Citations

AbstractIntron retention (IR) has been implicated in the pathogenesis of complex diseases such as cancers; its association with Alzheimer's disease (AD) remains unexplored. We performed genome‐wide analysis of IR through integrating genetic, transcriptomic, and proteomic data of AD subjects and mouse models from the Accelerating Medicines Partnership‐Alzheimer's Disease project. We identified 4535 and 4086 IR events in 2173 human and 1736 mouse genes, respectively. Quantitation of IR enabled the identification of differentially expressed genes that conventional exon‐level approaches did not reveal. There were significant correlations of intron expression within innate immune genes, like HMBOX1, with AD in humans. Peptides with a high probability of translation from intron‐retained mRNAs were identified using mass spectrometry. Further, we established AD‐specific intron expression Quantitative Trait Loci, and identified splicing‐related genes that may regulate IR. Our analysis provides a novel resource for the search for new AD biomarkers and pathological mechanisms.

AbstractBackground and purposeAlthough erenumab has demonstrated significant reduction in migraine frequency and improved quality of life in studies lasting 3 to 12 months, little is known about long‐term therapy.MethodsThis study was an open‐label, 5‐year treatment phase following a 12‐week, double‐blind, placebo‐controlled trial in adults with episodic migraine. Patients initially received open‐label erenumab 70 mg, which increased to 140 mg following a protocol amendment. Efficacy analyses included change from baseline in monthly migraine days (MMDs), monthly acute migraine‐specific medication (AMSM) days, and health‐related quality of life.ResultsOf 383 patients enrolled, 250 switched to 140 mg; 215 (56.1%) completed open‐label treatment. Mean (standard error) change in MMDs from baseline of 8.7 (0.2) days was −5.3 (0.3) days; an average reduction of 62.3% at year 5. Among patients using AMSM at baseline (6.3 [2.8] treatment days), mean change in monthly AMSM days was −4.4 (0.3) days at the end of 5 years. Patient‐reported outcomes indicated stable improvements in disability, headache impact, and migraine‐specific quality of life. Exposure‐adjusted patient incidence rates of adverse events (AEs) were 123.0/100 patient‐years; AEs were most frequently nasopharyngitis, upper respiratory tract infection, and influenza. Serious AEs (SAEs) reported by 49 patients (3.8/100 patient‐years) were mostly single occurrence. Two fatal adverse events were reported. There were no increases in incidence of AEs, SAEs, or AEs leading to treatment discontinuation over 5 years of exposure.ConclusionsTreatment with erenumab was associated with reductions in migraine frequency and improvements in health‐related quality of life that were maintained for at least 5 years. No new safety signals were observed.

AbstractBackgroundUsing an objective mathematical algorithm to assess corticolimbic involvement of neurofibrillary tangle pathology, we identified three AD subtypes: hippocampal sparing (HpSp) AD, typical AD, and limbic predominant AD. Typical AD brains were representative of the expected patterns of hippocampal and cortical involvement as outlined by Braak tangle stage. In contrast, we discovered an extreme phenotype that exists outside of the Braakian‐concept of neurofibrillary tangle patterns. Hippocampal sparing AD cases demonstrate unexpected sparing of the hippocampus relative to severely involved association cortices and limbic predominant AD cases demonstrate inundation of the hippocampus relative to mildly involved association cortices. Using these AD subtypes, our objective was to leverage our understanding of disease spectrum to uncover transcriptomic changes that underlie selective vulnerability of the hippocampus in AD.MethodWe performed RNA‐sequencing in 40 neuropathologically diagnosed AD cases (10 HpSp, 20 Typical and 10 Limbic) and 15 controls to uncover gene expression changes associated with phenotype differences allowing us to prioritize 44 genes for further exploration. Validation with NanoString and quantification with digital pathology were performed in an expanded cohort of 158 AD cases (36 HpSp, 79 Typical and 35 Limbic) and 32 controls. We quantified digital pathology measures of early tangle pathology (CP13), mature tangle pathology (Ab39), amyloid‐β (33.1.1). In addition, we measured cellular markers for microglia (CD68), endothelia (CD34), and astroglia (GFAP). Deep learning based prediction models were employed, which nominated the SLC38A2 gene as a predictor of AD.ResultThe SLC38A2 gene was originally prioritized in our RNA‐sequencing data based upon differential expression between controls and typical AD. We observed upregulation of SLC38A2 in all AD subtypes. RNA‐seq gene expression measures validated well with NanoString for SLC38A2 (R=0.97, p<0.001). Regression analysis identified early tangle (p=0.011) and late tangle measures (p=0.026) as significant predictors of gene expression, but not amyloid measures (p=0.74). Microgliosis approached significance (p=0.052), but with no contribution from endothelial cell burden (p=0.17) or astrogliosis (p=0.58). Age approached significance (p=0.06), but not sex (p=0.88) or APOE‐ε4 status (p=0.10).ConclusionOur data supports consideration of intra‐disease divergence with regard to case stratification and may reveal genes previously masked by heterogeneity of cohorts.

Background and Aims During liver fibrosis, liver sinusoidal endothelial cells (LSECs) release angiocrine signals to recruit inflammatory cells into the liver. p300, a master regulator of gene transcription, is associated with pathological inflammatory response. Therefore, we examined how endothelial p300 regulates angiocrine signaling and inflammation related to portal hypertension and fibrogenesis. Approach and Results CCl4 or partial inferior vena cava ligation (pIVCL) was used to induce liver injury. Mice with LSEC‐specific p300 deletion (p300 LSECΔ/Δ) or C‐C motif chemokine ligand 2 (Ccl2) deficiency, nuclear factor kappa B (NFκB)–p50 knockout mice, and bromodomain containing 4 (BRD4) inhibitors in wild‐type mice were used to investigate mechanisms of inflammation regulation. Leukocytes were analyzed by mass cytometry by time‐of‐flight. Epigenetic histone marks were modified by CRISPR endonuclease‐deficient CRISPR‐associated 9‐fused with the Krüppel associated box domain (CRISPR‐dCas9‐KRAB)–mediated epigenome editing. Portal pressure and liver fibrosis were reduced in p300 LSECΔ/Δ mice compared to p300 fl/fl mice following liver injury. Accumulation of macrophages was also reduced in p300 LSECΔ/Δ mouse livers. Ccl2 was the most up‐regulated chemokine in injured LSECs, but its increase was abrogated in p300 LSECΔ/Δ mice. While the macrophage accumulation was increased in NFκB‐p50 knockout mice with enhanced NFκB activity, it was reduced in mice with LSEC‐specific Ccl2 deficiency and mice treated with specific BRD4 inhibitors. In vitro, epigenome editing of CCL2 enhancer and promoter regions by CRISPR‐dCas9‐KRAB technology repressed TNFα‐induced CCL2 transcription through H3K9 trimethylation. In contrast, TNFα activated CCL2 transcription by promoting p300 interaction with NFκB and BRD4, leading to histone H3 lysine 27 acetylation at CCL2 enhancer and promoter regions. Conclusions In summary, endothelial p300 interaction with NFκB and BRD4 increases CCL2 expression, leading to macrophage accumulation, portal hypertension, and liver fibrosis. Inhibition of p300 and its binding partners might serve as therapy in the treatment of liver diseases.

Abstract ZUMA-1 demonstrated a high rate of durable response and a manageable safety profile with axicabtagene ciloleucel (axi-cel), an anti-CD19 chimeric antigen receptor (CAR) T-cell therapy, in patients with refractory large B-cell lymphoma. As previously reported, prespecified clinical covariates for secondary end point analysis were not clearly predictive of efficacy; these included Eastern Cooperative Oncology Group performance status (0 vs 1), age, disease subtype, disease stage, and International Prognostic Index score. We interrogated covariates included in the statistical analysis plan and an extensive panel of biomarkers according to an expanded translational biomarker plan. Univariable and multivariable analyses indicated that rapid CAR T-cell expansion commensurate with pretreatment tumor burden (influenced by product T-cell fitness), the number of CD8 and CCR7+CD45RA+ T cells infused, and host systemic inflammation, were the most significant determining factors for durable response. Key parameters differentially associated with clinical efficacy and toxicities, with both theoretical and practical implications for optimizing CAR T-cell therapy. This trial was registered at www.clinicaltrials.gov as #NCT02348216.

Abstract Background Microsatellite instability (MSI) is a molecular phenotype characterized by the lengthening or shortening of tandem DNA repeats within the human genome. MSI phenomenon has been reported in various solid tumors as the consequence of DNA mismatch repair deficiency. It is generally associated with good prognosis and effective immunotherapy treatment. The current MSI tests are based on various methodologies, including immunohistochemistry, polymerase chain reactions, or next-generation sequencing (NGS). Each testing methodology has its own technical advantages, but they either fail at liquid biopsy specimen or implement complicated and expensive experimental interventions. Methods A designated analysis pipeline was developed individually for tissue biopsy and liquid biopsy to evaluate the stability of MSI sites within a NGS panel testing targeting over 400 genes at a common depth of coverage (&lt;1000X for tissue biopsy and &lt;5000X for liquid biopsy). The liquid biopsy algorithm implements a signal enhancing module based on a pool of normal samples to boost assay performance. We validated the tissue biopsy testing with an exploratory cohort of 90 gastrointestinal tract cancer patients. Retrospective review of over 10,000 pan-cancer patient-matched tissue-cfDNA sample pairs permitted a comprehensive evaluation of the liquid biopsy MSI testing performance. Results For tissue biopsy specimen validation, the assay system displayed 0.97 area-under-the-curve (AUC) in receiver operative characteristics (ROC) curve analysis against the comparator assay, out-performing publicly available MSI analysis software. Unfortunately, the tissue MSI analysis pipeline and publicly available software fails with less than 10% sensitivity and 0.55 AUC when applied to liquid biopsy specimen with the patient-matched tissue MSI status used as the gold standard. In contrast, the dedicated cfDNA analysis pipeline significantly boosted the assay performance to 0.73 without additional experimental interventions, such as wild-type allele depletion or molecular barcode. Conclusions MSI testing in liquid biopsy specimen remains challenging due to the low fraction of tumor originated cfDNA and consequently weak signal intensity. The implementation of the signal enhancing MSI analysis algorithm demonstrated high proficiency at improving the signal-to-noise ratio. This bioinformatics approach offered a cost-effective and non-invasive alternative for MSI testing without significantly modifying the NGS library preparation protocols. Citation Format: Xiangyuan Ma, Hua Bao, Xiaoling Tong, Zhili Chang, Xunbiao Liu, Xiuyan Zhang, Ryan S. Robetyore, Xue Wu, Yang W. Shao. Validation of a NGS-based MSI testing workflow in liquid biopsy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 730.

Abstract Background Individual carrying germline mutations within BRCA1 or BRCA2 gene confers well-established increased risk of developing breast and ovarian cancers in the life-time than the general population. Recent advancement in clinical oncology and pharmacology discovered that poly ADP ribose polymerase (PARP) inhibitors are particularly effective in treating cancer that is homologous recombination deficient (HRD) through synthetic lethality, irrespective of the primary tumor site or the type of the BRCA1/2 inactivation variant. Methods A dedicated next-generation sequencing (NGS) panel was designed to interrogate the mutational status of BRCA1/2 and 18 other HRD-associated genes. Using a cohort of 522 clinical samples and artificially engineered cell line samples, we evaluated the mutation detection proficiency for single nucleotide variant (SNV) and small insertion or deletion (Indel) variants. BRCA1/2 inactivation is often the consequence of large genomic rearrangement (LGR) within the gene and phenotypically shown as exon-level copy number variant (CNV). Given the technical challenge of accurate exon-level CNV detection within a small panel, we developed a noise-reduction model based on a pool of normal (PON) samples for CNV result analysis. We further designed droplet digital polymerase chain reaction (ddPCR) for each exon of BRCA1/2 to verify the exonic CNV findings. Results Using a comparator assay as the gold standard, the BRCA1/2 panel achieved an accuracy of 96.7%, a precision of 98.9%, and a lower limit of detection of 3% variant allelic frequency (VAF) for SNV and Indel. The CNV pipeline achieved 97.8% sensitivity and 99.7% specificity for exonic CNV events that were supported by at least one piece of secondary evidence, out-performing publicly available software. The ddPCR verification further enhances assay accuracy and provides a significantly improved statistically confidence for the final CNV calls unparalleled by any other experimental methodologies. Conclusions Using a large cohort of clinical samples, the authors demonstrated the proficiency of a targeted NGS approach for variant and LGR detection of BRCA1/2 and the other genes of the DNA damage repair pathway. An effective method was developed to suppress the noise within the NGS-derived CNV profile, which is exaggerated by the small panel size and short targeted regions. The authors would also like to call the researcher's attention to a critical caveat within the CNV testing results generated from NGS-based methods. Citation Format: Xiangyuan Ma, Hua Bao, Zhili Chang, Yong Wu, Changwan Du, Rong Gao, Yu Song, Ryan S. Robetyore, Xue Wu, Yang W. Shao. Technical validation of a NGS-based analysis pipeline for BRCA1/2 variants and large genomic rearrangements detection [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4268.

Abstract Background: The intrinsic immune response in triple negative breast cancer (TNBC) has both prognostic and predictive utility, and immune-related biomarkers are now actionable targets in TNBC. Current modalities for immune profiling are limited by lack of spatial context and precise quantitation. We used quantitative digital spatial profiling (DSP) to 1) characterize the immune architecture of tumoral and stromal regions in TNBC and 2) identify prognostic immune signatures. Methods: From the FinXX trial of adjuvant capecitabine therapy, tumor sections (FFPE) matched for patient characteristics, treatment arm and RFS-based outcome (N=44) were assessed with Nanostring GeoMxTM DSP. Briefly, with 3-plex immunofluorescence (pan-cytokeratin, CD45, CD68 with SYTO-13 dye for nuclei), spatially-defined CD45-enriched or CD68-enriched stromal segments and adjacent tumor segments (N=950) were selected for digital quantitation (NanoString nCounter) of 39 immune proteins (e.g. immune cell profiling proteins: CD3, CD4, CD8, CD11c, CD20, CD56, CD68, HLA-DR, PD-1, PD-L1, Granzyme B), immune drug targets (e.g. VISTA, STING, IDO1), and activation status-related proteins (e.g. ICOS, PD-L2, CD40, CD40L, CD44). Normalized, differentially-expressed (DE) proteins were evaluated, and weighted gene co-expression network analysis (WGCNA) was performed to identify modules of highly co-expressing immune proteins, and association with RFS as a categorical variable was determined. Results: 20 DE proteins in tumor segments were associated with RFS. Among them, those with the highest RFS-associated fold changes (FC) included CD20, CD40, CD56, HLA-DR, Granzyme B, and PD-L2 (FC): 2.1-4.4) (p &lt; 0.05). In contrast, only 4 DE proteins were associated with RFS in stromal segments (Granzyme B, IDO-1, PD-L1, and PD-L2 (FC: 1.5-3.3) (p &lt; 0.05). By WGCNA, 2 tumor-based immune modules [IM1 (lymphocyte infiltration) and IM2 (lymphocyte activation)] were identified based on their co-expression. The top three co-expressed proteins in IM1 were CD4, CD40, and CD45 (r &gt;0.9). The three top co-expressed proteins in IM2 were PD-L1, PD-L2, and Granzyme B (r &gt; 0.85). Together with CD56 expression, IM1 and IM2 comprised a set of 3 intratumoral immune features that associated with RFS. For recurring tumors, all 3 immune features clustered with low scores . Among non-recurrent tumors, two distinct, non-overlapping clusters with inverse characteristics were identified: high IM2, low IM1, low CD56 vs. low IM2, high IM1, and high CD56. Overall, across all 3 immune features, PDL2, CD40 and CD56 expression most strongly associated with RFS. Conclusion: With spatially-defined, precise quantification of immune proteins, we identified 3 immune features, including two distinct immune modules, IM1 and IM2 that correlate with durable RFS. These immune modules of highly co-expressing proteins were associated with RFS only when present in the tumor compartment but not tumor-adjacent, immune cell-rich stroma. Citation Format: Jodi M. Carter, Saranya Chumsri, Yaohua Ma, Xue Wang, Jennifer M. Kachergus, Douglas Hinerfeld, Heather Ann Brauer, Sarah Warren, Heikki Joensuu, Edith A. Perez, Aubrey Thompson. Quantitative digital spatial profiling reveals novel prognostic intratumoral immune signatures in triple-negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1004.