Citations

Background Fremanezumab (formerly TEV‐48125) is a monoclonal antibody directed against calcitonin‐gene‐related peptide (CGRP), a validated target for migraine preventive therapy. In two previous phase 2 studies, fremanezumab administered once every 28 days for 12 weeks was found to be effective and safe as a preventive treatment for patients suffering from episodic migraine (EM) and chronic migraine (CM). Objective To evaluate the efficacy and safety of fremanezumab as an add‐on preventive therapy in individuals with EM and CM who are on stable doses of preventive migraine medications. Methods Two randomized placebo‐controlled studies tested once‐monthly subcutaneous injections of various dosing regimens of fremanezumab versus placebo in EM and CM. Headache information was captured daily using an electronic headache diary. For these post hoc analyses, data were pooled from patients who were on stable preventive medications and taking fremanezumab doses of 225 mg or 675/225 mg, or placebo. Results The sample consisted of 133 patients, (67 fremanezumab and 66 placebo). Total reduction in migraine days for the duration of the study was 12.4 for fremanezumab and 7.4 for placebo ( P = .0321). There were also decreases in moderate/severe headache days (12.5 vs 7.1, P = .0058), and days using acute medication for headaches relative to placebo (11.6 vs 7.5, P = .0414). Treatment emergent adverse events were generally mild and transient, and no serious adverse events were considered to be treatment‐related by the site investigators. Conclusions The findings from these post hoc analyses suggest that fremanezumab is a safe and effective add‐on treatment for migraine patients being concomitantly treated with other migraine preventive medications. Trials are registered at Clinicaltrials.gov NCT02025556 and NCT02021773.

AbstractThe purpose of this study is to identify regions of spinal column in which more than three contiguous vertebrae can be reliably and quickly aligned within 1 mm using a 6‐degree (6D) couch and full body immobilization device. We analyzed 45 cases treated over a 3‐month period. Each case was aligned using ExacTrac x‐ray positioning system with integrated 6D couch to be within 1° and 1 mm in all six dimensions. Cone‐Beam computed tomography (CBCT) with at least 17.5 cm field of view (FOV) in the superior–inferior direction was taken immediately after ExacTrac positioning. It was used to examine the residual error of five to nine contiguous vertebrae visible in the FOV. The residual error of each vertebra was determined by expanding/contracting the vertebrae contour with a margin in millimeter integrals on the planning CT such that the new contours would enclose the corresponding vertebrae contour on CBCT. Submillimeter initial setup accuracy was consistently achieved in 98% (40/41) cases for a span of five or more vertebrae starting from T2 vertebra and extending caudally to S5. The curvature of spinal column along the cervical region and cervicothoracic junction was not easily reproducible between treatment and simulation. Fifty‐seven percent (8/14) of cases in this region had residual setup error of more than 1 mm in nearby vertebrae after alignment using 6D couch with image guidance. In conclusion, 6D couch integrated with image guidance is convenient and accurately corrects small rotational shifts. Consequently, more than three contiguous vertebrae can be aligned within 1 mm with immobilization that reliably reproduces the curvature of the thoracic and lumbar spinal column. Ability of accurate setup is becoming less a concern in limiting the use of stereotactic radiosurgery or stereotactic body radiation therapy to treat multilevel spinal target.

BackgroundThe ability to isolate extracellular vesicles (EVs) such as exosomes or microparticles is an important method that is currently not standardized. While commercially available kits offer purification of EVs from biofluids, such purified EV samples will also contain non‐EV entities such as soluble protein and nucleic acids that could confound subsequent experimentation. Ideally, only EVs would be isolated and no soluble protein would be present in the final EV preparation.MethodsWe compared commercially available EV isolation kits with immunoaffinity purification techniques and evaluated our final EV preparations using atomic force microscopy (AFM) and nanoscale flow cytometry (NFC). AFM is the only modality capable of detecting distinguishing soluble protein from EVs which is important for downstream proteomics approaches. NFC is the only technique capable of quantitating the proportion of target EVs to non‐target EVs in the final EV preparation.ResultsTo determine enrichment of prostate derived EVs relative to non‐target MPs, anti‐PSMA (Prostate Specific Membrane Antigen) antibodies were used in NFC. Antibody‐based immunoaffinity purification generated the highest quality of prostate derived EV preparations due to the lack of protein and RNA present in the samples. All kits produced poor purity EV preparations that failed to deplete the sample of plasma protein.ConclusionsWhile attractive due to their ease of use, EV purification kits do not provide substantial improvements in isolation of EVs from biofluids such as plasma. Immunoaffinity approaches are more efficient and economical and will also eliminate a significant portion of plasma proteins which is necessary for downstream approaches.

Abstract Lung cancer is the leading cancer-related cause of death in the United States. We had previously performed multipoint linkage analysis on families with a strong family history of lung cancer and found significant linkage to the 6q25 region. In order to find the source of that linkage signal, we performed targeted sequencing 6q23-6q27 on 75 individuals from the 9 most highly linked families. We performed two types of parametric linkage analysis, using an autosomal dominant mode of inheritance with 10% penetrance for carriers and a 1% phenocopy rate using a disease allele frequency of 1%. The first type was a two-point analysis using an Elston-Stewart algorithm. This approach did not lead to any genome-wide significant results but demonstrated significant heterogeneity throughout the nine families.In an effort to recover the power from the original multipoint analysis, we performed a regional linkage analysis using SEQLinkage and MERLIN. SEQLinkage built regional haplotypes that corresponded to a gene or an intergenic region - the regions were based on a customized map of our design. The regional markers were multiallelic which allowed for greater information content and were similar to the microsatellites that were used in the original multipoint analysis. The regional markers were then analyzed in a two-point linkage analysis via MERLIN. This allowed us to identify two genome-wide significant signals at PACRG-AS1 at 6q26 (HLOD = 3.4) and SAMD5 at 6q24 (3.3). The PACRG-AS1 is novel, though it is associated with the known lung cancer gene PARK2. SAMD5 may be involved in lung cancer cell proliferation. The heterogeneity of the two signals was particularly interesting. The PACRG-AS1 was driven primarily by two families. The SAMD5 marker was not being strongly driven by any of the families and appears to be a small cumulative effect across the nine families. In addition, several families have large, but non-significant LOD scores at other loci across the region. This further reinforces the locus heterogeneity within the region, and it is likely that our two significant signals here are not the only variants affecting the phenotype. We further attempted to localize the signals by running SEQLinkage using a custom map where genes are broken into exons and introns. Though this resulted in no significant markers, the highest signal was located in the intronic region of SAMD5 (HLOD = 3) and several other suggestive signals were localized to intronic regions of good candidate genes like SASH1 and ARID1B. Examination of predicted effects of the candidate regulatory variants using eQTL databases is ongoing. This is a region full of promising candidates, and it is likely that the two significant signals found here are just part of many that could be affecting lung cancer risk. We plan to do further analysis within the individual families to elucidate any more genes affecting this signal. Citation Format: Anthony M. Musolf, Claire L. Simpson, Bilal A. Moiz, Candace Middlebrooks, Mariza de Andrade, Diptasri Mandal, Colette Gaba, Ping Yang, Yafang Li, Ming You, Elena Y. Kupert, Marshall W. Anderson, Ann G. Schwartz, Susan M. Pinney, Christopher I. Amos, Joan E. Bailey-Wilson. A study in locus heterogeneity: Targeted sequencing analysis of 6q reveals multiple significant loci as the source of a previous linkage peak in familial lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4290. doi:10.1158/1538-7445.AM2017-4290

Abstract Lung cancer (LC) is the leading cancer killer of Americans; an estimated 158,000 people will die in the U.S. from LC in 2016. While it is well-known that LC risk is affected by the environment, particularly tobacco smoking, there is a substantial genetic risk to LC also. We examined genotype data from Illumina HumanCore-12v1-0 array (297,000 SNPs) on 175 individuals in 25 extended families with a strong history of LC recruited by the Genetic Epidemiology of Lung Cancer Consortium. The purpose of this study is to determine which families are segregating a high-penetrance genetic risk haplotype so that the most informative families can be selected for DNA sequencing studies. Quality control was performed to remove SNPs and individuals with greater than 1% missingness as well as monomorphic SNPs. SNPs with Mendelian errors in more than one family were dropped. Identity-by-descent values were calculated to confirm correct familial relationships; 1 individual was dropped. After quality control we were left with approximately 245,000 SNPs for analysis. We performed three types of parametric linkage analyses using an autosomal dominant model with 40% penetrance in carriers and 1% penetrance in non-carriers. A disease allele frequency of 0.01 was used. Standard single variant two-point analysis between the disease and each marker was performed using TwoPointLods. Multipoint linkage analysis was performed using SimWalk2. We also performed regional-based linkage analyses using SEQLinkage and MERLIN. SEQLinkage builds a multiallelic regional marker (similar to a microsatellite) that corresponds to a gene or a portion of a gene. Two-point linkage analyses were then performed on the regional markers using MERLIN. We identified five loci that were genome-wide significant (HLOD score ≥ 3.3) from the regional-based linkage analyses on 18p11.23 (HLOD = 4.1), 2p22.2 (3.9), 14q13.1 (3.7), 16p13.1 (3.4), and 20q13.11 (3.4). It is particularly exciting that the scores centered on prospective cancer genes. Our highest score was centered on PTPRM, a protein tyrosine phosphatase on chromosome 18 that has been implicated as a LC oncogene. The signal on 20q13 also centered on another protein tyrosine phosphatase (PTPRT) that has been shown to be mutated in LC cells. The source of the signal on 16p13 was RNA binding protein RBFOX1, which has been shown to be deleted in LC cell lines and the source of the signal on 2p22 was LRP1B, a LDL receptor-related protein that is often inactivated in LC cells. The source of the final signal on 14q13 was NPAS3, a transcription factor that is a tumor suppressor in brain tumors. It should be noted that all previous evidence linking these genes to cancer was based on somatic mutations; this is the first time any of these genes has been shown to be significantly linked to germline disease risk in a family-based study. We plan to perform targeted sequencing on the linked regions to elucidate the exact causal variant. Citation Format: Joan E. Bailey-Wilson, Anthony M. Musolf, Claire L. Simpson, Mariza de Andrade, Diptasri Mandal, Colette G. Gaba, Ping Yang, Ming You, Elena Y. Kupert, Marshall W. Anderson, Ann G. Schwartz, Susan M. Pinney, Christopher I. Amos. Familial lung cancer is significantly linked to cancer-associated genes on five chromosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4268. doi:10.1158/1538-7445.AM2017-4268

Abstract Prostate cancer is the most frequently diagnosed male cancer. For prostate cancer that has progressed to an advanced or metastatic stage, androgen deprivation therapy (ADT) is the standard of care. ADT inhibits activity of the androgen receptor (AR), a master regulator transcription factor in normal and cancerous prostate cells. The major limitation of ADT is the development of castration-resistant prostate cancer (CRPC), which is almost invariably due to transcriptional re-activation of the AR. One mechanism of AR transcriptional re-activation is expression of AR-V7, a truncated, constitutively active AR variant (AR-V) arising from alternative AR pre-mRNA splicing. Noteworthy, AR-V7 is being developed as a predictive biomarker of primary resistance to androgen receptor (AR)-targeted therapies in CRPC. Multiple additional AR-V species are expressed in clinical CRPC, but the extent to which these may be co-expressed with AR-V7 or predict resistance is not known. Here we utilized long read sequencing to identify and quantify AR isoforms expressed in CRPC. To unambiguously characterize all AR isoforms, we prepared Iso-Seq™ libraries via 3’ rapid amplification of cDNA ends (RACE) with RNA isolated from prostate cancer cell lines and xenograft tissues using a forward primer anchored in AR exon 1. 3’RACE reactions were subjected to single molecule, real-time (SMRT®) long-read sequencing with a Pacific Biosciences RSII System. Our work identified AR-V9 as a truncated isoform that is frequently co-expressed with AR-V7 in CRPC. Mechanistically, our work re-annotated AR-V7 and AR-V9 mRNAs, showing these two species shared a common 3’ terminal exon containing separate splice acceptor sites. Taking into account this new information, novel siRNAs and antibodies which could distinguish between AR-V7 and AR-V9 were designed, validated and used to measure the relative expression of these two AR isoforms in CRPC cells with a view to determining the potential of AR-V9 as a predictive biomarker of primary resistance to AR-targeted therapies. Citation Format: Manish Kohli, Yeung Ho, David W. Hillman, Jamie L. Van Etten, Christine Henzler, Rendong Yang, Yingming Li, Elizabeth Tseng, Ting Hon, Tyson A. Clark, Liguo Wang, Kevin Silverstein, Liewei Wang, Scott M. Dehm. SMRT® Sequencing of full-length androgen receptor isoforms in prostate cancer reveals previously hidden drug resistant variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 425. doi:10.1158/1538-7445.AM2017-425

Abstract Purpose: The treatment of ovarian cancer poses a challenge since 70% of patients will relapse with incurable platinum resistant disease. Although ovarian cancers lack frequently-occurring driver mutations or amplifications, the study presented herein examines the concept of individualized therapy based on mate pair sequencing and protein expression in ovarian cancer patient-derived xenografts. Experimental Procedure: Macrodissection of patient tumor was followed by genomic DNA isolation and next-generation sequencing using an Illumina Mate Pair protocol to identify structural genomic changes and copy number variations. Secondary analyses were carried out to select potential therapeutic targets among those. The tumors were also propagated intraperitoneally in immunocompromised mice and treated with standard chemotherapy or/and targeted therapy chosen based on genomic analyses. Results: Mate pair analysis revealed that, in general, copy number changes (amplifications, gains and losses) were common in ovarian tumors and included genes potentially targetable. Tumors from early generation patient- derived xenografts showed a landscape of genomic aberrations identical to that of original patient tumor. One PDX model in which high expression of HER2 and copy number gains at RICTOR and AKT genetic loci were revealed, was treated with either chemotherapy and Pertuzumab/trastuzumab (HER2 inhibitors), MK-8669 (mTOR inhibitor) or MK-2206 (AKT inhibitor) or chemotherapy alone. Tumor size was measured by serial abdominal ultrasound and after 28 days, the final tumor size ratio compared to baseline was 0.23, 0.36, 0.55 and 0.56, respectively, indicating that the best response was seen in the combined chemotherapy and Pertuzumab/trastuzumab cohort and that was better than in the chemotherapy only cohort. Conclusions: Patient-derived xenografts models in conjunction with genomic and protein analyses of patient tumors are valuable for testing of combination therapies including specific targeting drugs. This approach also provides actionable information for more tailored treatment of ovarian cancer patients. Citation Format: Konstantinos Leventakos, Faye R. Harris, Lin Yang, Xiaonan Hou, Saravut Weroha, Irina V. Kovtun. Testing personalized therapies for ovarian cancer using mate pair genomic analysis and patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4814. doi:10.1158/1538-7445.AM2017-4814

Abstract Introduction: Terminal duct lobular unit (TDLU) involution, an age-related physiological process, is characterized by the reduction of size and numbers. Reduced involution is associated with higher mammographic density, a strong breast cancer risk factor. Both factors predict risk of developing breast cancer among women with benign disease. Given that prior studies have been conducted mainly in White women, we compared the extent of TDLU involution among Western and Asian breast cancer cases to describe potential racial heterogeneity in breast cancer etiology. Method: We obtained three TDLU involution metrics (count/100 mm2, mean span, and mean acini count/TDLU; all inversely correlated with TDLU involution) measured in benign breast tissue sections from 379 Chinese (254 luminal A and 125 core basal phenotype [CBP] cases) and 476 Polish (407 luminal A and 69 CBP) breast cancer cases. Polytomous logistic regression was performed using tertiles of TDLU measures as ordinal dependent variable and race as an independent variable. Covariates included age, body mass index, and parity. Analyses were performed separately by age group (<50, ≥50) and subtype (luminal A and CBP). Results: Among luminal A cases, Chinese had significantly greater TDLU count compared with Polish in both age groups (ORtrend= 11.0; 95% CI=2.80-43.2; Ptrend=0.001 for age<50; ORtrend= 12.1; 95% CI=5.12-28.7; Ptrend=1E-08 for age≥50). In addition, Chinese were more likely to have greater mean span and acini count among younger cases (ORtrend=2.5; 95% CI=1.44-4.28; Ptrend=0.001 for mean span and ORtrend=7.4; 95% CI=4.13-13.4; Ptrend=3E-11 for acini count) but not among older cases. Among CBP cases, Chinese were more likely to have greater acini count compared to Polish only among younger women (ORtrend=2.6; 95% CI=1.12-6.95; Ptrend=0.03 for age<50). Conclusion: We found Chinese were more likely to have reduced TDLU involution compared to Polish cases after accounting for potential confounders, with greater differences for luminal A and younger women. Studies of TDLU involution in diverse populations are needed to confirm and understand whether population difference in TDLU involution metrics are related to age at breast cancer onset and risks for specific tumor subtypes. Citation Format: Hyuna Sung, Changyuan Guo, Jennifer Guida, Shan Zheng, Erni Li, Jing Li, Nan Hu, Joseph Deng, Montserrat Garcia-Closas, Jonine Figueroa, Mark Sherman, Gretchen Gierach, Ning Lu, Xiaohong R. Yang. Racial variation in terminal duct lobular unit (TDLU) involution in Chinese and Polish breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5290. doi:10.1158/1538-7445.AM2017-5290

Abstract Elevated expression levels of MYC family genes are frequently observed in human cancer cells and correlate with tumor aggressiveness and poor prognosis. In neuroblastoma 40% of all cases are high-risk, of which 20% harbor amplification of the MYCN proto-oncogene. In high-risk cases lacking MYCN gene amplification, high expression levels of c-MYC (MYC) are often present and are associated with unfavorable histology and a poor survival. Unlike MYCN amplification, which is frequently observed in the presence of segmental chromosomal aberrations, MYC overexpression is not associated with genetic abnormalities or somatic mutations. In order to study this newly defined subgroup, we have created a novel transgenic zebrafish model in which overexpression of MYC alone in the peripheral sympathetic nervous system (PSNS) drives early-onset neuroblastoma in nearly every fish by seven weeks of age. The tumors resulting from MYC overexpression arise in the interrenal gland, which is the fish counterpart of the adrenal medulla, and are histologically identical to human neuroblastoma. We next performed the Assay for Transposase Accessible Chromatin (ATAC) sequencing and RNA-seq to identify open chromatin regions that correlate with activation of gene transcription. Lineage specific genes essential for neuronal precursor cell identity, such as PHOX2B, HAND2, and TFAP2A are highly expressed in both MYC-expressing and MYCN-amplified human neuroblastoma cell lines and are actively transcribed in zebrafish models of MYC and MYCN driven neuroblastoma. Furthermore, these studies reveal shared and differential regulatory of effects of MYC relative to MYCN activity in maintaining the malignant phenotype of neuroblastoma in vivo. Additional insight into the mechanisms of aberrant transcriptional regulation will inform the future design and use of therapeutic strategies targeting transcription in this high-risk malignancy of childhood. Citation Format: Mark W. Zimmerman, Shuning He, Shizhen Zhu, Song Yang, Yi Zhou, Leonard I. Zon, A Thomas Look. Modeling the chromatin and transcriptional landscape of MYC and MYCN driven neuroblastoma in zebrafish [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3871. doi:10.1158/1538-7445.AM2017-3871