib-resistant HCC by inhibiting PPAR constitutes a potential therapeutic strategy for overcoming sorafenib-resistance in HCC.

Implications: This study provides novel insight into the mechanism underlying sorafenib resistance and a potential therapeutic strategy targeting PPAR in advanced hepatocellular carcinoma. Mol Cancer Res; 15(9); 1230–42. ©2017 AACR.

http://ift.tt/2iMqsQt

Intratumor Heterogeneity: Novel Approaches for Resolving Genomic Architecture and Clonal Evolution

High-throughput genomic technologies have revealed a remarkably complex portrait of intratumor heterogeneity in cancer and have shown that tumors evolve through a reiterative process of genetic diversification and clonal selection. This discovery has challenged the classical paradigm of clonal dominance and brought attention to subclonal tumor cell populations that contribute to the cancer phenotype. Dynamic evolutionary models may explain how these populations grow within the ecosystem of tissues, including linear, branching, neutral, and punctuated patterns. Recent evidence in breast cancer favors branching and punctuated evolution driven by genome instability as well as nongenetic sources of heterogeneity, such as epigenetic variation, hierarchal tumor cell organization, and subclonal cell–cell interactions. Resolution of the full mutational landscape of tumors could help reconstruct their phylogenetic trees and trace the subclonal origins of therapeutic resistance, relapsed disease, and distant metastases, the major causes of cancer-related mortality. Real-time assessment of the tumor subclonal architecture, however, remains limited by the high rate of errors produced by most genome-wide sequencing methods as well as the practical difficulties associated with serial tumor genotyping in patients. This review focuses on novel approaches to mitigate these challenges using bulk tumor, liquid biopsies, single-cell analysis, and deep sequencing techniques. The origins of intratumor heterogeneity and the clinical, diagnostic, and therapeutic consequences in breast cancer are also explored. Mol Cancer Res; 15(9); 1127–37. ©2017 AACR.

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The Landscape of Isoform Switches in Human Cancers

Alternative usage of transcript isoforms from the same gene has been hypothesized as an important feature in cancers. However, differential usage of gene transcripts between conditions (isoform switching) has not been comprehensively characterized in and across cancer types. To this end, we developed methods for identification and visualization of isoform switches with predicted functional consequences. Using these methods, we characterized isoform switching in RNA-seq data from >5,500 cancer patients covering 12 solid cancer types. Isoform switches with potential functional consequences were common, affecting approximately 19% of multiple transcript genes. Among these, isoform switches leading to loss of DNA sequence encoding protein domains were more frequent than expected, particularly in pancancer switches. We identified several isoform switches as powerful biomarkers: 31 switches were highly predictive of patient survival independent of cancer types. Our data constitute an important resource for cancer researchers, available through interactive web tools. Moreover, our methods, available as an R package, enable systematic analysis of isoform switches from other RNA-seq datasets.

Implications: This study indicates that isoform switches with predicted functional consequences are common and important in dysfunctional cells, which in turn means that gene expression should be analyzed at the isoform level. Mol Cancer Res; 15(9); 1206–20. ©2017 AACR.

http://ift.tt/2iMyEA1

Infiltrating Myeloid Cells Exert Protumorigenic Actions via Neutrophil Elastase

Tissue infiltration and elevated peripheral circulation of granulocytic myeloid-derived cells is associated with poor outcomes in prostate cancer and other malignancies. Although myeloid-derived cells have the ability to suppress T-cell function, little is known about the direct impact of these innate cells on prostate tumor growth. Here, it is reported that granulocytic myeloid-derived suppressor cells (MDSC) are the predominant tumor-infiltrating cells in prostate cancer xenografts established in athymic nude mice. MDSCs significantly increased in number in the peripheral circulation as a function of xenograft growth and were successfully depleted in vivo by Gr-1 antibody treatment. Importantly, MDSC depletion significantly decreased xenograft growth. We hypothesized that granulocytic MDSCs might exert their protumorigenic actions in part through neutrophil elastase (ELANE), a serine protease released upon granulocyte activation. Indeed, it was determined that NE is expressed by infiltrating immune cells and is enzymatically active in prostate cancer xenografts and in prostate tumors of prostate-specific Pten-null mice. Importantly, treatment with sivelestat, a small-molecule inhibitor specific for NE, significantly decreased xenograft growth, recapitulating the phenotype of Gr-1 MDSC depletion. Mechanistically, NE activated MAPK signaling and induced MAPK-dependent transcription of the proliferative gene cFOS in prostate cancer cells. Functionally, NE stimulated proliferation, migration, and invasion of prostate cancer cells in vitro. IHC on human prostate cancer clinical biopsies revealed coexpression of NE and infiltrating CD33⁺ MDSCs.

Implications: This report suggests that MDSCs and NE are physiologically important mediators of prostate cancer progression and may serve as potential biomarkers and therapeutic targets. Mol Cancer Res; 15(9); 1138–52. ©2017 AACR.

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IKK{beta}-Mediated Resistance to Skin Cancer Development Is Ink4a/Arf-Dependent

IKKβ (encoded by IKBKB) is a protein kinase that regulates the activity of numerous proteins important in several signaling pathways, such as the NF-B pathway. IKKβ exerts a protumorigenic role in several animal models of lung, hepatic, intestinal, and oral cancer. In addition, genomic and proteomic studies of human tumors also indicate that IKBKB gene is amplified or overexpressed in multiple tumor types. Here, the relevance of IKKβ in skin cancer was determined by performing carcinogenesis studies in animal models overexpressing IKKβ in the basal skin layer. IKKβ overexpression resulted in a striking resistance to skin cancer development and an increased expression of several tumor suppressor proteins, such as p53, p16, and p19. Mechanistically, this skin tumor–protective role of IKKβ is independent of p53, but dependent on the activity of the Ink4a/Arf locus. Interestingly, in the absence of p16 and p19, IKKβ-increased expression favors the appearance of cutaneous spindle cell–like squamous cell carcinomas, which are highly aggressive tumors. These results reveal that IKKβ activity prevents skin tumor development, and shed light on the complex nature of IKKβ effects on cancer progression, as IKKβ can both promote and prevent carcinogenesis depending on the cell type or molecular context.

Implications: The ability of IKKβ to promote or prevent carcinogenesis suggests the need for further evaluation when targeting this protein. Mol Cancer Res; 15(9); 1255–64. ©2017 AACR.

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Near-Infrared Photoimmunotherapy Targeting Prostate Cancer with Prostate-Specific Membrane Antigen (PSMA) Antibody

Prostate-specific membrane antigen (PSMA) is a membrane protein that is overexpressed manifold in prostate cancer and provides an attractive target for molecular therapy. Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that employs an antibody-photoabsorber conjugate (APC). Here, we describe the efficacy of NIR-PIT, using a fully human IgG₁ anti-PSMA monoclonal antibody (mAb), conjugated to the photoabsorber, IR700DX, in a PSMA-expressing PC3 prostate cancer cell line. Anti-PSMA-IR700 showed specific binding and cell-specific killing was observed after exposure of the cells to NIR light in vitro. In the in vivo study, anti-PSMA-IR700 showed high tumor accumulation and high tumor–background ratio. Tumor-bearing mice were separated into 4 groups: (i) no treatment; (ii) 100 μg of anti-PSMA-IR700 i.v.; (iii) NIR light exposure; (iv) 100 μg of anti-PSMA-IR700 i.v., NIR light exposure was administered. These were performed every week for up to 3 weeks. Tumor growth was significantly inhibited by NIR-PIT treatment compared with the other control groups (P < 0.001), and significantly prolonged survival was achieved (P < 0.0001 vs. other control groups). More than two thirds of tumors were cured with NIR-PIT. In conclusion, the anti-PSMA antibody is suitable as an APC for NIR-PIT. Furthermore, NIR-PIT with the anti-PSMA-IR700 antibody is a promising candidate of the treatment of PSMA-expressing tumors and could be readily translated to humans.

Implications: NIR-infrared photoimmunotherapy (NIR-PIT) using a fully human anti-PSMA-IR700 conjugate showed potential therapeutic effects against a PSMA-expressing prostate cancer that is readily translated to humans. Mol Cancer Res; 15(9); 1153–62. ©2017 AACR.

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Highlights of This Issue

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Inhibition of the Cell Death Pathway in Nonalcoholic Steatohepatitis (NASH)-Related Hepatocarcinogenesis Is Associated with Histone H4 lysine 16 Deacetylation

Hepatocellular carcinoma (HCC) is one of the most aggressive human cancers, and its incidence is steadily increasing worldwide. Recent epidemiologic findings have suggested that the increased incidence of HCC is associated with obesity, type II diabetes mellitus, and nonalcoholic steatohepatitis (NASH); however, the mechanisms and the molecular pathogenesis of NASH-related HCC are not fully understood. To elucidate the underlying mechanisms of the development of NASH-related HCC, we investigated the hepatic transcriptomic and histone modification profiles in Stelic Animal Model mice, the first animal model of NASH-related HCC to resemble the disease pathogenesis in humans. The results demonstrate that the development of NASH-related HCC is characterized by progressive transcriptomic alterations, global loss of histone H4 lysine 20 trimethylation (H4K20me3), and global and gene-specific deacetylation of histone H4 lysine 16 (H4K16). Pathway analysis of the entire set of differentially expressed genes indicated that the inhibition of cell death pathway was the most prominent alteration, and this was facilitated by persistent gene-specific histone H4K16 deacetylation. Mechanistically, deacetylation of histone H4K16 was associated with downregulation of lysine acetyltransferase KAT8, which was driven by overexpression of its inhibitor nuclear protein 1 (Nupr1). The results of this study identified a reduction of global and gene-specific histone H4K16 acetylation as a key pathophysiologic mechanism contributing to the development of NASH-derived HCC and emphasized the importance of epigenetic alterations as diagnostic and therapeutic targets for HCC.

Implications: Histone H4K16 deacetylation induces silencing of genes related to the cell death that occurred during the development of NASH-related HCC. Mol Cancer Res; 15(9); 1163–72. ©2017 AACR.

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Real-Time Transferrin-Based PET Detects MYC-Positive Prostate Cancer

Noninvasive biomarkers that detect the activity of important oncogenic drivers could significantly improve cancer diagnosis and management of treatment. The goal of this study was to determine whether ⁶⁸Ga-citrate (which avidly binds to circulating transferrin) can detect MYC-positive prostate cancer tumors, as the transferrin receptor is a direct MYC target gene. PET imaging paired with ⁶⁸Ga-citrate and molecular analysis of preclinical models, human cell-free DNA (cfDNA), and clinical biopsies were conducted to determine whether ⁶⁸Ga-citrate can detect MYC-positive prostate cancer. Importantly, ⁶⁸Ga-citrate detected human prostate cancer models in a MYC-dependent fashion. In patients with castration-resistant prostate cancer, analysis of cfDNA revealed that all patients with ⁶⁸Ga-citrate avid tumors had a gain of at least one MYC copy number. Moreover, biopsy of two PET avid metastases showed molecular or histologic features characteristic of MYC hyperactivity. These data demonstrate that ⁶⁸Ga-citrate targets prostate cancer tumors with MYC hyperactivity. A larger prospective study is ongoing to demonstrate the specificity of ⁶⁸Ga-citrate for tumors with hyperactive MYC.

Implications: Noninvasive measurement of MYC activity with quantitative imaging modalities could substantially increase our understanding of the role of MYC signaling in clinical settings for which invasive techniques are challenging to implement or do not characterize the biology of all tumors in a patient. Moreover, measuring MYC activity noninvasively opens the opportunity to study changes in MYC signaling in patients under targeted therapeutic conditions thought to indirectly inhibit MYC. Mol Cancer Res; 15(9); 1221–9. ©2017 AACR.

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Notch Represses Transcription by PRC2 Recruitment to the Ternary Complex

It is well established that Notch functions as a transcriptional activator through the formation of a ternary complex that comprises Notch, Maml, and CSL. This ternary complex then serves to recruit additional transcriptional cofactors that link to higher order transcriptional complexes. The mechanistic details of these events remain unclear. This report reveals that the Notch ternary complex can direct the formation of a repressor complex to terminate gene expression of select target genes. Herein, it is demonstrated that p19^Arf and Klf4 are transcriptionally repressed in a Notch-dependent manner. Furthermore, results indicate that Notch recruits Polycomb Repressor Complex 2 (PRC2) and Lysine Demethylase 1 (KDM1A/LSD1) to these promoters, which leads to changes in the epigenetic landscape and repression of transcription. The demethylase activity of LSD1 is a prerequisite for Notch-mediated transcriptional repression. In addition, a stable Notch transcriptional repressor complex was identified containing LSD1, PRC2, and the Notch ternary complex. These findings demonstrate a novel function of Notch and provide further insight into the mechanisms of Notch-mediated tumorigenesis.

Implications: This study provides rationale for the targeting of epigenetic enzymes to inhibit Notch activity or use in combinatorial therapy to provide a more profound therapeutic response. Mol Cancer Res; 15(9); 1173–83. ©2017 AACR.

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Histone H3.3K27M Represses p16 to Accelerate Gliomagenesis in a Murine Model of DIPG

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brainstem tumor genetically distinguished from adult GBM by the high prevalence of the K27M mutation in the histone H3 variant H3.3 (H3F3A). This mutation reprograms the H3K27me3 epigenetic landscape of DIPG by inhibiting the H3K27-specific histone methyltransferase EZH2. This globally reduces H3K27me2/3, critical repressive marks responsible for cell fate decisions, and also causes focal gain of H3K27me3 throughout the epigenome. To date, the tumor-driving effects of H3.3K27M remain largely unknown. Here, it is demonstrated that H3.3K27M cooperates with PDGF-B in vivo, enhancing gliomagenesis and reducing survival of p53 wild-type (WT) and knockout murine models of DIPG. H3.3K27M expression drives increased proliferation of tumor-derived murine neurospheres, suggesting that cell-cycle deregulation contributes to increased malignancy in mutant tumors. RNA sequencing on tumor tissue from H3.3K27M-expressing mice indicated global upregulation of PRC2 target genes, and a subset of newly repressed genes enriched in regulators of development and cell proliferation. Strikingly, H3.3K27M induced targeted repression of the p16/ink4a (CDKN2A) locus, a critical regulator of the G₀–G₁ to S-phase transition. Increased levels of H3K27me3 were observed at the p16 promoter; however, pharmacologic reduction of methylation at this promoter did not rescue p16 expression. Although DNA methylation is also present at this promoter, it is not K27M dependent. Intriguingly, inhibition of DNA methylation restores p16 levels and is cytotoxic against murine tumor cells. Importantly, these data reveal that H3.3K27M-mediated p16 repression is an important mechanism underlying the proliferation of H3.3K27M tumor cells, as in vivo cdkn2a knockout eliminates the survival difference between H3.3K27M and H3.3WT tumor-bearing mice.

Implications: This study shows that H3.3K27M mutation and PDGF signaling act in concert to accelerate gliomagenesis in a genetic mouse model and identifies repression of p16 tumor suppressor as a target of H3.3K27M, highlighting the G₁–S cell-cycle transition as a promising therapeutic avenue. Mol Cancer Res; 15(9); 1243–54. ©2017 AACR.

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NR4A2 Promotes DNA Double-strand Break Repair Upon Exposure to UVR

Exposure of melanocytes to ultraviolet radiation (UVR) induces the formation of UV lesions that can produce deleterious effects in genomic DNA. Encounters of replication forks with unrepaired UV lesions can lead to several complex phenomena, such as the formation of DNA double-strand breaks (DSBs). The NR4A family of nuclear receptors are transcription factors that have been associated with mediating DNA repair functions downstream of the MC1R signaling pathway in melanocytes. In particular, emerging evidence shows that upon DNA damage, the NR4A2 receptor can translocate to sites of UV lesion by mechanisms requiring post-translational modifications within the N-terminal domain and at a serine residue in the DNA-binding domain at position 337. Following this, NR4A2 aids in DNA repair by facilitating chromatin relaxation, allowing accessibility for DNA repair machinery. Using A2058 and HT144 melanoma cells engineered to stably express wild-type or mutant forms of the NR4A2 proteins, we reveal that the expression of functional NR4A2 is associated with elevated cytoprotection against UVR. Conversely, knockdown of NR4A2 expression by siRNA results in a significant loss of cell viability after UV insult. By analyzing the kinetics of the ensuing 53BP1 and RAD51 foci following UV irradiation, we also reveal that the expression of mutant NR4A2 isoforms, lacking the ability to translocate, transactivate, or undergo phosphorylation, display compromised repair capacity.

Implications: These data expand the understanding of the mechanism by which the NR4A2 nuclear receptor can facilitate DNA DSB repair. Mol Cancer Res; 15(9); 1184–96. ©2017 AACR.

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KITD816V Induces SRC-Mediated Tyrosine Phosphorylation of MITF and Altered Transcription Program in Melanoma

The oncogenic D816V mutation of the KIT receptor is well characterized in systemic mastocytosis and acute myeloid leukemia. Although KIT^D816V has been found in melanoma, its function and involvement in this malignancy is not understood. Here we show that KIT^D816V induces tyrosine phosphorylation of MITF through a triple protein complex formation between KIT, MITF, and SRC family kinases. In turn, phosphorylated MITF activates target genes that are involved in melanoma proliferation, cell-cycle progression, suppression of senescence, survival, and invasion. By blocking the triple protein complex formation, thus preventing MITF phosphorylation, the cells became hypersensitive to SRC inhibitors. We have therefore delineated a mechanism behind the oncogenic effects of KIT^D816V in melanoma and provided a rationale for the heightened SRC inhibitor sensitivity in KIT^D816V transformed cells.

Implications: This study demonstrates that an oncogenic tyrosine kinase mutant, KIT^D816V, can alter the transcriptional program of the transcription factor MITF in melanoma Mol Cancer Res; 15(9); 1265–74. ©2017 AACR.

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Normal and Cancerous Tissues Release Extrachromosomal Circular DNA (eccDNA) into the Circulation

Cell-free circulating linear DNA is being explored for noninvasive diagnosis and management of tumors and fetuses, the so-called liquid biopsy. Previously, we observed the presence of small extrachromosomal circular DNA (eccDNA), called microDNA, in the nuclei of mammalian tissues and cell lines. Now, we demonstrate that cell-free microDNA derived from uniquely mapping regions of the genome is detectable in plasma and serum from both mice and humans and that they are significantly longer (30%–60% >250 bases) than cell-free circulating linear DNA (~150 bases). Tumor-derived human microDNA is detected in the mouse circulation in a mouse xenograft model of human ovarian cancer. Comparing the microDNA from paired tumor and normal lung tissue specimens reveals that the tumors contain longer microDNA. Consistent with human cancers releasing microDNA into the circulation, serum and plasma samples (12 lung and 11 ovarian cancer) collected prior to surgery are enriched for longer cell-free microDNA compared with samples from the same patient obtained several weeks after surgical resection of the tumor. Thus, circular DNA in the circulation is a previously unexplored pool of nucleic acids that could complement miRNAs and linear DNA for diagnosis and for intercellular communication.

Implications: eccDNA derived from chromosomal genomic sequence, first discovered in the nuclei of cells, are detected in the circulation, are longer than linear cell-free DNA, and are released from normal tissue and tumors into the circulation. Mol Cancer Res; 15(9); 1197–205. ©2017 AACR.

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Augmented TME O-GlcNAcylation Promotes Tumor Proliferation through the Inhibition of p38 MAPK

O-GlcNAcylation is a dynamic O-linked glycosylation event that plays a crucial role in regulating cellular signaling. Recent studies indicate that increased O-GlcNAcylation is a general feature in cancer and contributes to various cancer phenotypes, including cell proliferation, survival, invasion, metastasis, and energy metabolism. However, the role of O-GlcNAcylation in the tumor microenvironment (TME) is not fully elucidated. Here, B16 melanoma cells were subcutaneously transplanted into O-GlcNAc transferase transgenic (Ogt-Tg) mice exhibiting elevated O-GlcNAcylation to examine the effect of O-GlcNAcylation in the TME on tumor progression. In this model system, B16 tumor growth was significantly higher in Ogt-Tg/+ mice compared with wild-type (WT) mice. The tumors grown in Ogt-Tg/+ mice showed significant downregulation of p38 MAPK activity and upregulation of the ERK1/2 signaling pathway. In addition, proinflammatory cytokine production was significantly lower in the tumor tissues from Ogt-Tg/+ mice than in those from WT mice. Activation of NF-B, a key regulator in the cytokine production, was downregulated in the macrophages of the tumor tissues grown in Ogt-Tg/+ mice. These data reveal that elevated O-GlcNAcylation in the TME reduces the production of inflammatory cytokines and promotes cancer progression through downregulation of p38 MAPK activity and subsequent upregulation of the ERK1/2 signaling pathway.

Implications: The reduced production of inflammatory cytokines by augmented O-GlcNAcylation in the TME, mainly macrophages, promotes tumor proliferation through the inhibition of p38 MAPK and suggests a possible cause of increased morbidity and mortality rates for various cancers in diabetic patients. Mol Cancer Res; 15(9); 1287–98. ©2017 AACR.

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Factors Affecting Adjuvant Therapy in Stage III Pancreatic Cancer—Analysis of the National Cancer Database

Background: Adjuvant therapy after curative resection is associated with survival benefit in stage III pancreatic cancer. We analyzed the factors affecting the outcome of adjuvant therapy in stage III pancreatic cancer and compared overall survival with different modalities of adjuvant treatment. Methods: This is a retrospective study of patients with stage III pancreatic cancer listed in the National Cancer Database (NCDB) who were diagnosed between 2004 and 2012. Patients were stratified based on adjuvant therapy they received. Unadjusted Kaplan-Meier and multivariable Cox regression analysis were performed. Results: We analyzed a cohort included 1731 patients who were recipients of adjuvant therapy for stage III pancreatic cancer within the limits of our database. Patients who received adjuvant chemoradiation had the longest postdiagnosis survival time, followed by patients who received adjuvant chemotherapy, and finally patients who received no adjuvant therapy. On multivariate analysis, advancing age and patients with Medicaid had worse survival, whereas Spanish origin and lower Charlson comorbidity score had better survival. Conclusions: Our study is the largest trial using the NCDB addressing the effects of adjuvant therapy specifically in stage III pancreatic cancer. Within the limits of our study, survival benefit with adjuvant therapy was more apparent with longer duration from date of diagnosis.

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Recombinant rabies virus expressing IL-15 enhances immunogenicity through promoting the activation of dendritic cells in mice

Abstract

Rabies remains a public health threat that kills approximately 59,000 people worldwide each year, most of which are from the developing countries of Africa and Asia where dog rabies are endemic. Therefore, developing an affordable and efficacious vaccine is crucial for rabies control in these countries. Interleukin (IL)-15, an immunoregulatory cytokine, is a pluripotent molecule with therapeutic potential, which targets many cell types and links the innate and adaptive immune system. In this study, IL-15 gene was cloned and inserted into the genome of a recombinant rabies virus (RABV) strain LBNSE (designated as LBNSE-IL15), and the effect of over-expression of IL-15 on the immunogenicity of RABV was investigated. It was found that mice vaccinated with LBNSE-IL15 could induce significantly higher level of virus-neutralizing antibody (VNA) than those immunized with LBNSE, resulting in the higher protection after challenge. Further investigation was performed to find out the possible role of IL-15 plays in the process of antibody induction, and it was found that LBNSE-IL15 could enhance the maturation of dendritic cells (DCs) in immunized mice. Furthermore, the mice immunized with LBNSE-IL15 could promote the T_FH cells differentiation and the generation of germinal center B cells and plasma cells. Together, these data indicated that IL-15 could be a potential adjuvant in enhancing the immunogenicity of RABV, contributing to the development of more-efficacious rabies vaccines.

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An update on studies on etiological factors, disease progression, and malignant transformation in oral submucous fibrosis

Samiha Bari, Rashmi Metgud, Zankhana Vyas, Aniruddh Tak

Journal of Cancer Research and Therapeutics 2017 13(3):399-405

Worldwide estimates of oral submucous fibrosis (OSMF) show a confinement to Indians and Southeast Asians. In India, the prevalence of OSMF has increased over the past four decades from 0.03% to 6.42%. The condition is well recognized for its malignant potential rate of 7-30%. The condition has a multifactorial etiology and may remain either stationary or become severe, leaving an individual physically challenged both physically and psychologically. Hence, the study aims at reviewing studies done on various etiological factors leading to its onset. Their analysis may serve as an adjunct in defining the broad spectrum of the causation of this potentially malignant disorder.

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A study of the distribution of B-cell lymphoma/leukemia-2 in odontogenic cyst and tumors: Histochemical study

Khushboo Phull, Rashmi Metgud, Shrikant Patel

Journal of Cancer Research and Therapeutics 2017 13(3):570-575

Introduction: The odontogenic keratocyst is known for its aggressiveness, high recurrence rate, and transformation of keratinized epithelia to nonkeratinized squamous epithelium for which inflammation has been suggested to be responsible. B-cell lymphoma/leukemia-2 (Bcl-2) an anti-apoptotic protein, prolongs the life span of epithelial cells and allows proliferation, differentiation, and morphogenesis. Materials and Methods: Study was carried out comprising of 90 cases; (30 ameloblastoma, 30 keratocystic odontogenic tumor (KCOT), and 30 radicular cyst). Bcl-2 expression was determined with respect to localization, area (percentage) and intensity of stained cells in epithelium, and connective tissue stroma by counting the endothelial, round, and fusiform cells. Results: In epithelium bcl-2 expression in KCOTs was higher followed by ameloblastoma and lowest in the radicular cyst. Whereas, in connective tissue stroma bcl-2 expression was higher in KCOT and radicular cyst than ameloblastoma cases. Solid variants showed statistically higher expression as compared to the unicystic variants of ameloblastoma (P = 0.009, 0.033, 0.011, and 0.041). Conclusion: High expression of bcl-2 in KCOT supports the general agreement that some features of KCOT are those of a neoplasia. The bcl-2 expression in connective tissue cells suggests that these cells may also be important as epithelial cells in the biological behavior odontogenic keratocyst.

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Physical activity influences the immune system of breast cancer patients

Thorsten Schmidt, Marion van Mackelenbergh, Daniela Wesch, Christoph Mundhenke

Journal of Cancer Research and Therapeutics 2017 13(3):392-398

It has been suggested that physical activity in breast cancer patients can not only improve quality of life. Influences on physical and psychological levels have been evaluated, but effects on the immune system of breast cancer patients are hardly known. A PubMed search identified relevant trials and meta-analyses from 1970 to 2013. This review summarizes the results of international studies and the current discussion of effects of physical activity on the immune system of breast cancer patients. Highlighted are effects of physical activity on the immune system. Seven original articles and 14 reviews included in this review. Two original and the review articles includes other tumor entities besides breast cancer.Evaluated methods such as dose-response relationships for exercise in oncology, hardly exist. Increased immunological anti-cancer activity due to physical activity is probably mediated via an increase in number and cytotoxicity of monocytes and natural killer cells and cytokines.

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The impact of patient positioning and use of belly board on small bowel toxicity in patients receiving pelvic radiotherapy for gynecological malignancies

Milan Anjanappa, Rajeev Kavalakara Raghavan, Francis V James, Aswin Kumar, Susan Mathews, Sarin Bhaskaran, Shaiju Vasudevannair Sreedevi, Puthuveetil Govindan Jayaprakash

Journal of Cancer Research and Therapeutics 2017 13(3):519-523

Aim: To determine the efficacy of belly board device in patients receiving postoperative radiation for gynecological malignancies in terms of setup error and acute small bowel toxicity. Materials and Methods: Patients requiring postoperative radiation for gynecological malignancies were prospectively randomized to either treatment with supine position (supine arm) or prone position using belly board (prone arm). Each patient underwent computed tomography simulation in the assigned treatment position, and a three-dimensional conformal radiation was planned. Weekly two to three treatment sessions were verified using portal imaging and setup errors were noted. All patients were reviewed weekly to assess for symptoms and toxicity using a structured format. The systematic and random errors were calculated along the three axes. Results: Twenty-four patients were randomized and 22 patients were available for the final analysis. The systematic error in supine arm versus prone arm was 3.9 versus 3.5 mm, 2.1 versus 4.8 mm and 3.1 versus 3.1 mm along lateral, antero-posterior (AP) and cranio-caudal (CC) direction. The random error in supine arm versus prone arm was 5 versus 3.9 mm, 2.9 mm versus 4.4 mm and 4.3 versus 3.4 mm along lateral, AP and CC direction. The calculated planning target volume margin for supine arm was 1.3, 0.7, and 1.0 cm and margin for prone arm was 1.1, 1.5, and 1.0 cm along lateral, AP, and CC direction, respectively. One patient in supine arm developed Grade 3 toxicity. Conclusion: The systematic error and random error is more along AP direction for prone position. The acute small bowel toxicity was less using belly board.

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Comment on: Survey of cervical cancer survivors regarding quality of life and sexual function

Antonio Simone Laganà, Valentina Lucia La Rosa, Daniele Fanale, Salvatore Giovanni Vitale

Journal of Cancer Research and Therapeutics 2017 13(3):598-599



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Lactate – A new frontier in the immunology and therapy of prostate cancer

Iuliana Nenu, Grigore-Aristide Gafencu, Tiberiu Popescu, Gabriel Kacso

Journal of Cancer Research and Therapeutics 2017 13(3):406-411

Prostate cancer, one of the most common male malignancies with an increasing incidence in the recent years, requires the development of new methods of treatment. One of the most debated subjects is the tumor-associated macrophages (TAM). Although, the pathophysiological mechanisms are still a subject of intense research, TAM acts as procarcinogenic factors. It was also demonstrated that hypoxia-inducible factor 1 (HIF1) induces the expression of TAM genes involved in prostate carcinogenesis. Furthermore, it should be noted that the stromal extracellular lactate, the result of tumoral glycolysis process is one of the HIF1 activators. In addition, lactate inhibits the differentiation of monocytes and dendritic cells and also induces the inactivation of the cytotoxic T-lymphocytes. Through an analysis of recent studies, we conclude that lactate is a vital component of several ways of modulating the immune response at the stromal prostatic adenocarcinoma including TAM activation and cytotoxic T lymphocytes immunosuppression. Our review focuses on the impact of lactate on prostatic adenocarcinoma progression in terms of its immunology, and how this influences the therapy of this condition and the clinical outcome.

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Honey bee venom combined with 1,25-dihydroxyvitamin D3as a highly efficient inducer of differentiation in human acute myeloid leukemia cells

Homa Mohseni-Kouchesfahani, Mohammad Nabioni, Zahra Khosravi, Maryam Rahimi

Journal of Cancer Research and Therapeutics 2017 13(3):544-549

Purpose: Most cancer cells exhibit a defect in their capacity to mature into nonreplicating adult cells and existing in a highly proliferating state. Differentiation therapy by agents such as 1,25-dihydroxyvitamin D3(1,25-(OH)2 VD3) represents a useful approach for the treatment of cancer including acute myeloid leukemia. Human myeloid leukemia cell lines are induced to terminal differentiation into monocyte lineage by 1,25-(OH)2 VD3. However, usage of these findings in the clinical trials is limited by calcemic effects of 1,25-(OH)2 VD3. Attempts to overcome this problem have focused on a combination of low concentrations 1,25-(OH)2 VD3 with other compounds to induce differentiation of HL-60 cells. In this study, the effect of honey bee venom (BV) and 1,25-(OH)2 VD3, individually and in combination, on proliferation and differentiation of human myeloid leukemia HL-60 cells were assayed. Materials and Methods: In this in vitro study, toxic and nontoxic concentrations of BV and 1,25-(OH)2 VD3 were tested using Trypan blue stained cell counting and (3[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. In addition, differentiation of cells was assayed using a Wright-Giemsa staining and nitroblue tetrazolium reduction test. Data were analyzed by a one-way analysis of the variance test using SPSS software. Results: Our findings showed that both the BV and 1,25-(OH)2 VD3, in a dose and time-dependent manner, caused cell death at high concentrations and inhibited cell proliferation at lower concentrations. About 5 nM of 1,25-(OH)2 VD3 induced differentiation of HL-60 cells to monocytes after 72 h. 2.5 μg/ml of BV suppressed proliferation of HL-60 cells but had not any effects on their differentiation, whereas in combination with 5 nM of 1,25-(OH)2 VD3, it enhanced antiproliferative and differentiation potency of 1,25-(OH)2 VD3. Conclusions: These results indicate that BV potentiates the 1,25-(OH)2 VD3-induced HL-60 cell differentiation into monocytes

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