Rapid and highly sensitive approach for multiplexed somatic fusion detection.

Somatic gene translocations are key to making an accurate diagnosis in many cancers including many pediatric sarcomas. Currently available molecular diagnostic approaches to identifying somatic pathognomonic translocations have limitations such as minimal multiplexing, high cost, complex computational requirements, or slow turnaround times. We sought to develop a new fusion-detection assay optimized to mitigate these challenges. To accomplish this goal, we developed a highly sensitive multiplexed digital PCR-based approach that can identify the gene partners of multiple somatic fusion transcripts. This assay was validated for specificity with cell lines and synthetized DNA fragments. Assay sensitivity was optimized using a tiered amplification approach for fusion detection from low input and/or degraded RNA. The assay was then tested for the potential application of fusion detection from FFPE tissue and liquid biopsy samples. We found that this multiplexed PCR approach was able to accurately identify the presence of seven different targeted fusion transcripts with a turnaround time of 1 to 2 days. The addition of a tiered amplification step allowed the detection of targeted fusions from as little as 1 pg of RNA input. We also identified fusions from as little as two unstained slides of FFPE tumor biopsy tissue, from circulating tumor cells collected from tumor-bearing mice, and from liquid biopsy samples from patients with known fusion-positive cancers. We also demonstrated that the assay could be easily adapted for additional fusion targets. In summary, this novel assay detects multiple somatic fusion partners in biologic samples with low tumor content and low-quality RNA in less than two days. The assay is inexpensive and could be applied to surgical and liquid biopsies, particularly in places with inadequate resources for more expensive and expertise-dependent assays such as next-generation sequencing.

Rare FGFR Oncogenic Alterations in Sequenced Pediatric Solid and Brain Tumors Suggest FGFR Is a Relevant Molecular Target in Childhood Cancer.

PURPOSE: Multiple FGFR inhibitors are currently in clinical trials enrolling adults with different solid tumors, while very few enroll pediatric patients. We determined the types and frequency of FGFR alterations (FGFR1-4) in pediatric cancers to inform future clinical trial design. METHODS: Tumors with FGFR alterations were identified from two large cohorts of pediatric solid tumors subjected to targeted DNA sequencing: The Dana-Farber/Boston Children's Profile Study (n = 888) and the multi-institution GAIN/iCAT2 (Genomic Assessment Improves Novel Therapy) Study (n = 571). Data from the combined patient population of 1,395 cases (64 patients were enrolled in both studies) were reviewed and cases in which an FGFR alteration was identified by OncoPanel sequencing were further assessed. RESULTS: We identified 41 patients with tumors harboring an oncogenic FGFR alteration. Median age at diagnosis was 8 years (range, 6 months-26 years). Diagnoses included 11 rhabdomyosarcomas, nine low-grade gliomas, and 17 other tumor types. Alterations included gain-of-function sequence variants (n = 19), amplifications (n = 10), oncogenic fusions (FGFR3::TACC3 [n = 3], FGFR1::TACC1 [n = 1], FGFR1::EBF2 [n = 1], FGFR1::CLIP2 [n = 1], and FGFR2::CTNNA3 [n = 1]), pathogenic-leaning variants of uncertain significance (n = 4), and amplification in combination with a pathogenic-leaning variant of uncertain significance (n = 1). Two novel FGFR1 fusions in two different patients were identified in this cohort, one of whom showed a response to an FGFR inhibitor. CONCLUSION: In summary, activating FGFR alterations were found in approximately 3% (41/1,395) of pediatric solid tumors, identifying a population of children with cancer who may be eligible and good candidates for trials evaluating FGFR-targeted therapy. Importantly, the genomic and clinical data from this study can help inform drug development in accordance with the Research to Accelerate Cures and Equity for Children Act.

Molecular profiling identifies targeted therapy opportunities in pediatric solid cancer.

To evaluate the clinical impact of molecular tumor profiling (MTP) with targeted sequencing panel tests, pediatric patients with extracranial solid tumors were enrolled in a prospective observational cohort study at 12 institutions. In the 345-patient analytical population, median age at diagnosis was 12 years (range 0-27.5); 298 patients (86%) had 1 or more alterations with potential for impact on care. Genomic alterations with diagnostic, prognostic or therapeutic significance were present in 61, 16 and 65% of patients, respectively. After return of the results, impact on care included 17 patients with a clarified diagnostic classification and 240 patients with an MTP result that could be used to select molecularly targeted therapy matched to identified alterations (MTT). Of the 29 patients who received MTT, 24% had an objective response or experienced durable clinical benefit; all but 1 of these patients received targeted therapy matched to a gene fusion. Of the diagnostic variants identified in 209 patients, 77% were gene fusions. MTP with targeted panel tests that includes fusion detection has a substantial clinical impact for young patients with solid tumors.

The Immune System and Its Contribution to Variability in Regenerative Medicine.

The immune system plays a critical role in directing tissue repair and regeneration outcomes. Tissue engineering technologies that are designed to promote new tissue growth will therefore be impacted by immune factors that are present in patients both locally at the site of intervention and systemically. The immune state of patients can be influenced by many factors, including infection, nutrition, and other disease comorbidities. As a result, the immune state is highly variable and may be a source of variability in tissue-engineered products in the clinic, which is not found in preclinical models. In this review, we will summarize key immune cells and evidence of their activity in tissue repair and potential in tissue engineering systems. We also discuss how clinical translation of tissue engineering strategies, in particular stem cells, helped elucidate the importance of the immune system. With increased understanding of the immune system's role in repair and tissue engineering systems, it will likely become a therapeutic target and component of future therapies.

Biomaterials direct functional B cell response in a material-specific manner.

B cells are an adaptive immune target of biomaterials development in vaccine research but, despite their role in wound healing, have not been extensively studied in regenerative medicine. To probe the role of B cells in biomaterial scaffold response, we evaluated the B cell response to biomaterial materials implanted in a muscle wound using a biological extracellular matrix (ECM), as a reference for a naturally derived material, and synthetic polyester polycaprolactone (PCL), as a reference for a synthetic material. In the local muscle tissue, small numbers of B cells are present in response to tissue injury and biomaterial implantation. The ECM materials induced mature B cells in lymph nodes and antigen presentation in the spleen. The synthetic PCL implants resulted in prolonged B cell presence in the wound and induced an antigen-presenting phenotype. In summary, the adaptive B cell immune response to biomaterial induces local, regional, and systemic immunological changes.

Landscape of transcript isoforms in single T cells infiltrating in non-small-cell lung cancer.

Single-cell RNA sequencing (scRNA-seq) has enabled high-resolution characterization of molecular signatures of tumor-infiltrating lymphocytes. However, analyses at the transcript isoform level are rarely reported. As alternative splicing is critical to T-cell differentiation and activation, here, we proposed a computational method named IDEA (Isoform Detection, Enrichment, and functional Annotation) to comprehensively detect and annotate differentially used isoforms across cell subtypes. We applied IDEA on a scRNA-seq data set of 12,346 T cells from non-small-cell lung cancer (NSCLC). We found that most genes tend to dominantly express one isoform in single T cells, enabling typing T cells based on the isotypes, given a gene. Isotype analysis suggested that tumor-infiltrating T cells significantly preferred specific isotypes for 245 genes in CD8+ T cells and 456 genes in CD4+ T cells. Functional annotation suggests that the preferred isoforms involved in coding/noncoding switches, transcription start site changes, gains/losses of domains, and subcellular translocation. Clonal analysis revealed that isoform switching occurred during T-cell activation/differentiation. Our analysis provides precise characterization of the molecular events in tumor-infiltrating T cells and sheds new light on the regulatory mechanisms of tumor-infiltrating T cells.