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Introduction: Some patients have significant anatomic changes during radiotherapy, necessitating an adaptive repeat CT-simulation and re-planning. This yields two unique planning datasets that introduce uncertainty into total dose records. This study explored the impact of using deformable image registration (DIR) to spatially align repeat CT-simulation images and calculate total planned dose distributions., Materials & Methods: Data from 5 head-and-neck, 5 lung, and 5 sarcoma patients who had unanticipated re-planning during radiotherapy were analyzed in a treatment planning system (RayStation v6.1 RaySearch Laboratories). Total planned doses to normal tissues were calculated using two methods and the previously generated manual contours defined on each CT. The first method, termed 'parameter addition', simply sums the relevant DVH metrics from the initial and re-planned distributions without spatially registering the CTs. The second, termed 'dose accumulation', uses a validated hybrid contour/intensity-based DIR algorithm to deform initial CT and dose distribution onto the repeat CT and re-planning dose distribution. DVH metrics from the summed distribution on the repeat CT are then calculated. Dose differences for organs-at-risk between parameter addition and dose accumulation ≥100 cGy were assumed to be clinically relevant. To elucidate whether relevant differences were due to registration accuracy or contouring variability between CTs, the analysis was repeated using contours on the first CT and the same contours deformed to the repeat CT with DIR., Results: For all patients, high overall DIR accuracy was verified visually (qualitatively) and numerically (quantitatively) using image similarity and contour-based metrics. All head-and-neck and lung patients, and one sarcoma patient (11 of 15 total) had dose differences between parameter addition and dose accumulation ≥100 cGy, with absolute mean differences of 160 cGy (range 101-436 cGy) seen in 41 of 205 total DVH criteria. In 22 of these 41 criteria, these differences were attributed to contouring variability between CTs. After correcting for contouring variations using DIR, the mean absolute differences in 7 of these 22 criteria with a relevant result (across 6 patients) was 146 cGy (range 100-502 cGy). In only 4 DVH criteria, the DIR mapped contours had higher variations than the original contours. One lung patient had a DVH criteria exceeding the clinical dose constraint by 125 cGy with parameter addition, and with accurate DIR and dose accumulation, the criteria was actually 97 cGy lower than the constraint., Conclusions: The use of DIR to generate total planned dose records revealed substantial dose differences in most cases compared to commonly used clinical methods (i.e. parameter addition), and altered the planned acceptance criteria in a minority. DIR is recommended to be used for future adaptive re-plans to generate total planned dose records and facilitate accurate re-contouring. More accurate dose records may also improve our understanding of clinical outcomes., (Copyright © 2024. Published by Elsevier Inc.)

Purpose: To evaluate the use of blood cell-free DNA (cfDNA) to identify emerging mechanisms of resistance to PARP inhibitors (PARPi) in high-grade serous ovarian cancer (HGSOC)., Experimental Design: We used targeted sequencing (TS) to analyze 78 longitudinal cfDNA samples collected from 30 patients with HGSOC enrolled in a phase II clinical trial evaluating cediranib (VEGF inhibitor) plus olaparib (PARPi) after progression on PARPi alone. cfDNA was collected at baseline, before treatment cycle 2, and at end of treatment. These were compared with whole-exome sequencing (WES) of baseline tumor tissues., Results: At baseline (time of initial PARPi progression), cfDNA tumor fractions were 0.2% to 67% (median, 3.25%), and patients with high ctDNA levels (>15%) had a higher tumor burden (sum of target lesions; P = 0.043). Across all timepoints, cfDNA detected 74.4% of mutations known from prior tumor WES, including three of five expected BRCA1/2 reversion mutations. In addition, cfDNA identified 10 novel mutations not detected by WES, including seven TP53 mutations annotated as pathogenic by ClinVar. cfDNA fragmentation analysis attributed five of these novel TP53 mutations to clonal hematopoiesis of indeterminate potential (CHIP). At baseline, samples with significant differences in mutant fragment size distribution had shorter time to progression (P = 0.001)., Conclusions: Longitudinal testing of cfDNA by TS provides a noninvasive tool for detection of tumor-derived mutations and mechanisms of PARPi resistance that may aid in directing patients to appropriate therapeutic strategies. With cfDNA fragmentation analyses, CHIP was identified in several patients and warrants further investigation., (©2023 The Authors; Published by the American Association for Cancer Research.)

The Farnesoid X Receptor (FXR) belongs to the nuclear receptor superfamily and is an essential bile acid (BA) receptor that regulates the expression of genes involved in the metabolism of BAs. FXR protects the liver from BA overload, which is a major etiology of hepatocellular carcinoma. Herein, we investigated the changes in gene expression and chromatin accessibility in hepatocytes by performing RNA-seq in combination with the Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) using a novel FXR knockout mouse model ( Fxr ex5Δ : Nr1h4 ex5Δ/ex5Δ ) generated through CRISPR/Cas9. Consistent with previous Fxr knockout models, we found that Fxr ex5Δ mice develop late-onset HCC associated with increased serum and hepatic BAs. FXR deletion was associated with a dramatic loss of chromatin accessibility, primarily at promoter-associated transcription factor binding sites. Importantly, several genes involved in BA biosynthesis and circadian rhythm were downregulated following loss of FXR, also displayed reduced chromatin accessibility at their promoter regions. Altogether, these findings suggest that FXR helps to maintain a transcriptionally active state by regulating chromatin accessibility through its binding and recruitment of transcription factors and coactivators.

DNA methylation is an essential covalent modification that is required for growth and development. Once considered to be a relatively stable epigenetic mark, many studies have established that DNA methylation is dynamic. The 5-methylcytosine (5-mC) mark can be removed through active DNA demethylation in which 5-mC is converted to an unmodified cytosine through an oxidative pathway coupled to base excision repair (BER). The BER enzyme Thymine DNA Glycosylase (TDG) plays a key role in active DNA demethylation by excising intermediates of 5-mC generated by this process. TDG acts as a key player in transcriptional regulation through its interactions with various nuclear receptors and transcription factors, in addition to its involvement in classical BER and active DNA demethylation, which serve to protect the stability of the genome and epigenome, respectively. Recent animal studies have identified a connection between the loss of Tdg and the onset of tumorigenesis. In this review, we summarize the recent findings on TDG's function as a transcriptional regulator as well as the physiological relevance of TDG and active DNA demethylation in cancer.

Background: In high grade serous ovarian cancer (HGSOC), there is a spectrum of sensitivity to first line platinum-based chemotherapy. This study molecularly characterizes HGSOC patients from two distinct groups of chemotherapy responders (good vs. poor)., Methods: Following primary debulking surgery and intravenous carboplatin/paclitaxel, women with stage III-IV HGSOC were grouped by response. Patients in the good response (GR) and poor response (PR) groups respectively had a progression-free intervals (PFI) of ≥12 and ≤6 months. Analysis of surgical specimens interrogated genomic and immunologic features using whole exome sequencing. RNA-sequencing detected gene expression outliers and inference of immune infiltrate, with validation by targeted NanoString arrays. PD-L1 expression was scored by immunohistochemistry (IHC)., Results: A total of 39 patient samples were analyzed (GR = 20; PR = 19). Median PFI for GR and PR patient cohorts was 32 and 3 months, respectively. GR tumors were enriched for loss-of-function BRCA2 mutations and had a significantly higher nonsynonymous mutation rate compared to PR tumors (p = 0.001). Samples from the PR cohort were characterized by mutations in MGA and RAD51B and trended towards a greater rate of amplification of PIK3CA, MECOM, and ATR in comparison to GR tumors. Gene expression analysis by NanoString correlated increased PARP4 with PR and increased PD-L1 and EMSY with GR. There was greater tumor immune cell infiltration and higher immune cell PD-L1 protein expression in the GR group., Conclusions: Our research demonstrates that tumors from HGSOC patients responding poorly to first line chemotherapy have a distinct molecular profile characterized by actionable drug targets including PARP4., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

DREAM (Dp, Rb-like, E2F, and MuvB) is a transcriptional repressor complex that regulates cell proliferation, and its loss causes neonatal lethality in mice. To investigate DREAM function in adult mice, we used an assembly-defective p107 protein and conditional deletion of its redundant family member p130. In the absence of DREAM assembly, mice displayed shortened survival characterized by systemic amyloidosis but no evidence of excessive cellular proliferation. Amyloid deposits were found in the heart, liver, spleen, and kidneys but not the brain or bone marrow. Using laser-capture microdissection followed by mass spectrometry, we identified apolipoproteins as the most abundant components of amyloids. Intriguingly, apoA-IV was the most detected amyloidogenic protein in amyloid deposits, suggesting apoA-IV amyloidosis (AApoAIV). AApoAIV is a recently described form, whereby WT apoA-IV has been shown to predominate in amyloid plaques. We determined by ChIP that DREAM directly regulated Apoa4 and that the histone variant H2AZ was reduced from the Apoa4 gene body in DREAM's absence, leading to overexpression. Collectively, we describe a mechanism by which epigenetic misregulation causes apolipoprotein overexpression and amyloidosis, potentially explaining the origins of nongenetic amyloid subtypes.

In a recent publication, we demonstrated that conditional deletion of the gene encoding thymine DNA glycosylase (TDG) leads to a late onset of hepatocellular carcinoma (HCC). TDG loss causes disruption in active DNA demethylation in the liver and dysregulation of the farnesoid X receptor and small heterodimer partner (FXR-SHP) regulatory cascade. This leads to a loss of bile acid and glucose homeostasis, which predisposes mice to HCC., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Thymine DNA glycosylase (TDG) is a nuclear receptor coactivator that plays an essential role in the maintenance of epigenetic stability in cells. Here, we demonstrate that the conditional deletion of TDG in adult mice results in a male-predominant onset of hepatocellular carcinoma (HCC). TDG loss leads to a prediabetic state, as well as bile acid (BA) accumulation in the liver and serum of male mice. Consistent with these data, TDG deletion led to dysregulation of the farnesoid X receptor (FXR) and small heterodimer partner (SHP) regulatory cascade in the liver. FXR and SHP are tumor suppressors of HCC and play an essential role in BA and glucose homeostasis. These results indicate that TDG functions as a tumor suppressor of HCC by regulating a transcriptional program that protects against the development of glucose intolerance and BA accumulation in the liver., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

TBX3 is a member of the highly conserved family of T-box transcription factors involved in embryogenesis, organogenesis and tumor progression. While the functional role of TBX3 in tumorigenesis has been widely studied, less is known about the specific functions of the different isoforms (TBX3iso1 and TBX3iso2) which differ in their DNA-binding domain. We therefore sought to investigate the functional consequence of this highly conserved splice event as it relates to TBX3-induced tumorigenesis. By utilizing a nude mouse xenograft model, we have identified differential tumorigenic potential between TBX3 isoforms, with TBX3iso1 overexpression more commonly associated with invasive carcinoma and high tumor vascularity. Transcriptional analysis of signaling pathways altered by TBX3iso1 and TBX3iso2 overexpression revealed significant differences in angiogenesis-related genes. Importantly, osteopontin (OPN), a cancer-associated secreted phosphoprotein, was significantly up-regulated with TBX3iso1 (but not TBX3iso2) overexpression. This pattern was observed across three non/weakly-tumorigenic breast cancer cell lines (21PT, 21NT, and MCF7). Up-regulation of OPN in TBX3iso1 overexpressing cells was associated with induction of hyaluronan synthase 2 (HAS2) expression and increased retention of hyaluronan in pericellular matrices. These transcriptional changes were accompanied by the ability to induce endothelial cell vascular channel formation by conditioned media in vitro, which could be inhibited through addition of an OPN neutralizing antibody. Within the TCGA breast cancer cohort, we identified an 8.1-fold higher TBX3iso1 to TBX3iso2 transcript ratio in tumors relative to control, and this ratio was positively associated with high-tumor grade and an aggressive molecular subtype. Collectively, the described changes involving TBX3iso1-dependent promotion of angiogenesis may thus serve as an adaptive mechanism within breast cancer cells, potentially explaining differences in tumor formation rates between TBX3 isoforms in vivo. This study is the first of its kind to report significant functional differences between the two TBX3 isoforms, both in vitro and in vivo.

Background: The mechanism by which surgical innovation is spread in orthopaedic surgery is not well studied. The recent widespread transition from open to arthroscopic rotator cuff repair techniques provides us with the opportunity to study the spread of new technology; doing so would be important because it is unclear how novel orthopaedic techniques disseminate across time and geography, and previous studies of innovation in healthcare may not apply to the orthopaedic community., Questions/purposes: (1) How much regional variation was associated with the adoption of arthroscopic rotator cuff repair in the United States Medicare population between 2006 and 2014 and how did this change over time? (2) In which regions of the United States was arthroscopic rotator cuff repair first adopted and how did it spread geographically? (3) Which regional factors were associated with the adoption of this new technology?, Methods: We divided the United States into 306 hospital referral regions based upon referral patterns observed in the Centers for Medicare & Medicaid Services MedPAR database, which records all Medicare hospital admissions; this has been done in numerous previous studies using methodology introduced by the Dartmouth Atlas. The proportion of arthroscopic rotator cuff repairs versus open rotator cuff repairs in each hospital referral region was calculated using adjusted procedural rates from the Medicare Part B Carrier File from 2006 to 2014, as it provided a nationwide sample of patients, and was used as a measure of adoption. A population-weighted, multivariable linear regression analysis was used to identify regional characteristics independently associated with adoption., Results: There was substantial regional variation associated with the adoption of arthroscopy for rotator cuff repair as the percentage of rotator cuff repair completed arthroscopically in 2006 ranged widely among hospital referral regions with a high of 85.3% in Provo, UT, USA, and a low of 16.7% in Seattle, WA, USA (OR 30, 95% CI 17.6 to 52.2; p < 0.001). In 2006, regions in the top quartiles for Medicare spending (+9.1%; p = 0.008) independently had higher adoption rates than those in the bottom quartile, as did regions with a greater proportion of college-educated residents (+12.0%; p = 0.009). The Northwest region (-14.4%; p = 0.009) and the presence of an academic medical center (-5.8%; p = 0.026) independently had lower adoption than other regions and those without academic medical centers. In 2014, regions in the top quartiles for Medicare spending (+5.7%; p = 0.033) and regions with a greater proportion of college-educated residents (+9.4%; p = 0.005) independently had higher adoption rates than those in the bottom quartiles, while the Northwest (-9.6%; p = 0.009) and Midwest regions (-5.1%; p = 0.017) independently had lower adoption than other regions., Conclusion: The heterogeneous diffusion of arthroscopic rotator cuff repair across the United States highlights that Medicare beneficiaries across regions did not have equal access to these procedures and that these discrepancies continued to persist over time. A higher level of education and increased healthcare spending were both associated with greater adoption in a region and conversely suggest that regions with lower education and healthcare spending may pursue innovation more slowly. There was evidence that regions with academic medical centers adopted this technology more slowly and may highlight the role that private industry and physicians in nonacademic organizations play in surgical innovation. Future studies are needed to understand if this later adoption leads to inequalities in the quality and value of surgical care delivered to patients in these regions., Level of Evidence: Level III, therapeutic study.

Embryonal tumors with multilayered rosettes (ETMRs) are highly lethal infant brain cancers with characteristic amplification of Chr19q13.41 miRNA cluster (C19MC) and enrichment of pluripotency factor LIN28A. Here we investigated C19MC oncogenic mechanisms and discovered a C19MC-LIN28A-MYCN circuit fueled by multiple complex regulatory loops including an MYCN core transcriptional network and super-enhancers resulting from long-range MYCN DNA interactions and C19MC gene fusions. Our data show that this powerful oncogenic circuit, which entraps an early neural lineage network, is potently abrogated by bromodomain inhibitor JQ1, leading to ETMR cell death., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The acquisition of cellular invasiveness by breast epithelial cells and subsequent transition from ductal carcinoma in situ (DCIS) to invasive breast cancer is a critical step in breast cancer progression. Little is known about the molecular dynamics governing this transition. We have previously shown that overexpression of the transcriptional regulator TBX3 in DCIS-like cells increases survival, growth, and invasiveness. To explore this mechanism further and assess direct transcriptional targets of TBX3 in a high-resolution, isoform-specific context, we conducted genome-wide chromatin-immunoprecipitation (ChIP) arrays coupled with transcriptomic analysis. We show that TBX3 regulates several epithelial-mesenchymal transition (EMT)-related genes, including SLUG and TWIST1. Importantly, we demonstrate that TBX3 is a direct regulator of SLUG expression, and SLUG expression is required for TBX3-induced migration and invasion. Assessing TBX3 by immunohistochemistry in early-stage (stage 0 and stage I) breast cancers revealed high expression in low-grade lesions. Within a second independent early-stage non-high-grade cohort, we observed an association between TBX3 level in the DCIS and size of the invasive focus. Additionally, there was a positive correlation between TBX3 and SLUG, and TBX3 and TWIST1 in the invasive carcinoma. Pathway analysis revealed altered expression of several proteases and their inhibitors, consistent with the ability to degrade basement membrane in vivo. These findings strongly suggest the involvement of TBX3 in the promotion of invasiveness and progression of early-stage pre-invasive breast cancer to invasive carcinoma through the low-grade molecular pathway. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

Methylase-assisted bisulfite sequencing (MAB-seq) is a derivatization technique to evaluate the presence of 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) at base-pair resolution. Although MAB-seq was originally designed to study these metabolites under steady-state conditions, we have developed an alternative protocol to evaluate the dynamics of 5-fC/5-caC accumulation in response to agonists, such as all-trans retinoic acid (ATRA). In addition, this protocol utilizes a lower quantity of the M.SssI enzyme without compromising methylation efficiency and requires less bench time. Herein, we describe the use of MAB-seq assay to evaluate the generation of 5-fC/5-caC in response to ATRA in mouse embryonic fibroblasts, using the hypermethylated in cancer 1 (Hic1) locus as a model system.

Background: The estrogen receptor (ER) is a ligand-dependant transcription factor expressed in many breast cancers and is the target of many endocrine-based cancer therapies. Genome-wide studies have shown that the ER binds to gene-specific enhancer regions in response to β-estradiol (E2) which undergo transcription producing noncoding enhancer RNA (eRNA). While eRNAs are important for transcriptional activation of neighboring genes, the mechanism remains poorly understood., Results: Using ChIP-Seq we generate a global profile of thymine DNA glycosylase (TDG), an ER coactivator that plays an essential role in DNA demethylation, in response to E2 in the MCF7 breast cancer cell line. Remarkably, we found that in response to E2 TDG localized to enhancers which also recruit ERα, RNA Pol II and other coregulators and which are marked by histone modifications indicative of active enhancers. Importantly, depletion of TDG inhibits E2-mediated transcription of eRNAs and transcription of ER-target genes. Functionally, we find that TDG both sensitizes MCF7 cells to tamoxifen-mediated cytostasis and increases migration and invasion of MCF7 cells., Conclusions: Taken together we find that TDG plays a central role in mediating transcription at a subset of enhancers and governs how MCF7 cells respond to both estrogenic and anti-estrogenic compounds and may be an effective therapeutic target.

High-risk human papillomaviruses (HPV) cause cancer at multiple distinct anatomical locations. Regardless of the tissue of origin, most HPV positive (HPV+) cancers show highly upregulated expression of the p16 product of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene. Paradoxically, HPV+ tumor cells require continuous expression of this tumor suppressor for survival. Thus, restoration of normal p16 regulation has potential therapeutic value against HPV induced cancers. Normally, p16 transcription is tightly controlled at the epigenetic level via polycomb repressive complex-mediated tri-methylation of histone 3 lysine 27 (H3K27me3). Although a mechanism by which HPV induces p16 has been proposed based on tissue culture models, it has not been extensively validated in human tumors. In this study, we used data from over 800 human cervical and head and neck tumors from The Cancer Genome Atlas (TCGA) to test this model. We determined the impact of HPV status on expression from the CDKN2A locus, the adjacent CDKN2B locus, and transcript levels of key epigenetic regulators of these loci. As expected, HPV+ tumors from both anatomical sites exhibited high levels of p16. Furthermore, HPV+ tumors expressed higher levels of KDM6A, which demethylates H3K27me3. CpG methylation of the CDKN2A locus was also consistently altered in HPV+ tumors. This data validates previous tissue culture studies and identifies remarkable similarities between the effects of HPV on gene expression and DNA methylation in both cervical and oral tumors in large human cohorts. Furthermore, these results support a model whereby HPV-mediated dysregulation of CDKN2A transcription requires KDM6A, a potentially druggable target., Competing Interests: CONFLICTS OF INTEREST Authors declare no conflicts of interest.

While molecular subgrouping has revolutionized medulloblastoma classification, the extent of heterogeneity within subgroups is unknown. Similarity network fusion (SNF) applied to genome-wide DNA methylation and gene expression data across 763 primary samples identifies very homogeneous clusters of patients, supporting the presence of medulloblastoma subtypes. After integration of somatic copy-number alterations, and clinical features specific to each cluster, we identify 12 different subtypes of medulloblastoma. Integrative analysis using SNF further delineates group 3 from group 4 medulloblastoma, which is not as readily apparent through analyses of individual data types. Two clear subtypes of infants with Sonic Hedgehog medulloblastoma with disparate outcomes and biology are identified. Medulloblastoma subtypes identified through integrative clustering have important implications for stratification of future clinical trials., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Retinoic acid (RA) plays important roles in development, growth, and homeostasis through regulation of the nuclear receptors for RA (RARs). Herein, we identify Hypermethylated in Cancer 1 (Hic1) as an RA-inducible gene. HIC1 encodes a tumor suppressor, which is often silenced by promoter hypermethylation in cancer. Treatment of cells with an RAR agonist causes a rapid recruitment of an RAR/RXR complex consisting of TDG, the lysine acetyltransferase CBP, and TET 1/2 to the Hic1 promoter. Complex binding coincides with a transient accumulation of 5fC/5caC and concomitant upregulation of Hic1 expression, both of which are TDG dependent. Furthermore, conditional deletion of Tdg in vivo is associated with Hic1 silencing and DNA hypermethylation of the Hic1 promoter. These findings suggest that the catalytic and scaffolding activities of TDG are essential for RA-dependent gene expression and provide important insights into the mechanisms underlying targeting of TET-TDG complexes., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)