Cunningham, Julie M.

Abstract Introduction Discontinuation of opioid medications may be associated with emergence of restless legs syndrome (RLS), which may complicate opioid withdrawal. However, this has not been systematically studied. We aimed to prospectively determine the frequency of the occurrence and severity of RLS symptoms in patients undergoing physician supervised opioid tapering during a 3-week interdisciplinary pain rehabilitation program. Methods Adult patients undergoing prescription opioid taper were prospectively recruited during their participation in the Mayo Pain Rehabilitation Center from 2016 to 2021. Subjects completed the Cambridge-Hopkins RLS Questionnaire 13 and International Restless Legs Syndrome Study Group Rating Scale (IRLS) at baseline, midpoint, and dismissal from the program as well as 2 weeks, 1 and 3 months after dismissal from the program. Results 101 patients participated with 61% being female taking a mean morphine equivalent dose of 46.6 ± 49.0 mg. Baseline prevalence of RLS symptoms was 29% (29/101), increasing to 32% (32/101) at midpoint of treatment and further to 35% (34/97) at dismissal from the program. Frequency of RLS symptoms peaked 2 weeks after dismissal at 38% (30/78) and steadily declined to 35% (28/80) and 31% (21/68) at 1 month and 3 months after dismissal, respectively. Thirty-five percent of patients without baseline RLS symptoms reported de novo RLS symptoms at some point during their opioid taper. RLS severity score followed a similar trend as the presence of symptoms with a baseline of 16.6 ± 10.0. RLS severity score was maximum at 19.1 ± 7.3 one month following dismissal and decreased but remained elevated above baseline at 3 months following dismissal. Conclusion Symptoms of RLS occurred in over a third of patients on chronic opioids and increased during opioid withdrawal, appearing to peak in frequency and severity 2-4 weeks after discontinuation and gradually improved 3 months after discontinuation. Over one-third of patients in our cohort developed de novo symptoms of RLS at some point during their opioid withdrawal. Physicians supervising individuals undergoing opioid withdrawal should be aware of the potential development of RLS symptoms which may hinder successful opioid withdrawal. Support (If Any)

Abstract BackgroundFormalin-fixed, paraffin-embedded (FFPE) tissues have many advantages for identification of risk biomarkers, including wide availability and potential for extended follow-up endpoints. However, RNA derived from archival FFPE samples has limited quality. Here we identified parameters that determine which FFPE samples have the potential for successful RNA extraction, library preparation, and generation of usable RNAseq data.MethodsWe optimized library preparation protocols designed for use with FFPE samples using seven FFPE and Fresh Frozen replicate pairs, and tested optimized protocols using a study set of 130 FFPE biopsies from women with benign breast disease. Metrics from RNA extraction and preparation procedures were collected and compared with bioinformatics sequencing summary statistics. Finally, a decision tree model was built to learn the relationship between pre-sequencing lab metrics and qc pass/fail status as determined by bioinformatics metrics.ResultsSamples that failed bioinformatics qc tended to have low median sample-wise correlation within the cohort (Spearman correlation < 0.75), low number of reads mapped to gene regions (< 25 million), or low number of detectable genes (11,400 # of detected genes with CPM > 2). The median RNA concentration and pre-capture library Qubit values for qc failed samples were 18.7 ng/ul and 1.68 ng/ul respectively, which were significantly lower than those of qc pass samples (40.8 ng/ul and 5.58 ng/ul). We built a decision tree model based on input RNA concentration, input library qubit values, and achieved an F score of 0.803 in predicting QC status (pass/fail) of FFPE samples. ConclusionsWe provide a bioinformatics quality control recommendation for FFPE samples from breast tissue by evaluating bioinformatic and sample metrics. Our results suggest a minimum concentration of 25 ng/ul FFPE-extracted RNA for library preparation and 1.8 ng/ul pre-capture library output to achieve adequate RNA-seq data for downstream bioinformatics analysis.

Abstract Purpose: Cardiac toxicity is a serious potential complication of HER2-directed therapies and anthracyclines. HER2 codon 655 and SLC28A3 gene polymorphisms have been reported to be associated with cardiac toxicity from anti-HER2 and anthracycline therapy, respectively. Association of the polymorphism at HER2 codon 655 with prognosis has also been reported. Experimental Design: Whole blood samples from patients treated on a randomized adjuvant breast cancer trial (BCIRG-006) that compared chemotherapy with or without trastuzumab plus either anthracycline or nonanthracycline chemotherapy were tested for genetic polymorphisms in HER2 codon 655 and SLC28A3. Genotypes were correlated with cardiac function and disease-free survival (DFS) outcomes. Results: Of 3,222 patients enrolled in BCIRG-006, 662 patient samples were successfully genotyped for the rs1136201 allele in HER2 (codon 655): 424 (64%) were AA, 30 (4.5%) were GG, and 208 (31%) were AG genotype. In addition, 665 patient samples were successfully genotyped for the rs7853758 allele in the SLC28A3 gene: 19 (3%) were AA, 475 (71%) were GG, and 171 (26%) were AG genotype. Follow-up time was 10 years. No correlation between DFS, cardiac event rate, or mean left ventricular ejection fraction (LVEF) and rs1136201 genotype was seen in the trastuzumab-treated or non–trastuzumab-treated patients. Moreover, mean LVEF and cardiac event rates were similar in all rs7853758 genotype groups treated with anthracycline-based therapy. Conclusions: In the largest study to date to evaluate whether two polymorphisms are associated with DFS and/or cardiac toxicity in HER2-positive breast cancer treated with trastuzumab and/or anthracyclines, we observed no correlation.

Abstract Background: Ovarian clear cell carcinoma (OCCC) is a rare ovarian cancer histotype that tends to be resistant to standard platinum-based chemotherapeutics. We sought to better understand the role of DNA methylation in clinical and biological subclassification of OCCC. Methods: We interrogated genome-wide methylation using DNA from fresh frozen tumors from 271 cases, applied nonsmooth nonnegative matrix factorization (nsNMF) clustering, and evaluated clinical associations and biological pathways. Results: Two approximately equally sized clusters that associated with several clinical features were identified. Compared with Cluster 2 (N = 137), Cluster 1 cases (N = 134) presented at a more advanced stage, were less likely to be of Asian ancestry, and tended to have poorer outcomes including macroscopic residual disease following primary debulking surgery (P &lt; 0.10). Subset analyses of targeted tumor sequencing and IHC data revealed that Cluster 1 tumors showed TP53 mutation and abnormal p53 expression, and Cluster 2 tumors showed aneuploidy and ARID1A/PIK3CA mutation (P &lt; 0.05). Cluster-defining CpGs included 1,388 CpGs residing within 200 bp of the transcription start sites of 977 genes; 38% of these genes (N = 369 genes) were differentially expressed across cluster in transcriptomic subset analysis (P &lt; 10−4). Differentially expressed genes were enriched for six immune-related pathways, including IFNα and IFNγ responses (P &lt; 10−6). Conclusions: DNA methylation clusters in OCCC correlate with disease features and gene expression patterns among immune pathways. Impact: This work serves as a foundation for integrative analyses that better understand the complex biology of OCCC in an effort to improve potential for development of targeted therapeutics.

Abstract Benign breast disease (BBD) is an established risk factor for development of breast cancer (BC). However, little is known about the prevalence of somatic genetic alterations at the time of BBD diagnosis and relationship to subsequent BC. To characterize DNA variants in BBD and investigate association with BC risk, we isolated DNA from formalin-fixed paraffin-embedded (FFPE) BBD biopsies and assessed mutations using a panel of 93 breast cancer predisposition genes (Qiagen GeneRead). The study set consisted of 120 BBD patients from three age-matched groups defined based on 16 years of BC follow-up: BBD-controls (cancer-free after 16 years), BBD-ER+, or BBD-ER- breast cancer (within 16 years). Bioinformatics quality checks included comparisons of paired fresh-frozen and FFPE replicates to reduce false variant calls induced by FFPE artifacts. Gene-level variant burden differences among study groups were assessed using 12 statistical approaches, adjusted for patient age, year of biopsy, and histology. To investigate possible immune implications of increased variant burden, extent of leukocytes in the BBD biopsies was assessed using CD45 immunohistochemistry. To assess variant profile changes during the progression from BBD to BC, we also performed targeted sequencing of 12 BBD-matched subsequent tumor samples (six ER+ and six ER-) developed within 5 years of BBD diagnosis. Gene-level association results showed consistently skewed distributions suggesting a higher variant burden in BBD controls: among the top 10 genes associated with case-status (p&lt;0.05), all had BC-risk odds ratio estimates less than 1.0. Immune infiltrates, assessed by CD45 H-scores, were positively associated with overall variant burden (r=0.42, p=0.0031), and noticeably higher in cancer-free controls (r=0.50, p=0.005). Comparison of mutation variants in 12 BBD cases that developed cancer within 5 years of diagnosis and their subsequent tumors revealed that more than half of the non-SNP variants were shared between the BBD and the subsequent tumor (54.6%), whereas less than one quarter were unique to the tumor (20.6%) or unique to the BBD (24.9%). Through BBD-BC paired variant analysis, three subjects were found with newly emergent clones uniquely appearing in the tumors and four subjects had loss of clones in the tumor that were present in the BBD. These results show that somatic variant burden is lower in patients with BBD that progress to BC, mutation profiles of BC progression are heterogeneous, and BBD tissues with reduced variant burden also show reduced immune infiltrate. This implicates a potential protective role for immune activation in BBD. Future directions will define the characteristics of the potentially protective immune response and its role in variant heterogeneity in a larger sample set, integrated with paired RNA sequencing to better understand the pathways involved and the relationship to immune infiltration. Citation Format: Stacey J. Winham, Julie M. Cunningham, Yuanhang Liu, Aditya V. Bhagwate, Ethan P. Heinzen, Samantha J. McDonough, Melody L. Stallings-Mann, Robert A. Vierkant, Jodi M. Carter, Mark E. Sherman, Derek C. Radisky, Amy C. Degnim, Chen Wang. Somatic variant burdenin benign breast disease and association with risk of subsequent breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2274.

Abstract Background: Many loci have been found to be associated with risk of epithelial ovarian cancer (EOC). However, although there is considerable variation in progression-free survival (PFS), no loci have been found to be associated with outcome at genome-wide levels of significance. Methods: We carried out a genome-wide association study (GWAS) of PFS in 2,352 women with EOC who had undergone cytoreductive surgery and standard carboplatin/paclitaxel chemotherapy. Results: We found seven SNPs at 12q24.33 associated with PFS (P &lt; 5 × 10–8), the top SNP being rs10794418 (HR = 1.24; 95% CI, 1.15–1.34; P = 1.47 × 10–8). High expression of a nearby gene, ULK1, is associated with shorter PFS in EOC, and with poor prognosis in other cancers. SNP rs10794418 is also associated with expression of ULK1 in ovarian tumors, with the allele associated with shorter PFS being associated with higher expression, and chromatin interactions were detected between the ULK1 promoter and associated SNPs in serous and endometrioid EOC cell lines. ULK1 knockout ovarian cancer cell lines showed significantly increased sensitivity to carboplatin in vitro. Conclusions: The locus at 12q24.33 represents one of the first genome-wide significant loci for survival for any cancer. ULK1 is a plausible candidate for the target of this association. Impact: This finding provides insight into genetic markers associated with EOC outcome and potential treatment options. See related commentary by Peres and Monteiro, p. 1604

BACKGROUND: The adenosine triphosphate-binding cassette, subfamily B, member 1 gene ( ABCB1 ) encodes P-glycoprotein (P-gp) that influences the intracellular transport of solutes including endogenous opioid peptides. The primary objective of this study was to determine the effects of the ABCB1 polymorphism c.3435C>T (rs10454642) on heat pain (HP) perception in a group of opioid-free adults with chronic pain. METHODS: Opioid-free adults with chronic pain consecutively admitted to a pain rehabilitation program comprised the study cohort (N = 134). Individuals were genotyped for the c.3435C>T (rs10454642) polymorphism. The polymorphism was analyzed with nonparametric tests using a dominant (cytosine-cytosine [CC] versus cytosine-thymine [CT] + thymine-thymine [TT]) and recessive (CC + CT versus TT) model of allele effects. Quantitative sensory testing was performed using the Computer Aided Sensory Evaluator IV system. RESULTS: The distribution of genotypes was 22% (N = 29) for CC, 45% (N = 60) for CT, and 33% (N = 45) for TT (Hardy-Weinberg, P > .1). A significant association was observed between the recessive model and HP threshold. Standardized values of HP threshold were significantly greater in the TT group than the CC + CT group (median difference, −0.77; 95% confidence interval [CI], −1.49 to −0.23; P = .005), and the effect size estimate was small (Cliff delta = 0.30). In the dominant model, no significant difference in HP threshold was observed between the CC and CT + TT groups (median difference, −0.45; 95% CI, −1.15 to 0.00; P = .108). CONCLUSIONS: These results posit that the efflux of endogenous opioid peptides is reduced in individuals with the TT genotype due to lower expression of P-gp, which, in turn, results in higher HP threshold. This study contributes to the emerging understanding of how the ABCB1 c.3435C>T polymorphism contributes to pain perception in opioid-free adults with chronic pain and provides the foundation for investigating the potential effects of this polymorphism on the clinical course of chronic pain.

Abstract Background: Benign breast disease (BBD) is a risk factor for breast cancer (BC); however, little is known about the genetic alterations present at the time of BBD diagnosis, and how these relate to risk of incident BC. Methods: A subset of a long-term BBD cohort was selected to examine DNA variation across three BBD groups (42 future estrogen receptor-positive (ER+) BC, 36 future estrogen receptor-negative (ER-) BC and 42 controls cancer-free for at least 16 years post-BBD). DNA extracted from archival formalin fixed, paraffin-embedded (FFPE) tissue blocks was tested and analyzed for presence of DNA alterations using a targeted panel of 93 BC-associated genes. To address artifacts frequently observed in FFPE tissues (e.g., C>T changes), we applied three filtering strategies based on alternative allele frequencies and nucleotide substitution context. Gene-level associations were performed using two burden tests, SKAT-O and logistic regression, and adjusted for clinical and technical covariates including epithelial percentage, histologic impression, patient age and year of BBD biopsy. Results: After filtering, the variant frequency of SNPs in our samples was highly consistent with population allele frequencies reported in 1KG/ExAC (0.986, p <1e-16). Ten genes were found to be associated with later cancer status by four of 12 association methods, with nominal p-values <0.05: MED12, MSH2, BRIP1, PMS1, GATA3, MUC16, FAM175A, EXT2, MLH1 and TGFB1. Additionally, analysis revealed 11 and 19 gene-level associations with p-values <0.05 and OR<1 for all cancer cases and ER- cases, respectively. Comparing between the three case groups, BBD ER+ cases were closer to controls in mutation profile, while BBD ER- cases were distinct. Notably, the variant burden was significantly higher in controls than in either ER+ or ER- cases. CD45 expression was associated with mutational burden (p=0.003).Conclusions: Somatic mutations were more frequent in benign breast tissue from women who did not develop cancer, opening questions of clonal diversity or immune-mediated restraint on future cancer development. CD45 expression was positively associated with mutational burden, most strongly in controls. Further studies in both normal and premalignant tissues are needed to better understand the role of somatic gene mutations and their contribution to future cancer development.

Abstract Background: Accumulating evidence suggests a relationship between endometrial cancer and ovarian cancer. Independent genome-wide association studies (GWAS) for endometrial cancer and ovarian cancer have identified 16 and 27 risk regions, respectively, four of which overlap between the two cancers. We aimed to identify joint endometrial and ovarian cancer risk loci by performing a meta-analysis of GWAS summary statistics from these two cancers. Methods: Using LDScore regression, we explored the genetic correlation between endometrial cancer and ovarian cancer. To identify loci associated with the risk of both cancers, we implemented a pipeline of statistical genetic analyses (i.e., inverse-variance meta-analysis, colocalization, and M-values) and performed analyses stratified by subtype. Candidate target genes were then prioritized using functional genomic data. Results: Genetic correlation analysis revealed significant genetic correlation between the two cancers (rG = 0.43, P = 2.66 × 10−5). We found seven loci associated with risk for both cancers (PBonferroni &lt; 2.4 × 10−9). In addition, four novel subgenome-wide regions at 7p22.2, 7q22.1, 9p12, and 11q13.3 were identified (P &lt; 5 × 10−7). Promoter-associated HiChIP chromatin loops from immortalized endometrium and ovarian cell lines and expression quantitative trait loci data highlighted candidate target genes for further investigation. Conclusions: Using cross-cancer GWAS meta-analysis, we have identified several joint endometrial and ovarian cancer risk loci and candidate target genes for future functional analysis. Impact: Our research highlights the shared genetic relationship between endometrial cancer and ovarian cancer. Further studies in larger sample sets are required to confirm our findings.