The development of a custom RNA-sequencing panel for the identification of predictive and diagnostic biomarkers in glioma.

PURPOSE: Various molecular profiles are needed to classify malignant brain tumors, including gliomas, based on the latest classification criteria of the World Health Organization, and their poor prognosis necessitates new therapeutic targets. The Todai OncoPanel 2 RNA Panel (TOP2-RNA) is a custom-target RNA-sequencing (RNA-seq) using the junction capture method to maximize the sensitivity of detecting 455 fusion gene transcripts and analyze the expression profiles of 1,390 genes. This study aimed to classify gliomas and identify their molecular targets using TOP2-RNA. METHODS: A total of 124 frozen samples of malignant gliomas were subjected to TOP2-RNA for classification based on their molecular profiles and the identification of molecular targets. RESULTS: Among 55 glioblastoma cases, gene fusions were detected in 11 cases (20%), including novel MET fusions. Seven tyrosine kinase genes were found to be overexpressed in 15 cases (27.3%). In contrast to isocitrate dehydrogenase (IDH) wild-type glioblastoma, IDH-mutant tumors, including astrocytomas and oligodendrogliomas, barely harbor fusion genes or gene overexpression. Of the 34 overexpressed tyrosine kinase genes, MDM2 and CDK4 in glioblastoma, 22 copy number amplifications (64.7%) were observed. When comparing astrocytomas and oligodendrogliomas in gene set enrichment analysis, the gene sets related to 1p36 and 19q were highly enriched in astrocytomas, suggesting that regional genomic DNA copy number alterations can be evaluated by gene expression analysis. CONCLUSIONS: TOP2-RNA is a highly sensitive assay for detecting fusion genes, exon skipping, and aberrant gene expression. Alterations in targetable driver genes were identified in more than 50% of glioblastoma. Molecular profiling by TOP2-RNA provides ample predictive, prognostic, and diagnostic biomarkers that may not be identified by conventional assays and, therefore, is expected to increase treatment options for individual patients with glioma.

Detection of Novel Tyrosine Kinase Fusion Genes as Potential Therapeutic Targets in Bone and Soft Tissue Sarcomas Using DNA/RNA-based Clinical Sequencing.

BACKGROUND: Approximately 1% of clinically treatable tyrosine kinase fusions, including anaplastic lymphoma kinase, neurotrophic tyrosine receptor kinase, RET proto-oncogene, and ROS proto-oncogene 1, have been identified in soft tissue sarcomas via comprehensive genome profiling based on DNA sequencing. Histologic tumor-specific fusion genes have been reported in approximately 20% of soft tissue sarcomas; however, unlike tyrosine kinase fusion genes, these fusions cannot be directly targeted in therapy. Approximately 80% of tumor-specific fusion-negative sarcomas, including myxofibrosarcoma and leiomyosarcoma, that are defined in complex karyotype sarcomas remain genetically uncharacterized; this mutually exclusive pattern of mutations suggests that other mutually exclusive driver oncogenes are yet to be discovered. Tumor-specific, fusion-negative sarcomas may be associated with unique translocations, and oncogenic fusion genes, including tyrosine kinase fusions, may have been overlooked in these sarcomas. QUESTIONS/PURPOSES: (1) Can DNA- or RNA-based analysis reveal any characteristic gene alterations in bone and soft tissue sarcomas? (2) Can useful and potential tyrosine kinase fusions in tumors from tumor-specific, fusion-negative sarcomas be detected using an RNA-based screening system? (3) Do the identified potential fusion tumors, especially in neurotrophic tyrosine receptor kinase gene fusions in bone sarcoma, transform cells and respond to targeted drug treatment in in vitro assays? (4) Can the identified tyrosine kinase fusion genes in sarcomas be useful therapeutic targets? METHODS: Between 2017 and 2020, we treated 100 patients for bone and soft tissue sarcomas at five institutions. Any biopsy or surgery from which a specimen could be obtained was included as potentially eligible. Ninety percent (90 patients) of patients were eligible; a further 8% (8 patients) were excluded because they were either lost to follow-up or their diagnosis was changed, leaving 82% (82 patients) for analysis here. To answer our first and second questions regarding gene alterations and potential tyrosine kinase fusions in eight bone and 74 soft tissue sarcomas, we used the TruSight Tumor 170 assay to detect mutations, copy number variations, and gene fusions in the samples. To answer our third question, we performed functional analyses involving in vitro assays to determine whether the identified tyrosine kinase fusions were associated with oncogenic abilities and drug responses. Finally, to determine usefulness as therapeutic targets, two pediatric patients harboring an NTRK fusion and an ALK fusion were treated with tyrosine kinase inhibitors in clinical trials. RESULTS: DNA/RNA-based analysis demonstrated characteristic alterations in bone and soft tissue sarcomas; DNA-based analyses detected TP53 and copy number alterations of MDM2 and CDK4 . These single-nucleotide variants and copy number variations were enriched in specific fusion-negative sarcomas. RNA-based screening detected fusion genes in 24% (20 of 82) of patients. Useful potential fusions were detected in 19% (11 of 58) of tumor-specific fusion-negative sarcomas, with nine of these patients harboring tyrosine kinase fusion genes; five of these patients had in-frame tyrosine kinase fusion genes ( STRN3-NTRK3, VWC2-EGFR, ICK-KDR, FOXP2-MET , and CEP290-MET ) with unknown pathologic significance. The functional analysis revealed that STRN3-NTRK3 rearrangement that was identified in bone had a strong transforming potential in 3T3 cells, and that STRN3-NTRK3 -positive cells were sensitive to larotrectinib in vitro. To confirm the usefulness of identified tyrosine kinase fusion genes as therapeutic targets, patients with well-characterized LMNA-NTRK1 and CLTC-ALK fusions were treated with tyrosine kinase inhibitors in clinical trials, and a complete response was achieved. CONCLUSION: We identified useful potential therapeutic targets for tyrosine kinase fusions in bone and soft tissue sarcomas using RNA-based analysis. We successfully identified STRN3-NTRK3 fusion in a patient with leiomyosarcoma of bone and determined the malignant potential of this fusion gene via functional analyses and drug effects. In light of these discoveries, comprehensive genome profiling should be considered even if the sarcoma is a bone sarcoma. There seem to be some limitations regarding current DNA-based comprehensive genome profiling tests, and it is important to use RNA testing for proper diagnosis and accurate identification of fusion genes. Studies on more patients, validation of results, and further functional analysis of unknown tyrosine kinase fusion genes are required to establish future treatments. CLINICAL RELEVANCE: DNA- and RNA-based screening systems may be useful for detecting tyrosine kinase fusion genes in specific fusion-negative sarcomas and identifying key therapeutic targets, leading to possible breakthroughs in the treatment of bone and soft tissue sarcomas. Given that current DNA sequencing misses fusion genes, RNA-based screening systems should be widely considered as a worldwide test for sarcoma. If standard treatments such as chemotherapy are not effective, or even if the sarcoma is of bone, RNA sequencing should be considered to identify as many therapeutic targets as possible.

[Mesalazine-induced myocarditis].

A 30-year-old man presented with constipation and abdominal pain. He was suspected of having ulcerative colitis, and administration of 2400mg/day of oral mesalazine was initiated. After 10 days of treatment, he experienced fever and chest pain. Blood examination, electrocardiography, and cardiac ultrasonography revealed elevated cardiac enzymes, ST-segment elevation, and diffuse hypokinesis, respectively. Mesalazine-induced acute myocarditis was diagnosed based on a positive drug-induced lymphocyte stimulation test and the absence of other myocarditis-causing conditions. Prompt cessation of mesalazine quickly improved his heart function and test results. Although rare, clinicians should consider the possibility of cardiac adverse events caused by mesalazine.

Case of severe alcoholic hepatitis following acute pancreatitis.

This report describes the clinical course of a 41 year-old African woman who presented with an episode of acute alcoholic pancreatitis followed next by severe alcoholic hepatitis (SAH). Initially admitted for pancreatitis, the patient responded promptly to comprehensive treatment with strict abstinence from alcohol. However, remarkable elevations in white blood cell count to 44,000/µL and total bilirubin level to 12.4 mg/dL were observed 5-7 weeks later. Contrast-enhanced computed tomography revealed rapidly progressing hepatosplenomegaly. Histological analysis of a liver biopsy detected ballooned hepatocytes with Mallory-Denk bodies and significant neutrophilic infiltration in the hepatic parenchyma, which confirmed the diagnosis of SAH. The patient's hepatosplenomegaly and overall condition improved with supportive care alone. The reported case reveals the unexpected fact that SAH can develop after alcoholic acute pancreatitis.

A molecular glue RBM39-degrader induces synthetic lethality in cancer cells with homologous recombination repair deficiency.

E7820 and Indisulam (E7070) are sulfonamide molecular glues that modulate RNA splicing by degrading the splicing factor RBM39 via ternary complex formation with the E3 ligase adaptor DCAF15. To identify biomarkers of the antitumor efficacy of E7820, we treated patient-derived xenograft (PDX) mouse models established from 42 patients with solid tumors. The overall response rate was 38.1% (16 PDXs), and tumor regression was observed across various tumor types. Exome sequencing of the PDX genome revealed that loss-of-function mutations in genes of the homologous recombination repair (HRR) system, such as ATM, were significantly enriched in tumors that responded to E7820 (p = 4.5 × 103). Interestingly, E7820-mediated double-strand breaks in DNA were increased in tumors with BRCA2 dysfunction, and knockdown of BRCA1/2 transcripts or knockout of ATM, ATR, or BAP1 sensitized cancer cells to E7820. Transcriptomic analyses revealed that E7820 treatment resulted in the intron retention of mRNAs and decreased transcription, especially for HRR genes. This induced HRR malfunction probably leads to the synthetic lethality of tumor cells with homologous recombination deficiency (HRD). Furthermore, E7820, in combination with olaparib, exerted a synergistic effect, and E7820 was even effective in an olaparib-resistant cell line. In conclusion, HRD is a promising predictive biomarker of E7820 efficacy and has a high potential to improve the prognosis of patients with HRD-positive cancers.

High-Throughput Functional Evaluation of MAP2K1 Variants in Cancer.

Activating mutations in mitogen-activated protein kinase kinase 1 (MAP2K1) are involved in a variety of cancers and may be classified according to their RAF dependence. Sensitivity to combined BRAF and MEK treatments is associated with co-mutations of MAP2K1 and BRAF; however, the significance of less frequent MAP2K1 mutations is largely unknown. The transforming potential and drug sensitivity of 100 MAP2K1 variants were evaluated using individual assays and the mixed-all-nominated-in-one method. In addition, A375, a melanoma cell line harboring the BRAF V600E mutation, was used to evaluate the function of the MAP2K1 variants in combination with active RAF signaling. Among a total of 67 variants of unknown significance, 16 were evaluated as oncogenic or likely oncogenic. The drug sensitivity of the individual variants did not vary with respect to BRAF inhibitors, MEK inhibitors (MEKi), or their combination. Sensitivity to BRAF inhibitors was associated with the RAF dependency of the MAP2K1 variants, whereas resistance was higher in RAF-regulated or independent variants compared with RAF-dependent variants. Thus, the synergistic effect of BRAF and MEKis may be observed in RAF-regulated and RAF-dependent variants. MAP2K1 variants exhibit differential sensitivity to BRAF and MEKis, suggesting the importance of individual functional analysis for the selection of optimal treatments for each patient. This comprehensive evaluation reveals precise functional information and provides optimal combination treatment for individual MAP2K1 variants.

Preclinical assessment of combination therapy of EGFR tyrosine kinase inhibitors in a highly heterogeneous tumor model.

The development of tyrosine kinase inhibitors (TKIs) has improved the treatment of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations. The current research priority is to provide viable treatments for patients who have drug-resistant EGFR mutations. We evaluated the drug sensitivity of various EGFR mutants to monotherapies and combination therapies of EGFR-TKIs. In vitro, the transforming potential and drug sensitivity of 357 EGFR variants were assessed. In vivo, we tested the sensitivity of EGFR variants to different regimens of EGFR-TKIs by examining changes in the proportion of each variant within the tumor. Out of 357 variants thoroughly examined for transforming activities, 144 (40.3%) and 282 (79.0%) transformed 3T3 and Ba/F3 cells, respectively. Among the latter variants, 50 (17.7%) were found to be resistant or only partly resistant to osimertinib or afatinib. Four of 25 afatinib-resistant variants (16%) were sensitive to osimertinib, whereas 25 of 46 osimertinib-resistant variants (54.3%) were sensitive to afatinib. Despite the lack of a synergistic impact, TKI combination treatment effectively reduced in vivo the heterogeneous tumors composed of 3T3 cells with different EGFR variants. Regimens starting with afatinib and subsequently switched to osimertinib suppressed tumor development more efficiently than the opposite combination. Combination EGFR-TKI treatment may decrease tumor growth and prevent the development of resistant variants. This work created an experimental model of a heterogeneous tumor to find the best combination therapy regimen and proposes a basic notion of EGFR-TKI combination therapy to enhance the prognosis of NSCLC patients.

Comprehensive functional evaluation of variants of fibroblast growth factor receptor genes in cancer.

Various genetic alterations of the fibroblast growth factor receptor (FGFR) family have been detected across a wide range of cancers. However, inhibition of FGFR signaling by kinase inhibitors demonstrated limited clinical effectiveness. Herein, we evaluated the transforming activity and sensitivity of 160 nonsynonymous FGFR mutations and ten fusion genes to seven FGFR tyrosine kinase inhibitors (TKI) using the mixed-all-nominated-in-one (MANO) method, a high-throughput functional assay. The oncogenicity of 71 mutants was newly discovered in this study. The FGFR TKIs showed anti-proliferative activities against the wild-type FGFRs and their fusions, while several hotspot mutants were relatively resistant to those TKIs. The drug sensitivities assessed with the MANO method were well concordant with those evaluated using in vitro and in vivo assays. Comprehensive analysis of published FGFR structures revealed a possible mechanism through which oncogenic FGFR mutations reduce sensitivity to TKIs. It was further revealed that recurrent compound mutations within FGFRs affect the transforming potential and TKI-sensitivity of corresponding kinases. In conclusion, our study suggests the importance of selecting suitable inhibitors against individual FGFR variants. Moreover, it reveals the necessity to develop next-generation FGFR inhibitors, which are effective against all oncogenic FGFR variants.

Synthesis and bioassay of a boron-dipyrromethene derivative of estradiol for fluorescence imaging in vivo.

C7α-substituted estradiols bind to estrogen receptors in cell nuclei, yet these derivatives remain little used in bioimaging. Here, we describe a fluorescent derivative of estradiol (E2) with a boron-dipyrromethene (BODIPY) moiety attached to C7α, synthesized by olefin metathesis reaction of 7α-allylestradiol and 9-decenyl-BODIPY. In ovariectomized rats and non-ovariectomized mice, E2-BODIPY promoted the growth of uterine tissue similar to the effect of estradiol. Twenty-four hours after subcutaneous injection of E2-BODIPY in non-ovariectomized mice, we observed fluorescence of E2-BODIPY in the nuclei of uterine epithelial cells. Our findings suggest that fluorescence microscopy can localize this derivative in E2-responsive cells during normal development and tumorigenesis in vivo.