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Chemotherapy-induced peripheral neuropathy (CIPN) is a common and debilitating condition associated with a number of chemotherapeutic agents. Drugs commonly implicated in the development of CIPN include platinum agents, taxanes, vinca alkaloids, bortezomib, and thalidomide analogues. As a drug response can vary between individuals, it is hypothesized that an individual's specific genetic variants could impact the regulation of genes involved in drug pharmacokinetics, ion channel functioning, neurotoxicity, and DNA repair, which in turn affect CIPN development and severity. Variations of other molecular markers may also affect the incidence and severity of CIPN. Hence, the objective of this review was to summarize the known biological (molecular and genomic) predictors of CIPN and discuss the means to facilitate progress in this field.

Simple Summary Chemotherapy-induced peripheral neuropathy (CIPN) is a common debilitating complication of treatment with platinum-based compounds and taxanes. CIPN is predominantly a sensory symptom, causing numbness, tingling, and pain in the hands and/or feet. We performed a genome-wide association study on two independent study groups (N08Cx comprised of NCCTG clinical trial participants in the N08C1, N08CA, and N08CB studies; and Mayo Clinic Breast Disease Registry (MCBDR)) to find genetic variants that are associated with sensory symptoms during or after paclitaxel, paclitaxel and carboplatin, or oxaliplatin receipt. A genetic variant (single nucleotide polymorphism, SNP) rs56360211 near PDE6C had a very strong association with CIPN in N08Cx but not in the MCBDR, while the variant rs113807868 near TMEM150C was significantly associated with CIPN in the MCBDR but not in N08Cx. This lack of replication suggests that neither is actually strongly associated with CIPN. Abstract Chemotherapy-induced peripheral neuropathy (CIPN) is a common and potentially permanent adverse effect of chemotherapeutic agents including taxanes such as paclitaxel and platinum-based compounds such as oxaliplatin and carboplatin. Previous studies have suggested that genetics may impact the risk of CIPN. We conducted genome-wide association studies (GWASs) for CIPN in two independent populations who had completed European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ)-CIPN20 assessments (a CIPN-specific 20-item questionnaire which includes three scales that evaluate sensory, autonomic, and motor symptoms). The study population N08Cx included 692 participants from three clinical trials (North Central Cancer Treatment Group (NCCTG) N08C1, N08CA, and N08CB) who had been treated with paclitaxel, paclitaxel plus carboplatin, or oxaliplatin. The primary endpoint for the GWAS was the change from pre-chemotherapy CIPN20 sensory score to the worse score over the following 18 weeks. Study population The Mayo Clinic Breast Disease Registry (MCBDR) consisted of 381 Mayo Clinic Breast Disease Registry enrollees who had been treated with taxane or platinum-based chemotherapy. The primary endpoint for the GWAS assessed was the earliest CIPN20 sensory score available after the completion of chemotherapy. In multivariate model analyses, chemotherapy regimen (p = 3.0 × 10−8) and genetic ancestry (p = 0.007) were significantly associated with CIPN in the N08Cx population. Only age (p = 0.0004) was significantly associated with CIPN in the MCBDR population. The SNP most associated with CIPN was rs56360211 near PDE6C (p =7.92 × 10−8) in N08Cx and rs113807868 near TMEM150C in the MCBDR (p = 1.27 × 10−8). Due to a lack of replication, we cannot conclude that we identified any genetic predictors of CIPN.

Aim: This study explored whether inherited variants in genes causing the hereditary neuropathy condition Charcot–Marie–Tooth disease are associated with sensitivity to paclitaxel-induced peripheral neuropathy (PN). Patients & methods: Hereditary neuropathy genes previously associated with risk of paclitaxel-induced PN were sequenced in paclitaxel-treated patients. Eight putative genetic predictors in five hereditary neuropathy genes ( ARHGEF10, SBF2, FGD4, FZD3 and NXN) were tested for association with PN sensitivity after accounting for systemic exposure and clinical variables. Results: FZD3 rs7833751, a proxy for rs7001034, decreased PN sensitivity (additive model, β = -0.41; 95% CI: -0.66 to -0.17; p = 0.0011). None of the other genetic predictors were associated with PN sensitivity. Conclusion: Our results support prior evidence that FZD3 rs7001034 is protective of PN and may be useful for individualizing paclitaxel treatment to prevent PN.

Knee osteoarthritis is one of the most common causes of chronic pain worldwide, and several animal models have been developed to investigate disease mechanisms and treatments to combat associated morbidities. Here we describe a novel method for assessment of locomotor pain behavior in Yucatan swine. We used monosodium iodoacetate (MIA) to induce osteoarthritis in the hindlimb knee, and then conducted live observation, quantitative gait analysis, and quantitative weight-bearing stance analysis. We used these methods to test the hypothesis that locomotor pain behaviors after osteoarthritis induction would be detected by multiparameter quantitation for at least 12 wk in a novel large animal model of osteoarthritis. MIA-induced knee osteoarthritis produced lameness quantifiable by all measurement techniques, with onset at 2 to 4 wk and persistence until the conclusion of the study at 12 wk. Both live observation and gait analysis of kinetic parameters identified mild and moderate osteoarthritis phenotypes corresponding to a binary dose relationship. Quantitative stance analysis demonstrated the greatest sensitivity, discriminating between mild osteoarthritis states induced by 1.2 and 4.0 mg MIA, with stability of expression for as long as 12 wk. The multiparameter quantitation used in our study allowed rejection of the null hypothesis. This large animal model of quantitative locomotor pain resulting from MIA-induced osteoarthritis may support the assessment of new analgesic strategies for human knee osteoarthritis.

Aims Chemotherapy-induced peripheral neuropathy (PN) is a treatment limiting toxicity of paclitaxel. We evaluated if EPHA genetic variation (EPHA4, EPHA5, EPHA6, and EPHA8) is associated with PN sensitivity by accounting for variability in systemic paclitaxel exposure (time above threshold). Methods Germline DNA from 60 patients with breast cancer was sequenced. PN was measured using the 8-item sensory subscale (CIPN8) of the patient-reported CIPN20. Associations for 3 genetic models were tested by incorporating genetics into previously published PN prediction models integrating measured paclitaxel exposure and cumulative treatment. Significant associations were then tested for association with PN-related treatment disruption. Results EPHA5 rs7349683 (minor allele frequency = 0.32) was associated with increased PN sensitivity (β-coefficient = 0.39, 95% confidence interval 0.11-0.67, p = 0.007). Setting a maximum tolerable threshold of CIPN8 = 30, optimal paclitaxel exposure target is shorter for rs7349683 homozygous (11.6 h) than heterozygous (12.6 h) or wild-type (13.6 h) patients. Total number of missense variants (median = 0, range 0-2) was associated with decreased PN sensitivity (β-coefficient: -0.42, 95% confidence interval -0.72 to -0.12, P = .006). No association with treatment disruption was detected for the total number of missense variants or rs7349683. Conclusion Isolating toxicity sensitivity by accounting for exposure is a novel approach, and rs7349683 represents a promising marker for PN sensitivity that may be used to individualize paclitaxel treatment.

Purpose Discrepancies between clinicians’ assessment of chemotherapy-induced peripheral neuropathy (CIPN) and patient-reported outcomes (PRO) have been described, though the underlying reasons are unknown. Our objective was to identify potential patient-specific factors associated with under-describing of CIPN to clinicians in women with non-metastatic breast cancer treated with paclitaxel. Methods Patients enrolled in an observational study (n = 60) completed weekly CIPN PRO using the EORTC CIPN20. Clinician-documented CIPN using the NCI CTCAE were abstracted from the electronic medical record and paired with CIPN20 data at weeks 7 and 10. Patients were classified as under-describers if their CIPN20 was above the 80th percentile of the CIPN20 distribution for that CTCAE grade from an independent clinical trial (N08CA). Demographics, Assessment of Survivor Concerns (ASC), Trust in Oncologist Scale (TiOS), and health literacy assessment were collected post-treatment via survey. Repeated measures cumulative logistic regression models were used to identify factors associated with under-describing CIPN. Results Forty-two women completed the survey (response rate 70%). Three and 9 patients were categorized as under-describers at weeks 7 and 10, respectively. Women who were not working (OR = 9.00, 95%CI 1.06–76.15), had lower income (OR = 7.04, 95%CI 1.5–32.99), and displayed higher trust in their oncologist’s competence (OR = 1.29, 95%CI 1.03–1.62 for a 0.1-unit increase in score) were more likely to under-describe CIPN symptoms. Conclusions This preliminary study identified non-working status, low income and trust in oncologist’s competence as potential factors influencing under-description of CIPN to the clinical team. Further work is needed to clarify these relationships and test additional factors., Highlights • This pilot study examined factors associated with under-describing of neuropathy. • Patient-reported and clinician-documented neuropathy severity were compared. • Non-working status and low income were associated with neuropathy under-describing. • Trust in oncologist’s competence was associated with neuropathy under-describing. • Recording patient-clinician interactions would confirm under-describing behavior.

PURPOSE: Clinical practice guidelines on chemotherapy-induced peripheral neuropathy (CIPN) use the NCI Common Terminology Criteria for Adverse Events (CTCAE), while recent clinical trials employ a potentially superior measure, the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-CIPN twenty-item scale (QLQ-CIPN20), a patient-reported outcome (PRO). Practitioners and researchers lack guidance, regarding how QLQ-CIPN20 results relate to the traditional CTCAE during the serial assessment of patients undergoing chemotherapy.METHODS: Two large CIPN clinical trial datasets (538 patients) pairing QLQ-CIPN20 and CTCAE outcomes were analyzed using a multivariable linear mixed model with QLQ-CIPN20 score as the outcome variable, CTCAE grade as the main effect, and patient as random effect (accounting for internal correlation of serial measures).RESULTS: The association between QLQ-CIPN20 scores and CTCAE grades was strong (p < 0.0001), whereby patients with higher CTCAE grade had worse QLQ-CIPN20 scores. Some variation of QLQ-CIPN20 scores was observed based on drug, treatment, and cycle. While there was a marked difference in the mean QLQ-CIPN20 scores between CTCAE grades, the ranges of QLQ-CIPN20 scores within each CTCAE grade were large, leading to large overlap in CIPN20 scores across CTCAE grades.CONCLUSIONS: A strong positive association of QLQ-CIPN20 scores and CTCAE grade provides evidence of convergent validity as well as practical guidance, as to how to quantitatively interpret QLQ-CIPN20 scores at the study level in terms of the traditional CTCAE. The present results also highlight an important clinical caveat, specifically, that conversion of a specific QLQ-CIPN20 score to a specific CTCAE score may not be reliable at the level of an individual patient.

Oxaliplatin therapy can be complicated by chemotherapy-induced peripheral neuropathy (CIPN). Other neurotoxic chemotherapies have been linked to single nucleotide variants (SNV) in Charcot-Marie-Tooth disease (CMT) genes. Whether oxaliplatin carries increased risks of CIPN due to SNV in CMT-associated genes is unknown. 353 patients receiving oxaliplatin in NCCTG N08CB were serially evaluated for CIPN using a validated patient-reported outcome (PRO) instrument, the CIPN20 questionnaire (sensory scale). 49 canonical CMT-associated genes were analyzed for rare and common SNV by nextgen sequencing. The 157 patients with the highest and lowest susceptibility to CIPN (cases and controls) harbored 270 non-synonymous SNV in CMT-associated genes (coding regions). 143 of these were rare, occurring only once (“singletons”). CIPN cases had 0.84 singletons per patient compared with 0.98 in controls. An imbalance in favor of cases was noted only in few genes including PRX, which was previously highlighted as a candidate CIPN gene in patients receiving paclitaxel. However, the imbalance was only modest (5 singleton SNV in cases and 2 in controls). Therefore, while singleton SNV were common, they did overall not portend an increased risk of CIPN. Furthermore, testing CMT-associated genes using recurrent non-synonymous SNV did not reveal any significant association with CIPN. Genetic analysis of patients from N08CB provides clinical guidance that oxaliplatin chemotherapy decisions should not be altered by the majority of SNV that may be encountered in CMT-associated genes when common genetic tests are performed, such as exome or genome sequencing. Oxaliplatin's CIPN risk appears unrelated to CMT-associated genes.

Purpose This pilot trial aimed to assess if cooling hands and feet with crushed ice during receipt of paclitaxel helps prevent peripheral neuropathy. Methods This prospective, randomized trial compared cryotherapy to standard care in patients initiating paclitaxel weekly x 12. For those on cryotherapy, hands and feet were cooled starting 15 min prior to and ending 15 min after each paclitaxel dose. EORTC QLQ-CIPN20 was completed at baseline, weekly x12, then monthly x6. Area under the curve (AUC) was calculated for subscale scores, adjusting for baseline, and compared between arms (Wilcoxon rank-sum test). Cross-study comparisons used data from 2 prior similarly-conducted neuropathy trials. Results Forty-six patients were accrued. Three withdrew and one was ineligible. Of the remaining 42 (21 cryotherapy, 21 control), 39 (19 cryotherapy, 20 control) were analyzable for AUC. Cryotherapy was well tolerated, but the AUC of the CIPN20 sensory scores over 12 weeks of paclitaxel was not found to differ between the study arms (mean difference 3.45, 95% CI -3.13 to 10.02, p = 0.26). However, the control arm of the current trial experienced less neuropathy than did the placebo arms of two previous similar trials. When our cryotherapy arm was compared to the combined control arms from all three trials, the cryotherapy arm had less neuropathy (Wilcoxon Rank-Sum p = 0.01). Conclusion While there was no difference in CIPN20 scores identified between the 2 study arms in the current phase II trial, further investigation is needed given that the control arm experienced less neuropathy than was expected.

Health-related quality of life (HRQOL) is an important prognostic patient-reported outcome in oncology. Because prior studies suggest that HRQOL is, in part, heritable, we performed a GWAS to elucidate genetic factors associated with HRQOL in breast cancer survivors. Physical and mental HRQOL were measured via paper surveys that included the PROMIS-10 physical and mental health domain scales in 1442 breast cancer survivors participating in the Mayo Clinic Breast Disease Registry (MCBDR). In multivariable regression analyses, age and financial concerns were significantly associated with global physical health (age: p = 1.6 × 10−23, financial concerns: p = 4.8 × 10−40) and mental health (age: p = 3.5 × 10−7, financial concerns: p = 2.0 × 10−69). Chemotherapy was associated with worse global mental health (p = 0.01). In the GWAS, none of the SNPs reached the genome-wide association significance threshold of 5 × 10−8 for associations with either global physical or global mental health, however, a cluster of SNPs in SCN10A, particularly rs112718371, appeared to be linked to worse global physical health (p = 5.21 × 10−8). Additionally, SNPs in LMX1B, SGCD, PARP12 and SEMA5A were also moderately associated with worse physical and mental health (p &lt, 10−6). These biologically plausible candidate SNPs warrant further study as possible predictors of HRQOL.

Chronic pain is a widespread and debilitating condition, encountered by physicians in a variety of practice settings. Although many pharmacologic and behavioral strategies exist for the management of this condition, treatment is often unsatisfactory. Scrambler Therapy is a novel, non-invasive pain modifying technique that utilizes trans-cutaneous electrical stimulation of pain fibers with the intent of re-organizing maladaptive signaling pathways. This review was conducted to further evaluate what is known regarding the mechanisms and mechanics of Scrambler Therapy and to investigate the preliminary data pertaining to the efficacy of this treatment modality.The PubMed/Medline, SCOPUS, EMBASE, and Google Scholar databases were searched for all articles published on Scrambler Therapy prior to November 2015. All case studies and clinical trials were evaluated and reported in a descriptive manner.To date, 20 reports, of varying scientific quality, have been published regarding this device; all but one small study, published only as an abstract, provided results that appear positive.The positive findings from preliminary studies with Scrambler Therapy support that this device provides benefit for patients with refractory pain syndromes. Larger, randomized studies are required to further evaluate the efficacy of this approach.

Treatment for chronic, locoregional pain ranks among the most prevalent unmet medical needs. The failure of systemic analgesic drugs, such as opioids, is often due to their off-target toxicity, development of tolerance, and abuse potential. Interventional pain procedures provide target specificity but lack pharmacologically selective agents with long-term efficacy. Gene therapy vectors are a new tool for the development of molecularly selective pain therapies, which have already been proved to provide durable analgesia in preclinical models. Taken together, advances in image-guided delivery and gene therapy may lead to a new class of dual selective analgesic treatments integrating the molecular selectivity of analgesic genes with the anatomic selectivity of interventional delivery techniques.

Background: Chemotherapy-related amenorrhea (CRA) impacts quality of life and reproductive options, and may play a role in treatment decision-making for young breast cancer patients. Although age, increasing doses of alkylating agents, and tamoxifen are known to increase the risk of chemotherapy-related amenorrhea (CRA), it is difficult to predict which women will develop CRA in order to inform fertility preservation and treatment decisions. We sought to investigate whether single nucleotide polymorphisms (SNPs) that have been associated with a younger age at natural menopause and ovarian reserve in non-oncologic populations were predictive of CRA in two large German clinical trials that administered gonadotoxic chemotherapy. Methods: A total of 1322 premenopausal participants under age 50 who enrolled in SUCCESS B (a phase 3 trial of FEC-docetaxel-trastuzumab +/- gemcitabine for high risk early stage breast cancer) or SUCCESS C (a phase 3 trial of FEC-docetaxel-cyclophosphamide vs. docetaxel-cyclophosphamide that also tested the efficacy of a weight loss intervention in early stage breast cancer patients) were eligible for this study. We excluded patients who were receiving ovarian suppression. On these trials, menstrual status was self-reported as amenorrheic or not every three months after chemotherapy. Bloods were stored and patients provided consent for DNA analysis. We genotyped nine candidate SNPs in six genes (rs2002555 and rs11170547 in AMHR2, rs6166 in FSHR, rs10852344 Results: Of the 554 eligible women with SNP data and menstrual data at 12 months, 70% reported CRA; of the 458 eligible women with SNP data and menstrual data at 24 months, 69% reported CRA. The two SNPs in AMHR2 showed suggestive associations with CRA at 12 and 24 months. [Final results pending]. Conclusions: Genetic variation in AMHR2 may be associated with CRA. Additional research will be needed to validate these findings in other breast cancer survivors and to identify additional SNPs that may be associated with CRA. Together with other clinical factors, genetic variation may improve our ability to predict who will experience CRA, and may inform reproductive and treatment decision-making in cancer patients. Citation Format: Ruddy KJ, Rack B, Schwitulla J, Lambrechts D, Haeberle L, Schramm A, Trapp E, Scholz C, Beutler AS, Ginsburg E, Couch F, Partridge AH, Wang L, Weinshilboum RM, Janni W, Vachon C, Fasching P. Genetic predictors of chemotherapy-related amenorrhea. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-10-07.

Paclitaxel‐induced peripheral neuropathy (PIPN) cannot be predicted from clinical parameters and might have a pharmacogenomic basis. Previous studies identified single nucleotide variants (SNV) associated with PIPN. However, only a subset of findings has been confirmed to date in more than one study, suggesting a need for further re‐testing and validation in additional clinical cohorts. Candidate PIPN‐associated SNVs were identified from the literature. SNVs were retested in 119 patients selected by extreme phenotyping from 269 in NCCTG N08C1 (Alliance) as previously reported. SNV genotyping was performed by a combination of short‐read sequencing analysis and Taqman PCR. These 22 candidate PIPN SNVs were genotyped. Two of these, rs7349683 in the EPHA5 and rs3213619 in ABCB1 were found to be significantly associated with PIPN with an Odds ratios OR = 2.07 (P = 0.02) and OR = 0.12 (P = 0.03), respectively. In addition, three SNVs showed a trend toward a risk‐ or protective effect that was consistent with previous reports. The rs10509681 and rs11572080 in the gene CYP2C8*3 showed risk effect with an OR = 1.49 and rs1056836 in CYP1B1 showed a protective effect with an OR = 0.66. None of the other results supported the previously reported associations, including some SNVs displaying an opposite direction of effect from previous reports, including rs1058930 in CYP2C8, rs17222723 and rs8187710 in ABCC2, rs10771973 in FGD4, rs16916932 in CACNB2 and rs16948748 in PITPNA. Alliance N08C1 validated or supported a minority of previously reported SNV‐PIPN associations. Associations previously reported by multiple studies appeared to have a higher likelihood to be validated by Alliance N08C1.

Background Development of new analgesic drugs or gene therapy vectors for spinal delivery will be facilitated by “hyperlocal” targeting of small therapeutic injectate volumes if spine imaging technology can be used that is ready for future clinical translation. New Method This study provides methods for MRI-guided drug delivery to the periganglionic epidural space and the dorsal root ganglion (DRG) in the Yucatan swine. Results Phantom studies showed artifact-corrected needle localization with frequency encoding parallel to the needle shaft, while maximizing bandwidth (125 KHz) minimized needle artifact. A custom constructed 8–12 element surface coil (phased array) wrapped over the spine in conjunction with lateral recumbent positioning achieved diagnostic quality signal to noise ratio at the depth of the DRG and afforded transforaminal access via anterolateral or posterolateral vectors, as well as interlaminar access. Swine epidural anatomy was homologous with human anatomy. Injectate containing 2% gadolinium allowed imaging of injectate volumes in increments as small as 10 microliters and discrimination of epidural flow from intraparenchymal injectate delivery into a DRG. All technical and technological elements of the procedure appear clinically translatable. Comparison with Existing Methods Computed tomographic or fluoroscopic guidance cannot directly visualize drug delivery into the DRG due to contrast medium toxicity, nor reliably identify epidural injection volumes of Conclusions MRI-guided hyperlocal delivery in swine provides a translatable and faithful model of future human spinal novel drug- or gene therapy vector delivery.

INTRODUCTION: Intrathecal interleukin-10 delivered by plasmid or viral gene vectors has been proposed for clinical testing because it is effective for chronic pain in rodents, a potential therapeutic for various human diseases, and was found to be non-toxic in dogs, when the human interleukin-10 ortholog was tested. However, recent studies in swine testing porcine interleukin-10 demonstrated fatal neurotoxicity. AIMS: To deliver vector-encoded human interleukin-10 in swine, measure expression of the transgene in cerebrospinal fluid, and monitor animals for signs of neurotoxicity. RESULTS: Human interleukin-10 levels peaked 2 weeks after vector administration followed by a rapid decline that occurred concomitant with the emergence of anti-human interleukin-10 antibodies in the cerebrospinal fluid and serum. Animals remained neurologically healthy throughout the study period. CONCLUSIONS: This study suggests that swine are not idiosyncratically sensitive to intrathecal interleukin-10 because, recapitulating previous reports in dogs, they suffered no clinical neurotoxicity from the human ortholog. These results strongly infer that toxicity of intrathecal interleukin-10 in large animal models was previously overlooked because of a species mismatch between transgene and host. The present study further suggests that swine were protected from interleukin-10 by a humoral immune response against the xenogeneic cytokine. Future safety studies of interleukin-10 or related therapeutics may require syngeneic large animal models.

Introduction Osteoarthritis (OA) is the most common form of arthritis. Medical and surgical treatments have yet to substantially diminish the global health and economic burden of OA. Due to recent advances in clinical imaging, including magnetic resonance imaging (MRI), a correlation has been established between structural joint damage and OA-related pain and disability. Existing preclinical animal models of OA are useful tools but each suffers specific roadblocks when translating structural MRI data to humans. Intraarticular injection of mono-iodoacetate (MIA) is a reliable, well-studied method to induce OA in small animals but joint size discrepancy precludes the use of clinical grade MRI to study structural disease. The porcine knee is suited for clinical MRI and demonstrates homology with humans. We set out to establish the first large animal model of MIA-induced knee OA in swine characterized by structural MRI. Materials and methods Yucatan swine (n = 27) underwent ultrasound-guided injection of knees with 1.2, 4, 12, or 40 mg MIA. MRI was performed at several time points over 12 weeks (n = 54 knees) and images were assessed according to a modified clinical grading scheme. Knees were harvested and graded up to 35 weeks after injection. Results MIA-injected knees (n = 25) but not control knees (n = 29) developed gross degeneration. A total of n = 6,000 MRI measurements were recorded by two radiologists. MRI revealed progressive cartilage damage, bone marrow edema, erosions, and effusions in MIA-injected knees. Lesion severity and progression was influenced by time, dose, and inter-individual variability. Conclusions Intraarticular injection of MIA produced structural knee degradation that was reliably characterized using clinical MRI in swine. Destruction was progressive and, similar to human OA, lesion severity was heterogeneous between and within treatment groups.

Purpose Given that the clinical course of oxaliplatin-induced neuropathy is not well defined, the current study was performed to better understand clinical parameters associated with its presentation. Methods Acute and chronic neuropathy was evaluated in patients receiving adjuvant FOLFOX (fluorouracil, leucovorin, and oxaliplatin) on study N08CB (North Central Cancer Treatment Group, Alliance). Acute neuropathy was assessed by having patients complete daily questionnaires for 6 days with each cycle of FOLFOX. Before each dose of FOLFOX and as long as 18 months after chemotherapy cessation, chronic neurotoxicity was assessed with use of the 20-item, European Organisation for Research and Treatment of Cancer quality-of-life questionnaire for patients with chemotherapy-induced peripheral neuropathy. Results Three hundred eight (89%) of the 346 patients had at least one symptom of acute neuropathy with the first cycle of FOLFOX; these symptoms included sensitivity to touching cold items (71%), sensitivity to swallowing cold items (71%), throat discomfort (63%), or muscle cramps (42%). Acute symptoms peaked at day 3 and improved, although they did not always resolve completely between treatments. These symptoms were about twice as severe in cycles 2 through 12 as they were in cycle 1. For chronic neurotoxicity, tingling was the most severe symptom, followed by numbness and then pain. During chemotherapy, symptoms in the hands were more prominent than they were in the feet; by 18 months, symptoms were more severe in the feet than they were in the hands. Patients with more severe acute neuropathy during the first cycle of therapy experienced more chronic sensory neurotoxicity (P < .0001). Conclusion Acute oxaliplatin-induced neuropathy symptoms do not always completely resolve between treatment cycles and are only half as severe on the first cycle as compared with subsequent cycles. There is a correlation between the severities of acute and chronic neuropathies.

Substantial research efforts have focused on methods of treating and preventing oxaliplatin-associated neuropathy, the dose-limiting toxicity associated with this drug. Administration of intravenous calcium and magnesium (CaMg) before and after oxaliplatin has been the most studied approach to preventing oxaliplatin-induced neuropathy. Although early reports demonstrated potential benefit, subsequent larger trials failed to confirm the efficacy of CaMg in preventing this adverse effect. This article explores how accumulating evidence for and against the use of CaMg for preventing oxaliplatin-induced neuropathy has impacted clinical practice.

The response of the peripheral nervous system (PNS) to injury may go together with alterations in epigenetics, a conjecture that has not been subjected to a comprehensive, genome-wide test. Using reduced representation bisulfite sequencing, we report widespread remodeling of DNA methylation in the rat dorsal root ganglion (DRG) occurring within 24 h of peripheral nerve ligation, a neuropathy model of allodynia. Significant (P < 10−4) cytosine hyper- and hypo-methylation was found at thousands of CpG sites. Remodeling occurred outside of CpG islands. Changes affected genes with known roles in the PNS, yet methylome remodeling also involved genes that were not linked to neuroplasticity by prior evidence. Consistent with emerging models relying on genome-wide methylation and RNA-seq analysis of promoter regions and gene bodies, variation of methylation was not tightly linked with variation of gene expression. Furthermore, approximately 44% of the dynamically changed CpGs were located outside of genes. We compared their positions with the intergenic, tissue-specific differentially methylated CpGs (tDMCs) of an independent experimental set consisting of liver, spleen, L4 control DRG, and muscle. Dynamic changes affected those intergenic CpGs that were different between tissues (P < 10−15) and almost never the invariant portion of the methylome (those CpGs that were identical across all tissues). Our findings—obtained in mixed tissue—show that peripheral nerve injury leads to methylome remodeling in the DRG. Future studies may address which of the cell types found in the DRG, such as specific groups of neurons or non-neuronal cells are affected by which aspect of the observed methylome remodeling.

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4',6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

Interleukin-10 (IL-10) delivered by intrathecal (i.t.) gene vectors is a candidate investigational new drug (IND) for several chronic neurological disorders such as neuropathic pain. We performed a preclinical safety study of IL-10. A syngeneic large animal model was used delivering porcine IL-10 (pIL-10) to the i.t. space in swine by adeno-associated virus serotype 8 (AAV8), a gene vector that was previously found to be nontoxic in the i.t. space. Unexpectedly, animals became ill, developing ataxia, seizures, and an inability to feed and drink, and required euthanasia. Necropsy demonstrated lymphocytic meningitis without evidence of infection in the presence of normal laboratory findings for body fluids and normal histopathology of peripheral organs. Results were replicated in a second animal cohort by a team of independent experimenters. An extensive infectious disease and neuropathology workup consisting of comprehensive testing of tissues and body fluids in a specialized research veterinary pathology environment did not identify a pathogen. These observations raise the concern that i.t. IL-10 therapy may not be benign, that previously used xenogeneic models testing the human homolog of IL-10 may not have been sensitive enough to detect toxicity, and that additional preclinical studies may be needed before clinical testing of IL-10 can be considered.

Paclitaxel is associated with both an acute pain syndrome (P-APS) and chronic chemotherapy-induced peripheral neuropathy (CIPN). Given that extensive animal data suggest that minocycline may prevent chemotherapy-induced neurotoxicity, the purpose of this pilot study was to investigate the efficacy of minocycline for the prevention of CIPN and the P-APS. Patients with breast cancer were enrolled prior to initiating neoadjuvant or adjuvant weekly paclitaxel for 12 weeks and were randomized to receive minocycline 200 mg on day 1 followed by 100 mg twice daily or a matching placebo. Patients completed (1) an acute pain syndrome questionnaire daily during chemotherapy to measure P-APS and (2) the EORTC QLQ-CIPN20 questionnaire at baseline, prior to each dose of paclitaxel, and monthly for 6 months post treatment, to measure CIPN. Forty-seven patients were randomized. There were no remarkable differences noted between the minocycline and placebo groups for the overall sensory neuropathy score of the EORTC QLQ-CIPN20 or its individual components, which evaluate tingling, numbness and shooting/burning pain in hands and feet. However, patients taking minocycline had a significant reduction in the daily average pain score attributed to P-APS (p = 0.02). Not only were no increased toxicities reported with minocycline, but there was a significant reduction in fatigue (p = 0.02). Results of this pilot study do not support the use of minocycline to prevent CIPN, but suggest that it may reduce P-APS and decrease fatigue; further study of the impact of this agent on those endpoints may be warranted.

Context Oxaliplatin-induced peripheral neuropathy (OIPN) is a dose-limiting toxicity of oxaliplatin and affects most colorectal cancer patients. OIPN is commonly evaluated by patient symptom report, using scales to reflect impairment. They do not discriminate between unique grouping of symptoms and signs, which impedes prompt identification of OIPN. Objective The objective of this study was to identify clusters of symptoms and signs that differentiated underlying clinical severity and segregated patients within our population into OIPN subgroups. Methods Chemotherapy-naive colorectal cancer patients ( N = 148) receiving oxaliplatin were administered the Total Neuropathy Score clinical (TNSc © ), which includes symptom report (sensory, motor, autonomic) and sensory examination (pin sense, vibration, reflexes). The TNSc was administered before chemotherapy initiation (T0) and after cumulative doses of oxaliplatin 510–520 mg/m 2 (T1) and 1020–1040 mg/m 2 of oxaliplatin (T2). Using mean T2 TNSc scores, latent class analysis grouped patients into OIPN severity cohorts. Results Latent class analysis categorized patients into four distinct OIPN groups: low symptoms and low signs ( n = 54); low symptoms and intermediate signs ( n = 44); low symptoms and high signs ( n = 21); and high symptoms and high signs ( n = 29). No differences were noted among OIPN groups on age, sex, chemotherapy regimen, or cumulative oxaliplatin dose. Conclusion We identified OIPN patient groups with distinct symptoms/signs, demonstrating variability of OIPN presentation regardless of cumulative oxaliplatin dose. Over half of the sample had positive findings on OIPN examination despite little or no symptoms. Sensory examination of all patients receiving oxaliplatin is indicated for timely identification of OIPN, which will allow earlier symptom management.

Dorsal root ganglia (DRG) are critical anatomical structures involved in nociception. Intraganglionic (IG) drug delivery is therefore an important route of administration for novel analgesic therapies. Although IG injection in large animal models is highly desirable for preclinical biodistribution and toxicology studies of new drugs, no method to deliver pharmaceutical agents into the DRG has been reported in any large species. The present study describes a minimally invasive technique of IG agent delivery in domestic swine, one of the most common large animal models. The technique utilizes CT guidance for DRG targeting and a custom-made injection assembly for convectionenhanced delivery (CED) of therapeutic agents directly into DRG parenchyma. The DRG were initially visualized by CT myelography to determine the optimal access route to the DRG. The subsequent IG injection consisted of 3 steps. First, a commercially available guide needle was advanced to a position dorsolateral to the DRG, and the dural root sleeve was punctured, leaving the guide needle contiguous with, but not penetrating, the DRG. Second, the custom-made stepped stylet was inserted through the guide needle into the DRG parenchyma. Third, the stepped stylet was replaced by the custom-made stepped needle, which was used for the IG CED. Initial dye injections performed in pig cadavers confirmed the accuracy of DRG targeting under CT guidance. Intraganglionic administration of adeno-associated virus in vivo resulted in a unilateral transduction of the injected DRG, with 33.5% DRG neurons transduced. Transgene expression was also found in the dorsal root entry zones at the corresponding spinal levels. The results thereby confirm the efficacy of CED by the stepped needle and a selectivity of DRG targeting. Imaging-based modeling of the procedure in humans suggests that IG CED may be translatable to the clinical setting.

Objective Mutations in Charcot-Marie-Tooth disease (CMT) genes are the cause of rare familial forms of polyneuropathy. Whether allelic variability in CMT genes is also associated with common forms of polyneuropathy—considered “acquired” in medical parlance—is unknown. Chemotherapy induced peripheral neuropathy (CIPN) occurs commonly in cancer patients and is individually unpredictable. We used CIPN as clinical model to investigate the association of non-CMT polyneuropathy with CMT genes.

Purpose of review this review aims at summarizing progress in clinical trials and basic science redefining the diagnosis and treatment of well differentiated small intestine neuroendocrine tumors (SI-NET). Recent findings Two clinical trials demonstrated antitumor activity of the long-acting somatostatin analogues octreotide LAR and lanreotide for advanced SI-NET. The mTOR inhibitor everolimus is another treatment option for patients with SI-NET, but awaits definitive proof of benefit in the ongoing RADIANT-4 study. Two whole exome/genome-sequencing studies reported in the past year provided the first genome-wide analysis of large sets of SI-NET at nucleotide resolution. Candidate therapeutically relevant alterations were found to affect SRC, SMAD genes, AURKA, EGFR, HSP90, and PDGFR as well as mutually exclusive amplification of AKT1 or AKT2 and other alterations of PI3K/Akt/mTOR signaling genes. The gene CDKN1B is inactivated by small insertions/deletions in 8% of patients with SI-NET suggesting cell cycle inhibitors as new candidate drugs for SI-NET. Circulating tumor cells and tumor-derived RNA in the blood are promising clinical tests for SI-NET.

Gene therapy with adeno-associated virus (AAV) has advanced in the last few years from promising results in animal models to >100 clinical trials (reported or under way). While vector availability was a substantial hurdle a decade ago, innovative new production methods now routinely match the scale of AAV doses required for clinical testing. These advances may become relevant to translational research in the chronic pain field. AAV for pain targeting the peripheral nervous system was proven to be efficacious in rodent models several years ago, but has not yet been tested in humans. The present review addresses the steps needed for translation of AAV for pain from the bench to the bedside focusing on pre-clinical toxicology. We break the potential toxicities into three conceptual categories of risk: First, risks related to the delivery procedure used to administer the vector. Second, risks related to AAV biology, i.e., effects of the vector itself that may occur independently of the transgene. Third, risks related to the effects of the therapeutic transgene. To identify potential toxicities, we consulted the existing evidence from AAV gene therapy for other nervous system disorders (animal toxicology and human studies) and from the clinical pharmacology of conventional analgesic drugs. Thereby, we identified required preclinical studies and charted a hypothetical path towards a future phase I/II clinical trial in the oncology-palliative care setting.

532 Background: Chemotherapy induced peripheral neuropathy (CIPN) is a common, debilitating paclitaxel side effect that has been primarily attributed to cumulative systemic paclitaxel exposure. Paclitaxel dose-adjustment to achieve target systemic exposure decreases but does not eliminate severe CIPN, suggesting some patients are inherently CIPN-sensitive. Ephrin ( EPHA) polymorphisms have been reported to increase CIPN occurrence (Baldwin 2012, Leandro-Garcia 2013, Boora 2016) but replication has been challenging, perhaps due to the inability to isolate CIPN-sensitivity by accounting for systemic exposure differences. The study purpose was to determine if EPHA genetic variants previously associated with CIPN occurrence are associated with CIPN-sensitivity. Methods: PN was assessed at baseline and weekly using the 8-item sensory subscale (CIPN8) of the patient-reported EORTC CIPN20 in patients receiving paclitaxel 80 mg/m2 weekly x 12. EPHA4, EPHA5, EPHA6, and EPHA8 were sequenced in germline DNA. Associations with higher PN-sensitivity were tested for three genetic models (total variants, coding variants, and rs7349683) by incorporating genetics into previously published CIPN8 predictive models that included measured paclitaxel exposure. Significant associations were tested for association with higher risk of PN-related treatment disruption (i.e. dose decrease, delay, or discontinuation). Results: In the 59 included patients, carrying EPHA5 rs7349683 was associated with greater CIPN sensitivity (beta coefficient: 0.40, standard error: 0.14, p = 0.005), indicating these patients had a greater increase in CIPN8 during treatment than would be predicted based on cumulative paclitaxel exposure. Rs7349683 was not associated with increased PN-induced treatment disruption, perhaps due to the low number of events (n = 19). Conclusions: Using a novel approach that isolates CIPN-sensitivity by accounting for measured paclitaxel exposure, our results provide further evidence that EPHA5 rs7349683 may be a promising marker for CIPN. If additional validation studies confirm this association, genetic testing could enable personalized treatment strategies to prevent CIPN in patients with breast cancer.

The diagnosed incidence of small intestine neuroendocrine tumors (SI-NETs) is increasing, and the underlying genomic mechanisms have not been defined for these tumors. Using exome/genome sequence analysis of SI-NETs, we identified recurrent somatic mutations and deletions in CDKN1B, the cyclin-dependent kinase inhibitor gene, which encodes p27. We observed frameshift mutations of CDKN1B in 14 of 180 SI-NETs, and we detected hemizygous deletions encompassing CDKN1B in 7 out of 50 SI-NETs, nominating p27 as a tumor suppressor and implicating cell cycle dysregulation in the etiology of SI-NET.

Chemotherapy induced peripheral neuropathy (CIPN) is a dose limiting toxicity of paclitaxel, termed here paclitaxel induced peripheral neuropathy (PIPN). PIPN affects a minority of patients and cannot be predicted from clinical patient characteristics. Therefore, a phamacogenomic basis has been proposed for PIPN (1). Genome wide association studies (GWAS) provide an unbiased approach for the identification of novel genetic loci associated with complex traits (2). Schneider et al. performed the first PIPN GWAS in 2011, (3) finding that the risk of PIPN was linked to two single nucleotide variants (SNV) in two genes that had previously not been known to be associated with this condition or with CIPN due to any other drug. The SNV rs2296308 in RWDD3 was reported to be associated with PIPN with a HR=1.5 and a significance level of p=8.5 × 10−8. The rs1829 SNV in TECTA was reported to be associated with PIPN with a HR= 2.1 and a significance level of p=3.2×10−7. Both SNV thereby passed the threshold set by the authors for genome-wide significance thereby representing important novel PIPN gene candidates. Bergmann et al. investigated rs2296308 and rs1829 in a Scandinavian ovarian cancer patient cohort (4). Their study failed to corroborate the report by Schneider et al., thereby calling that GWAS result into question. The original GWAS consisted of a very large cohort, 2204 patients, while the validation attempt by Bergmann et al. was limited to a smaller cohort of 241 patients. Both studies used similar phenotyping and analyses relying on Common Toxicity Criteria Adverse Effects (CTCAE) reporting of toxicity and time-to-grade 2 or worse scores as the primary endpoint. These conflicting results thereby created uncertainty in the field regarding the role of RWWD3 and TECTA. We recently reported a new PIPN cohort, N08C1, for which improved phenotyping was available using serial assessments of PIPN symptoms with the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire (QLQ) CIPN-20 (CIPN20) (5). Here, we report testing of the association of rs2296308 in RWWD3 and rs1829 in TECTA with PIPN in N08C1.

Purpose: Oxaliplatin and paclitaxel are commonly used chemotherapies associated with acute and chronic neuropathies. There is a need to better understand the similarities and differences of these clinical syndromes. Methods: Neuropathy data were pooled from patients receiving adjuvant oxaliplatin and weekly paclitaxel or every 3weeks of paclitaxel. Patients completed daily questionnaires after each chemotherapy dose and the European Organization for Research and Treatment of Cancer quality-of-life questionnaire for patients with chemotherapy-induced peripheral neuropathy before each chemotherapy cycle and for 12months post-treatment. Results: Acute neuropathy symptoms from both drugs peaked around day 3. Acute symptoms experienced in cycle 1 predicted occurrence in subsequent cycles. Paclitaxel-induced acute symptoms were similar in intensity in each cycle and largely resolved between cycles. Oxaliplatin-induced acute symptoms were about half as severe in the first cycle as in later cycles and did not resolve completely between cycles. Both drugs caused a predominantly sensory chronic neuropathy (with numbness and tingling being more common than pain). Oxaliplatin-induced neuropathy worsened after the completion of treatment and began to improve 3months post-treatment. In contrast, paclitaxel-induced neuropathy began improving immediately after chemotherapy cessation. During treatment, the incidence of paclitaxel sensory symptoms was similar in the hands and feet; with oxaliplatin, the hands were affected more than the feet. Both paclitaxel- and oxaliplatin-induced acute neurotoxicity appeared to predict the severity of chronic neuropathy, more prominently with oxaliplatin. Conclusions: Knowledge of the similarities and differences between neuropathy syndromes may provide insight into their underlying pathophysiology and inform future research to identify preventative treatment approaches.

Background DNA methylation is an important epigenetic modification involved in many biological processes. Reduced representation bisulfite sequencing (RRBS) is a cost-effective method for studying DNA methylation at single base resolution. Although several tools are available for RRBS data processing and analysis, it is not clear which strategy performs the best and there has not been much attention to the contamination issue from artificial cytosines incorporated during the end repair step of library preparation. To address these issues, we describe a new method, Targeted Alignment and Artificial Cytosine Elimination for RRBS (TRACE-RRBS), which aligns bisulfite sequence reads to MSP1 digitally digested reference and specifically removes the end repair cytosines. We compared this approach on a simulated and a real dataset with 7 other RRBS analysis tools and Illumina 450 K microarray platform. Results TRACE-RRBS aligns sequence reads to a small fraction of the genome where RRBS protocol targets on and was demonstrated as the fastest, most sensitive and specific tool for the simulated dataset. For the real dataset, TRACE-RRBS took about the same time as RRBSMAP, a third to a sixth of time needed for BISMARK and NOVOALIGN. TRACE-RRBS aligned more reads uniquely than other tools and achieved the highest correlation with 450 k microarray data. The end repair artificial cytosine removal increased correlation between nearby CpGs and accuracy of methylation quantification. Conclusions TRACE-RRBS is fast and more accurate tool for RRBS data analysis. It is freely available for academic use at http://bioinformaticstools.mayo.edu/. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2494-8) contains supplementary material, which is available to authorized users.

DNA methylation can control some CpG-poor genes but unbiased studies have not found a consistent genome-wide association with gene activity outside of CpG islands or shores possibly due to use of cell lines or limited bioinformatics analyses. We performed reduced representation bisulfite sequencing (RRBS) of rat dorsal root ganglia encompassing postmitotic primary sensory neurons (n = 5, r > 0.99; orthogonal validation p < 10−19). The rat genome suggested a dichotomy of genes previously reported in other mammals: low CpG content (< 3.2%) promoter (LCP) genes and high CpG content (≥ 3.2%) promoter (HCP) genes. A genome-wide integrated methylome-transcriptome analysis showed that LCP genes were markedly hypermethylated when repressed, and hypomethylated when active with a 40% difference in a broad region at the 5′ of the transcription start site (p < 10−87 for -6000 bp to -2000 bp, p < 10−73 for -2000 bp to +2000 bp, no difference in gene body p = 0.42). HCP genes had minimal TSS-associated methylation regardless of transcription status, but gene body methylation appeared to be lost in repressed HCP genes. Therefore, diametrically opposite methylome-transcriptome associations characterize LCP and HCP genes in postmitotic neural tissue in vivo.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major dose limiting side effect that can lead to long-term morbidity. Approximately one-third of patients receiving chemotherapy with taxanes, vinca alkaloids, platinum compounds or proteasome inhibitors develop this toxic side effect. It is not possible to predict who will get CIPN, however, genetic susceptibility may play a role. We explored this hypothesis using an established in vitro dorsal root ganglia neurite outgrowth (DRG-NOG) assay to assess possible genetic influences for cisplatin- and bortezomib-induced neurotoxicity. Almost all previous in vitro studies have used rats or mice. We compared DRG-NOG between four genetically defined, inbred mouse strains (C57BL/6J, DBA/2J, BALB/cJ, and C3H/HeJ) and one rat strain (Sprague Dawley). Our studies found differences in cisplatin and bortezomib-induced neurotoxicity between mouse and rat strains and between the different mouse strains. C57BL/6J and Balb/cJ DRG-NOG was more sensitive to cisplatin than DBA/2J and C3H/HeJ DRG-NOG, and all mouse strains were more sensitive to cisplatin than rat. Bortezomib induced a biphasic dose response in DBA/2J and C3H/H3J mice. C57BL/6J DRG-NOG was most sensitive and Balb/cJ DRG-NOG was least sensitive to bortezomib. Our animal data supports the hypothesis that genetic background may play a role in CIPN and care must be taken when rodent models are used to better understand the contribution of genetics in patient susceptibility to CIPN.

10016Background: Pilot data support that Scrambler therapy may benefit patients with Chemotherapy-induced peripheral neuropathy (CIPN). Methods: Patients with CIPN for at least 3 months were eligib...

e22075Background: Cooling the extremities during chemotherapy has been hypothesized to reduce subsequent peripheral neuropathy. Methods: A randomized pilot trial was conducted comparing cryotherapy...

mRNA-seq is a paradigm-shifting technology because of its superior sensitivity and dynamic range and its potential to capture transcriptomes in an agnostic fashion, i.e., independently of existing genome annotations. Implementation of the agnostic approach, however, has not yet been fully achieved. In particular, agnostic mapping of pre-mRNA splice sites has not been demonstrated. The present study pursued dual goals: (1) to advance mRNA-seq bioinformatics toward unbiased transcriptome capture and (2) to demonstrate its potential for discovery in neuroscience by applying the approach to an in vivo model of neurological disease. We have performed mRNA-seq on the L4 dorsal root ganglion (DRG) of rats with chronic neuropathic pain induced by spinal nerve ligation (SNL) of the neighboring (L5) spinal nerve. We found that 12.4% of known genes were induced and 7% were suppressed in the dysfunctional (but anatomically intact) L4 DRG 2 wk after SNL. These alterations persisted chronically (2 mo). Using a read cluster classifier with strong test characteristics (ROC area 97%), we discovered 10,464 novel exons. A new algorithm for agnostic mapping of pre-mRNA splice junctions (SJs) achieved a precision of 97%. Integration of information from all mRNA-seq read classes including SJs led to genome reannotations specifically relevant for the species used (rat), the anatomical site studied (DRG), and the neurological disease considered (pain); for example, a 64-exon coreceptor for the nociceptive transmitter substance P was identified, and 21.9% of newly discovered exons were shown to be dysregulated. Thus, mRNA-seq with agnostic analysis methods appears to provide a highly productive approach for in vivo transcriptomics in the nervous system.

Recombinant adeno-associated virus (rAAV) is a promising vector for applications in the central nervous system (CNS). Clinical phase I/II studies have demonstrated safety of rAAV-expressing therapeutic transgenes for a variety of benign (i.e., noncancer) CNS disorders, namely, Canavan disease,1 Parkinson’s disease,2,3,4 and Batten disease.5 Human studies were also recently reported targeting the retina.6,8,9,9 From the early stages of preclinical development, rAAV was appealing because it was found to be neurotrophic and capable of long-term activity without immune interference or toxicity when injected into the CNS.10

The zinc finger protein 217 (ZNF217) is an important oncogene based on the high frequency of amplification and overexpression in many cancer types, but its molecular mode of gene regulation is poorly understood. We purified a complex of nuclear ZNF217-binding proteins by affinity chromatography and identified its components by mass spectrometry as Jarid1b/Plu-1, G9a, LSD1, CoREST and CtBP1. Individual binding of these with ZNF217 was confirmed by co-immunoprecipiation (IP). Known activities of these proteins suggested a role of the ZNF217 complex in histone modification. Using in vitro assays the following activities were demonstrated: Histone H3 lysine 4 trimethyl (H3K4me3) demethylase activity, which co-fractionated with Jarid1b/Plu-1 in anion-exchange chromatography; H3K9 methylation, the known principal activity of G9a; and H3K27 methylation. The latter suggested EZH2 as another ZNF217 binding candidate, which could be confirmed by co-IP. Taken together, these findings suggest that ZNF217 assembles a distinct set of histone modifying proteins at target DNA sites that act synergistically in transcriptional repression.

Lumbar puncture (LP) is an attractive route to deliver drugs to the nervous system because it is a safe bedside procedure. Its use for gene therapy has been complicated by poor vector performance and failure to target neurons. Here we report highly effective gene transfer to the primary sensory neurons of the dorsal root ganglia (DRGs) with self-complementary recombinant adeno-associated virus serotype 8 (sc-rAAV8) modeling an LP. Transgene expression was selective for these neurons outlining their cell bodies in the DRGs and their axons projecting into the spinal cord. Immunohistochemical studies demonstrated transduction of cells positive for the nociceptive neuron marker vanilloid receptor subtype 1, the small peptidergic neuron markers substance P and calcitonin gene-related peptide, and the nonpeptidergic neuron marker griffonia simplicifolia isolectin B4. We tested the efficacy of the approach in a rat model of chronic neuropathic pain. A single administration of sc-rAAV8 expressing the analgesic gene prepro-β-endorphin (ppβEP) led to significant ( P < 0.0001) reversal of mechanical allodynia for ≥3 months. The antiallodynic effect could be reversed by the μ-opioid antagonist naloxone 4 months after gene transfer ( P < 0.001). Testing of an alternative nonopioid analgesic gene, IL-10, alone or in combination with ppβEP was equally effective ( P < 0.0001). All aspects of the procedure, such as the use of an atraumatic injection technique, isotonic diluent, a low-infusion pressure, and a small injection volume, are consistent with clinical practice of intrathecal drug use. Therefore, gene transfer by LP may be suitable for developing gene therapy-based treatments for chronic pain.

The predisposition of patients to develop polyneuropathy in response to toxic exposure may have a genetic basis. The previous study Alliance N08C1 found an association of the Charcot–Marie–Tooth disease (CMT) gene ARHGEF10 with paclitaxel chemotherapy induced peripheral neuropathy (CIPN) related to the three non-synonymous, recurrent single nucleotide variants (SNV), whereby rs9657362 had the strongest effect, and rs2294039 and rs17683288 contributed only weakly. In the present report, Alliance N08CA was chosen to attempt to replicate the above finding. N08CA was chosen because it is the methodologically most similar study (to N08C1) performed in the CIPN field to date. N08CA enrolled patients receiving the neurotoxic chemotherapy agent paclitaxel. Polyneuropathy was assessed by serial repeat administration of the previously validated patient reported outcome instrument CIPN20. A study-wide, Rasch type model was used to perform extreme phenotyping in n = 138 eligible patients from which “cases” and “controls” were selected for genetic analysis of SNV performed by TaqMan PCR. A significant association of ARHGEF10 with CIPN was found under the pre-specified primary endpoint, with a significance level of p = 0.024. As in the original study, the strongest association of a single SNV was seen for rs9657362 (odds ratio = 3.56, p = 0.018). To further compare results across the new and the previous study, a statistical “classifier” was tested, which achieved a ROC area under the curve of 0.60 for N08CA and 0.66 for N08C1, demonstrating good agreement. Retesting of the primary endpoint of N08C1 in the replication study N08CA validated the association of ARHGEF10 with CIPN.

Neuroendocrine cancer cell lines are used to investigate therapeutic targets in neuroendocrine tumors (NET) and have been instrumental in the design of clinical trials targeting the PI3K/AKT/mTOR pathways, VEGF inhibitors, and somatostatin analogues. It remains unknown, however, whether the genomic makeup of NET cell lines reflect that of primary NET since comprehensive unbiased genome sequencing has not been performed on the cell lines. Four bronchopulmonary NET (BP-NET)—NCI-H720, NCI-H727, NCI-H835, and UMC11—and two pancreatic neuroendocrine tumors (panNET)—BON-1 and QGP1—were cultured. DNA was isolated, and exome sequencing was done. GATK and EXCAVATOR were used for bioinformatic analysis. We detected a total of 1,764 nonsynonymous single nucleotide variants at a rate of 8 per Mb in BP-NET and 4.3 per Mb in panNET cell lines, including 52 mutated COSMIC cancer genes in these cell lines, such as TP53, BRCA1, RB1, TSC2, NOTCH1, EP300, GNAS, KDR, STK11, and APC but not ATRX, DAXX, nor MEN1 . Our data suggest that mutation rate, the pattern of copy number variations, and the mutational spectra in the BP-NET cell lines are more similar to the changes observed in small cell lung cancer than those found in primary BP-NET. Likewise, mutation rate and pattern including the absence of mutations in ATRX/DAXX, MEN1, and YY1 in the panNET cell lines BON1 and QGP1 suggest that these cell lines do not have the genetic signatures of a primary panNET. These results suggest that results from experiments with BP-NET and panNET cell lines need to be interpreted with caution.

Krüppel-like factor 6 (KLF6) is a cancer gene (www.sanger.ac.uk/genetics/CGP/Census/). Here, we demonstrate that KLF6 protein is rapidly degraded when apoptosis is induced via the intrinsic pathway by cisplatin, adriamycin, or UVB irradiation in multiple cell lines (HCT116, SW40, HepG2, PC3-M, Skov3, NIH-3T3, 293T, GM09706, and MEF, IMR-90). KLF6 degradation occurred in the presence or absence of p53, was associated with ubiquitination, mediated by the proteasome (half-life 16min, unstimulated), and independent of caspases and calpain. KLF6 was unchanged by apoptosis via the extrinsic/death-receptor pathway. Deregulation of KLF6 stability may alter its tumor suppressor function and/or the response of tumors to chemotherapeutics.

Carbamazepine (CBZ) is a common antiepileptic drug (AED) that acts through multiple mechanisms including blockade and potentiation of cation channels and modulation of neurotransmitter levels. Whether it affects any component of the gene transcription machinery is unknown. Histone deacetylases (HDAC) are important in the regulation of gene expression and are currently considered a potential target for drug development. Using a high-throughput screening assay based on acetylation-dependent gene expression, we identified CBZ as a candidate and proceeded to characterize its effects on HDAC. CBZ induced acetylation of histone H4 in the HepG2 liver carcinoma cell line. CBZ inhibited HDAC 3 and HDAC 7, which are representatives of HDAC class I and II respectively. Quantitative testing in an in vitro assay demonstrated HDAC inhibition with an IC50 of 2 μM. The major active metabolite of CBZ, CBZ-10,11-epoxide, was found to have the same HDAC inhibitory activity. The IC50 is considerably lower than therapeutic plasma levels that are typically achieved in patients (25–51 μM). CBZ shares important clinical characteristics (teratogenicity, activity as a mood stabilizer) with valproic acid, another AED that was recently identified as an inhibitor of HDAC. These observations raise the possibility that HDAC inhibition may contribute to the pharmacological profile of CBZ.

Hepatocellular carcinoma (HCC) is not only common, but often presents at a stage when potentially curative therapies are not feasible. Although hepatic artery chemoembolization likely confers survival benefit in unresectable HCC, the associated toxicities are substantial and warrant investigation of more efficacious and safe therapies. Many patients who present with unresectable HCC are not chemoembolization candidates, either because of extensive disease or severely impaired hepatic function. We reviewed 44 randomized trials investigating non-embolization-based therapies in unresectable HCC. Hepatic artery infusion of [ 1 3 1 I]lipiodol appears safe; initial studies suggest a survival benefit and efficacy comparable to more toxic embolization-based therapies. Some cytotoxic chemotherapy may confer a modest survival benefit in advanced HCC (including oral fluoropyrimidines, and hepatic arterial or i.v. cisplatin and doxorubicin). Tamoxifen does not confer survival benefit, either in advanced or limited HCC. Other therapies warranting further study include interferon (in optimally cytoreduced HCC), megestrol in patients with variant estrogen receptors, octreotide and pravastatin. More adequately powered, rigorously conducted studies will hopefully identify useful chemo-, radio-, immuno-, embolization-based and biologically targeted therapies during the next decade.

Intrathecal (IT) gene transfer using adeno-associated virus (AAV) may be clinically promising as a treatment for chronic pain if it can produce sufficiently high levels of a transgene product in the cerebrospinal fluid (CSF). Although this strategy was developed in rodents, no studies investigating CSF levels of an analgesic or antiallodynic protein delivered by IT AAV have been performed in large animals. Interleukin-10 (IL-10) is an antiallodynic cytokine for which target therapeutic levels have been established in rats. The present study tested IT AAV8 encoding either human IL-10 (hIL-10) or enhanced green fluorescent protein (EGFP) in a dog model of IT drug delivery. AAV8/hIL-10 at a dose of 3.5 × 10(12) genome copies induced high hIL-10 levels in the CSF, exceeding the target concentration previously found to be antiallodynic in rodents by >1000-fold. AAV8/EGFP targeted the primary sensory and motor neurons and the meninges. hIL-10, a xenogeneic protein in dogs, induced anti-hIL-10 antibodies detectable in the CSF and serum of dogs. The high hIL-10 levels demonstrate the efficacy of AAV for delivery of secreted transgenes into the IT space of large animals, suggesting a strong case for further development toward clinical testing.