Long-read sequencing of an advanced cancer cohort resolves rearrangements, unravels haplotypes, and reveals methylation landscapes.

The Long-Read Personalized OncoGenomics (POG) dataset comprises a cohort of 189 patient tumors and 41 matched normal samples sequenced using the Oxford Nanopore Technologies PromethION platform. This dataset from the POG program and the Marathon of Hope Cancer Centres Network includes DNA and RNA short-read sequence data, analytics, and clinical information. We show the potential of long-read sequencing for resolving complex cancer-related structural variants, viral integrations, and extrachromosomal circular DNA. Long-range phasing facilitates the discovery of allelically differentially methylated regions (aDMRs) and allele-specific expression, including recurrent aDMRs in the cancer genes RET and CDKN2A. Germline promoter methylation in MLH1 can be directly observed in Lynch syndrome. Promoter methylation in BRCA1 and RAD51C is a likely driver behind homologous recombination deficiency where no coding driver mutation was found. This dataset demonstrates applications for long-read sequencing in precision medicine and is available as a resource for developing analytical approaches using this technology.

Whole genome and transcriptome integrated analyses guide clinical care of pediatric poor prognosis cancers.

The role for routine whole genome and transcriptome analysis (WGTA) for poor prognosis pediatric cancers remains undetermined. Here, we characterize somatic mutations, structural rearrangements, copy number variants, gene expression, immuno-profiles and germline cancer predisposition variants in children and adolescents with relapsed, refractory or poor prognosis malignancies who underwent somatic WGTA and matched germline sequencing. Seventy-nine participants with a median age at enrollment of 8.8 y (range 6 months to 21.2 y) are included. Germline pathogenic/likely pathogenic variants are identified in 12% of participants, of which 60% were not known prior. Therapeutically actionable variants are identified by targeted gene report and whole genome in 32% and 62% of participants, respectively, and increase to 96% after integrating transcriptome analyses. Thirty-two molecularly informed therapies are pursued in 28 participants with 54% achieving a clinical benefit rate; objective response or stable disease ≥6 months. Integrated WGTA identifies therapeutically actionable variants in almost all tumors and are directly translatable to clinical care of children with poor prognosis cancers.

Defining the heterogeneity of unbalanced structural variation underlying breast cancer susceptibility by nanopore genome sequencing.

Germline structural variants (SVs) are challenging to resolve by conventional genetic testing assays. Long-read sequencing has improved the global characterization of SVs, but its sensitivity at cancer susceptibility loci has not been reported. Nanopore long-read genome sequencing was performed for nineteen individuals with pathogenic copy number alterations in BRCA1, BRCA2, CHEK2 and PALB2 identified by prior clinical testing. Fourteen variants, which spanned single exons to whole genes and included a tandem duplication, were accurately represented. Defining the precise breakpoints of SVs in BRCA1 and CHEK2 revealed unforeseen allelic heterogeneity and informed the mechanisms underlying the formation of recurrent deletions. Integrating read-based and statistical phasing further helped define extended haplotypes associated with founder alleles. Long-read sequencing is a sensitive method for characterizing private, recurrent and founder SVs underlying breast cancer susceptibility. Our findings demonstrate the potential for nanopore sequencing as a powerful genetic testing assay in the hereditary cancer setting.

Optimising clinical care through CDH1-specific germline variant curation: improvement of clinical assertions and updated curation guidelines.

BACKGROUND: Germline pathogenic variants in CDH1 are associated with increased risk of diffuse gastric cancer and lobular breast cancer. Risk reduction strategies include consideration of prophylactic surgery, thereby making accurate interpretation of germline CDH1 variants critical for physicians deciding on these procedures. The Clinical Genome Resource (ClinGen) CDH1 Variant Curation Expert Panel (VCEP) developed specifications for CDH1 variant curation with a goal to resolve variants of uncertain significance (VUS) and with ClinVar conflicting interpretations and continues to update these specifications. METHODS: CDH1 variant classification specifications were modified based on updated genetic testing clinical criteria, new recommendations from ClinGen and expert knowledge from ongoing CDH1 variant curations. The CDH1 VCEP reviewed 273 variants using updated CDH1 specifications and incorporated published and unpublished data provided by diagnostic laboratories. RESULTS: Updated CDH1-specific interpretation guidelines include 11 major modifications since the initial specifications from 2018. Using the refined guidelines, 97% (36 of 37) of variants with ClinVar conflicting interpretations were resolved to benign, likely benign, likely pathogenic or pathogenic, and 35% (15 of 43) of VUS were resolved to benign or likely benign. Overall, 88% (239 of 273) of curated variants had non-VUS classifications. To date, variants classified as pathogenic are either nonsense, frameshift, splicing, or affecting the translation initiation codon, and the only missense variants classified as pathogenic or likely pathogenic have been shown to affect splicing. CONCLUSIONS: The development and evolution of CDH1-specific criteria by the expert panel resulted in decreased uncertain and conflicting interpretations of variants in this clinically actionable gene, which can ultimately lead to more effective clinical management recommendations.

Immune modulation by molecularly targeted photothermal ablation in a mouse model of advanced hepatocellular carcinoma and cirrhosis.

Immunotherapy is a promising new treatment approach for hepatocellular carcinoma (HCC), but there are numerous barriers to immunotherapy in HCC, including an immunosuppressive microenvironment and the "immunotolerance" of the liver. Hyperthermia treatment modalities are standard of care for early stage HCC, and hyperthermia is known to have immunomodulatory effects. We have developed a molecularly targeted photothermal ablation (MTPA) technology that provides thermally tunable, tumor-specific heat generation. The purpose of this study was to evaluate the morphologic and immunologic effects of MTPA in an immunotherapy-resistant syngeneic mouse model of HCC in a background of toxin-induced cirrhosis. We found that the anatomic, cellular, and molecular features of this model recapitulate the characteristics of advanced human HCC. MTPA as a monotherapy and in combination with immune checkpoint therapy significantly increased intratumoral CD3+ and activated CD8+ T cells while decreasing regulatory T cells relative to control or immune checkpoint therapy alone based on immunohistochemistry, flow cytometry, and single cell RNA sequencing data. Furthermore, we identified evidence of MTPA's influence on systemic tumor immunity, with suppression of remote tumor growth following treatment of orthotopic tumors. The results of this study suggest that tumor-specific hyperthermia may help overcome resistance mechanisms to immunotherapy in advanced HCC.

Integrating Tumor Sequencing Into Clinical Practice for Patients With Mismatch Repair-Deficient Lynch Syndrome Spectrum Cancers.

INTRODUCTION: Uninformative germline genetic testing presents a challenge to clinical management for patients suspected to have Lynch syndrome, a cancer predisposition syndrome caused by germline variants in the mismatch repair (MMR) genes or EPCAM. METHODS: Among a consecutive series of MMR-deficient Lynch syndrome spectrum cancers identified through immunohistochemistry-based tumor screening, we investigated the clinical utility of tumor sequencing for the molecular diagnosis and management of suspected Lynch syndrome families. MLH1-deficient colorectal cancers were prescreened for BRAF V600E before referral for genetic counseling. Microsatellite instability, MLH1 promoter hypermethylation, and somatic and germline genetic variants in the MMR genes were assessed according to an established clinical protocol. RESULTS: Eighty-four individuals with primarily colorectal (62%) and endometrial (31%) cancers received tumor-normal sequencing as part of routine clinical genetic assessment. Overall, 27% received a molecular diagnosis of Lynch syndrome. Most of the MLH1-deficient tumors were more likely of sporadic origin, mediated by MLH1 promoter hypermethylation in 54% and double somatic genetic alterations in MLH1 (17%). MSH2-deficient, MSH6-deficient, and/or PMS2-deficient tumors could be attributed to pathogenic germline variants in 37% and double somatic events in 28%. Notably, tumor sequencing could explain 49% of cases without causal germline variants, somatic MLH1 promoter hypermethylation, or somatic variants in BRAF. DISCUSSION: Our findings support the integration of tumor sequencing into current Lynch syndrome screening programs to improve clinical management for individuals whose germline testing is uninformative.

Modelling hereditary diffuse gastric cancer initiation using transgenic mouse-derived gastric organoids and single-cell sequencing.

Hereditary diffuse gastric cancer (HDGC) is a cancer syndrome caused by germline variants in CDH1, the gene encoding the cell-cell adhesion molecule E-cadherin. Loss of E-cadherin in cancer is associated with cellular dedifferentiation and poor prognosis, but the mechanisms through which CDH1 loss initiates HDGC are not known. Using single-cell RNA sequencing, we explored the transcriptional landscape of a murine organoid model of HDGC to characterize the impact of CDH1 loss in early tumourigenesis. Progenitor populations of stratified squamous and simple columnar epithelium, characteristic of the mouse stomach, showed lineage-specific transcriptional programs. Cdh1 inactivation resulted in shifts along the squamous differentiation trajectory associated with aberrant expression of genes central to gastrointestinal epithelial differentiation. Cytokeratin 7 (CK7), encoded by the differentiation-dependent gene Krt7, was a specific marker for early neoplastic lesions in CDH1 carriers. Our findings suggest that deregulation of developmental transcriptional programs may precede malignancy in HDGC. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Influence of injection technique, drug formulation and tumor microenvironment on intratumoral immunotherapy delivery and efficacy.

BACKGROUND: Intratumoral delivery of immunotherapeutics represents a compelling solution to directly address local barriers to tumor immunity. However, we have previously shown that off-target delivery is a substantial problem during intratumoral injections; this can lead to diminished drug efficacy and systemic toxicities. We have identified three variables that influence intratumoral drug delivery: injection technique, drug formulation and tumor microenvironment. The purpose of this study was to characterize the impact of modifications in each variable on intratumoral drug delivery and immunotherapy efficacy. METHODS: Intratumoral injections were performed in a hybrid image-guided intervention suite with ultrasound, fluoroscopy and CT scanning capabilities in both rat and mouse syngeneic tumor models. Intratumoral drug distribution was quantified by CT volumetric imaging. The influence of varying needle design and hydrogel-based drug delivery on the immune response to a stimulator of interferon genes (STING) agonist was evaluated using flow cytometry and single cell RNA sequencing. We also evaluated the influence of tumor stiffness on drug injection distribution. RESULTS: Variations in needle design, specifically with the use of a multiside hole needle, led to approximately threefold improvements in intratumoral drug deposition relative to conventional end-hole needles. Likewise, delivery of a STING agonist through a multiside hole needle led to significantly increased expression of type I interferon-associated genes and 'inflammatory' dendritic cell gene signatures relative to end-hole STING agonist delivery. A multidomain peptide-based hydrogel embedded with a STING agonist led to substantial improvements in intratumoral deposition; however, the hydrogel was noted to generate a strong immune response against itself within the target tumor. Evaluation of tumor stroma on intratumoral drug delivery revealed that there was a greater than twofold improvement in intratumoral distribution in soft tumors (B16 melanoma) compared with firm tumors (MC38 colorectal). CONCLUSIONS: Injection technique, drug formulation and tumor stiffness play key roles in the accurate delivery of intratumoral immunotherapeutics.

A novel irinotecan-lipiodol nanoemulsion for intravascular administration: pharmacokinetics and biodistribution in the normal and tumor bearing rat liver.

Colorectal cancer is one of the most common cancers in the United States and treatment options are limited for patients who develop liver metastases. Several chemotherapeutic regimens have been used for transvascular liver-directed therapy in the treatment of colorectal liver metastases without clear evidence of superiority of one therapy over another. We describe the development of a novel nanoemulsion through combining irinotecan (IRI), a first line systemic agent used for the treatment of colon cancer, with lipiodol, an oily contrast medium derived from poppy seed oil, and evaluated its pharmacokinetic and biodistribution profile as a function of portal venous chemoembolization (PVCE) versus transarterial chemoembolization (TACE) delivery. The Tessari technique was used to create a stable emulsion (20 mg IRI mixed with 2 mL lipiodol) with resultant particle size ranging from 28.9 nm to 56.4 nm. Pharmacokinetic profile established through venous sampling in Buffalo rats demonstrate that the area under the curve (AUC0-∞) of IRI was significantly less after PVCE with IRI-lipiodol as compared to IRI alone (131 vs. 316 µg*min/mL, p-value = .023), suggesting significantly higher amounts of IRI retention in the liver with the IRI-lipiodol nanoemulsion via first-pass extraction. Subseqent biodistribution studies in tumor-bearing WAG/Rjj rats revealed more IRI present in the tumor following TACE versus PVCE (29.19 ± 12.33 µg/g versus 3.42 ± 1.62; p-value = .0033) or IV (29.19 ± 12.33 µg/g versus 1.05 ± 0.47; p-value = .0035). The IRI-lipiodol nanoemulsion demonstrated an acceptable hepatotoxicity profile in all routes of administration. In conclusion, the IRI-lipiodol nanoemulsion via TACE showed promise and warrants further investigation as an option for the treatment of metastatic colorectal cancer.

Molecularly targeted photothermal ablation improves tumor specificity and immune modulation in a rat model of hepatocellular carcinoma.

Thermal ablation is a standard therapy for patients with hepatocellular carcinoma (HCC). Contemporary ablation devices are imperfect, as they lack tumor specificity. An ideal ablation modality would generate thermal energy only within tumoral tissue. Furthermore, as hyperthermia is known to influence tumor immunity, such a tumor-specific ablation modality may have the ability to favorably modulate the tumor immune landscape. Here we show a clinically relevant thermal ablation modality that generates tumor-specific hyperthermia, termed molecularly targeted photothermal ablation (MTPA), that is based upon the excellent localization of indocyanine green to HCC. In a syngeneic rat model, we demonstrate the tumor-specific hyperthermia generated by MTPA. We also show through spatial and transcriptomic profiling techniques that MTPA favorably modulates the intratumoral myeloid population towards tumor immunogenicity and diminishes the systemic release of oncogenic cytokines relative to conventional ablation modalities.

Establishing a Framework for the Clinical Translation of Germline Findings in Precision Oncology.

Inherited genetic variation has important implications for cancer screening, early diagnosis, and disease prognosis. A role for germline variation has also been described in shaping the molecular landscape, immune response, microenvironment, and treatment response of individual tumors. However, there is a lack of consensus on the handling and analysis of germline information that extends beyond known or suspected cancer susceptibility in large-scale cancer genomics initiatives. As part of the Personalized OncoGenomics program in British Columbia, we performed whole-genome and transcriptome sequencing in paired tumor and normal tissues from advanced cancer patients to characterize the molecular tumor landscape and identify putative targets for therapy. Overall, our experience supports a multidisciplinary and integrative approach to germline data management. This includes a need for broader definitions and standardized recommendations regarding primary and secondary germline findings in precision oncology. Here, we propose a framework for identifying, evaluating, and returning germline variants of potential clinical significance that may have indications for health management beyond cancer risk reduction or prevention in patients and their families.

Endometrial Cancer Molecular Risk Stratification is Equally Prognostic for Endometrioid Ovarian Carcinoma.

PURPOSE: Endometrioid ovarian carcinoma (ENOC) is generally associated with a more favorable prognosis compared with other ovarian carcinomas. Nonetheless, current patient treatment continues to follow a "one-size-fits-all" approach. Even though tumor staging offers stratification, personalized treatments remain elusive. As ENOC shares many clinical and molecular features with its endometrial counterpart, we sought to investigate The Cancer Genome Atlas-inspired endometrial carcinoma (EC) molecular subtyping in a cohort of ENOC. EXPERIMENTAL DESIGN: IHC and mutation biomarkers were used to segregate 511 ENOC tumors into four EC-inspired molecular subtypes: low-risk POLE mutant (POLEmut), moderate-risk mismatch repair deficient (MMRd), high-risk p53 abnormal (p53abn), and moderate-risk with no specific molecular profile (NSMP). Survival analysis with established clinicopathologic and subtype-specific features was performed. RESULTS: A total of 3.5% of cases were POLEmut, 13.7% MMRd, 9.6% p53abn, and 73.2% NSMP, each showing distinct outcomes (P < 0.001) and survival similar to observations in EC. Median OS was 18.1 years in NSMP, 12.3 years in MMRd, 4.7 years in p53abn, and not reached for POLEmut cases. Subtypes were independent of stage, grade, and residual disease in multivariate analysis. CONCLUSIONS: EC-inspired molecular classification provides independent prognostic information in ENOC. Our findings support investigating molecular subtype-specific management recommendations for patients with ENOC; for example, subtypes may provide guidance when fertility-sparing treatment is desired. Similarities between ENOC and EC suggest that patients with ENOC may benefit from management strategies applied to EC and the opportunity to study those in umbrella trials.

Improved structural variant interpretation for hereditary cancer susceptibility using long-read sequencing.

PURPOSE: Structural variants (SVs) may be an underestimated cause of hereditary cancer syndromes given the current limitations of short-read next-generation sequencing. Here we investigated the utility of long-read sequencing in resolving germline SVs in cancer susceptibility genes detected through short-read genome sequencing. METHODS: Known or suspected deleterious germline SVs were identified using Illumina genome sequencing across a cohort of 669 advanced cancer patients with paired tumor genome and transcriptome sequencing. Candidate SVs were subsequently assessed by Oxford Nanopore long-read sequencing. RESULTS: Nanopore sequencing confirmed eight simple pathogenic or likely pathogenic SVs, resolving three additional variants whose impact could not be fully elucidated through short-read sequencing. A recurrent sequencing artifact on chromosome 16p13 and one complex rearrangement on chromosome 5q35 were subsequently classified as likely benign, obviating the need for further clinical assessment. Variant configuration was further resolved in one case with a complex pathogenic rearrangement affecting TSC2. CONCLUSION: Our findings demonstrate that long-read sequencing can improve the validation, resolution, and classification of germline SVs. This has important implications for return of results, cascade carrier testing, cancer screening, and prophylactic interventions.

Endobronchial ultrasound-guided injection of NBTXR3 radio-enhancing nanoparticles into mediastinal and hilar lymph nodes: a swine model to evaluate feasibility, injection technique, safety, nanoparticle retention and dispersion.

BACKGROUND: Loco-regionally advanced lung cancer is typically treated with a combination of chemotherapy and radiation therapy, but overall survival and local control remain poor. Radio-enhancing nanoparticles such as NBTXR3 activated by radiotherapy results in increased cell death and potentially an anti-tumor immune response. The goal of this study was to assess the feasibility and safety of endobronchial ultrasound (EBUS)-guided injection of NBTXR3 into mediastinal and hilar lymph nodes (LN), as well as assess nanoparticle retention in the LN post-injection. METHODS: Animals underwent bronchoscopy under general anesthesia with EBUS-guided injection of NBTXR3 into hilar and mediastinal LN. LN and injection volumes were calculated based on pre-injection computed tomography (CT) scans. CT scans were repeated at 5 min, 30 min, and 8 days post-injection. Blood-draws were also obtained at baseline and post-injection. Animals were then housed, monitored, and sacrificed 8 days post-injection. Necropsy was then performed with gross and histologic analysis of LN. RESULTS: A total of 20 LN were injected in 5 pigs (4 LN per animal). Nanoparticles were retained in 100% of LN at 30 min, and 90% of LN at 8 days. Extravasation of nanoparticles was seen in 4 out of the 20 LN. There were no cases of nanoparticle embolization visible by CT in distant organs. Small air-bubbles were introduced in the targets and surrounding tissue in 3 out of 20 LN. Of note, at 8 days, none of these air-bubbles were present on CT scan. There were no intra-procedural or post-procedural complications in either CT scans or necropsy findings. Pigs remained clinically stable and neither laboratory values nor necropsy showed evidence of inflammation. CONCLUSIONS: EBUS-guided injection of NBTXR3 radio-enhancing nanoparticles can be safely performed achieving a high rate of nanoparticle retention, low extravasation, and no visible nanoparticle embolization.

Burden of hereditary cancer susceptibility in unselected patients with pancreatic ductal adenocarcinoma referred for germline screening.

BACKGROUND: Recent guidelines recommend consideration of germline testing for all newly diagnosed pancreatic ductal adenocarcinoma (PDAC). The primary aim of this study was to determine the burden of hereditary cancer susceptibility in PDAC. A secondary aim was to compare genetic testing uptake rates across different modes of genetic counselling. PATIENTS AND METHODS: All patients diagnosed with PDAC in the province of British Columbia, Canada referred to a population-based hereditary cancer program were eligible for multi-gene panel testing, irrespective of cancer family history. Any healthcare provider or patients themselves could refer. RESULTS: A total of 305 patients with PDAC were referred between July 2016 and January 2019. Two hundred thirty-five patients attended a consultation and 177 completed index germline genetic testing. 25/177 (14.1%) of unrelated patients had a pathogenic variant (PV); 19/25 PV were in known PDAC susceptibility genes with cancer screening or risk-reduction implications. PDAC was significantly associated with PV in ATM (OR, 7.73; 95% CI, 3.10 to 19.33, P = 6.14E-05) when comparing age and gender and ethnicity-matched controls tested on the same platform. The overall uptake rate for index testing was 59.2% and was significantly higher with 1-on-1 consultations and group consultations compared to telehealth consultations (88.9% vs 82.9% vs 61.8%, P < .001). CONCLUSION: In a prospective clinic-based cohort of patients with PDAC referred for testing irrespective of family history, germline PV were detected in 14.1%. PV in ATM accounted for half of all PVs and were significantly associated with PDAC. These findings support recent guidelines and will guide future service planning in this population.

Pan-cancer analysis of advanced patient tumors reveals interactions between therapy and genomic landscapes.

Advanced and metastatic tumors with complex treatment histories drive cancer mortality. Here we describe the POG570 cohort, a comprehensive whole-genome, transcriptome and clinical dataset, amenable for exploration of the impacts of therapies on genomic landscapes. Previous exposure to DNA-damaging chemotherapies and mutations affecting DNA repair genes, including POLQ and genes encoding Polζ, were associated with genome-wide, therapy-induced mutagenesis. Exposure to platinum therapies coincided with signatures SBS31 and DSB5 and, when combined with DNA synthesis inhibitors, signature SBS17b. Alterations in ESR1, EGFR, CTNNB1, FGFR1, VEGFA and DPYD were consistent with drug resistance and sensitivity. Recurrent noncoding events were found in regulatory region hotspots of genes including TERT, PLEKHS1, AP2A1 and ADGRG6. Mutation burden and immune signatures corresponded with overall survival and response to immunotherapy. Our data offer a rich resource for investigation of advanced cancers and interpretation of whole-genome and transcriptome sequencing in the context of a cancer clinic.

Specifications of the ACMG/AMP variant curation guidelines for the analysis of germline CDH1 sequence variants.

The variant curation guidelines published in 2015 by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) provided the genetics community with a framework to assess variant pathogenicity; however, these rules are not gene specific. Germline pathogenic variants in the CDH1 gene cause hereditary diffuse gastric cancer and lobular breast cancer, a clinically challenging cancer predisposition syndrome that often requires a multidisciplinary team of experts to be properly managed. Given this challenge, the Clinical Genome Resource (ClinGen) Hereditary Cancer Domain prioritized the development of the CDH1 variant curation expert panel (VCEP) to develop and implement rules for CDH1 variant classifications. Here, we describe the CDH1 specifications of the ACMG/AMP guidelines, which were developed and validated after a systematic evaluation of variants obtained from a cohort of clinical laboratory data encompassing ∼827,000 CDH1 sequenced alleles. Comparing previously reported germline variants that were classified using the 2015 ACMG/AMP guidelines to the CDH1 VCEP recommendations resulted in reduced variants of uncertain significance and facilitated resolution of variants with conflicted assertions in ClinVar. The ClinGen CDH1 VCEP recommends the use of these CDH1-specific guidelines for the assessment and classification of variants identified in this clinically actionable gene.

Development of an Electroporation and Nanoparticle-based Therapeutic Platform for Bone Metastases.

Purpose To assess for nanopore formation in bone marrow cells after irreversible electroporation (IRE) and to evaluate the antitumoral effect of IRE, used alone or in combination with doxorubicin (DOX)-loaded superparamagnetic iron oxide (SPIO) nanoparticles (SPIO-DOX), in a VX2 rabbit tibial tumor model. Materials and Methods All experiments were approved by the institutional animal care and use committee. Five porcine vertebral bodies in one pig underwent intervention (IRE electrode placement without ablation [n = 1], nanoparticle injection only [n = 1], and nanoparticle injection followed by IRE [n = 3]). The animal was euthanized and the vertebrae were harvested and evaluated with scanning electron microscopy. Twelve rabbit VX2 tibial tumors were treated, three with IRE, three with SPIO-DOX, and six with SPIO-DOX plus IRE; five rabbit VX2 tibial tumors were untreated (control group). Dynamic T2*-weighted 4.7-T magnetic resonance (MR) images were obtained 9 days after inoculation and 2 hours and 5 days after treatment. Antitumor effect was expressed as the tumor growth ratio at T2*-weighted MR imaging and percentage necrosis at histologic examination. Mixed-effects linear models were used to analyze the data. Results Scanning electron microscopy demonstrated nanopores in bone marrow cells only after IRE (P , .01). Average volume of total tumor before treatment (503.1 mm3 ± 204.6) was not significantly different from those after treatment (P = .7). SPIO-DOX was identified as a reduction in signal intensity within the tumor on T2*-weighted images for up to 5 days after treatment and was related to the presence of iron. Average tumor growth ratios were 103.0% ± 75.8 with control treatment, 154.3% ± 79.7 with SPIO-DOX, 77% ± 30.8 with IRE, and -38.5% ± 24.8 with a combination of SPIO-DOX and IRE (P = .02). The percentage residual viable tumor in bone was significantly less for combination therapy compared with control (P = .02), SPIO-DOX (P , .001), and IRE (P = .03) treatment. The percentage residual viable tumor in soft tissue was significantly less with IRE (P = .005) and SPIO-DOX plus IRE (P = .005) than with SPIO-DOX. Conclusion IRE can induce nanopore formation in bone marrow cells. Tibial VX2 tumors treated with a combination of SPIO-DOX and IRE demonstrate enhanced antitumor effect as compared with individual treatments alone. © RSNA, 2017 Online supplemental material is available for this article.

Insights into interplay between rexinoid signaling and myogenic regulatory factor-associated chromatin state in myogenic differentiation.

While skeletal myogenesis is tightly coordinated by myogenic regulatory factors including MyoD and myogenin, chromatin modifications have emerged as vital mechanisms of myogenic regulation. We have previously established that bexarotene, a clinically approved agonist of retinoid X receptor (RXR), promotes the specification and differentiation of skeletal muscle lineage. Here, we examine the genome-wide impact of rexinoids on myogenic differentiation through integral RNA-seq and ChIP-seq analyses. We found that bexarotene promotes myoblast differentiation through the coordination of exit from the cell cycle and the activation of muscle-related genes. We uncovered a new mechanism of rexinoid action which is mediated by the nuclear receptor and largely reconciled through a direct regulation of MyoD gene expression. In addition, we determined a rexinoid-responsive residue-specific histone acetylation at a distinct chromatin state associated to MyoD and myogenin. Thus, we provide novel molecular insights into the interplay between RXR signaling and chromatin states pertinent to myogenic programs in early myoblast differentiation.