Non-invasive monitoring of neoadjuvant radiation therapy response in soft tissue sarcomas by multiparametric MRI and quantification of circulating tumor DNA-A study protocol.

BACKGROUND: Wide resection remains the cornerstone of localized soft-tissue sarcomas (STS) treatment. Neoadjuvant radiation therapy (NRT) may decrease the risk of local recurrences; however, its effectiveness for different histological STS subtypes has not been systematically investigated. The proposed prospective study evaluates the NRT response in STS using liquid biopsies and the correlation of multiparametric magnetic resonance imaging (mpMRI) with histopathology and immunohistochemistry. METHODS: Patients with localized high-grade STS, who qualify for NRT, are included in this study. LIQUID BIOPSIES: Quantification of circulating tumor DNA (ctDNA) in patient blood samples is performed by targeted next-generation sequencing. Soft-tissue sarcoma subtype-specific panel sequencing in combination with patient-specific exome sequencing allows the detection of individual structural variants and point mutations. Circulating free DNA is isolated from peritherapeutically collected patient plasma samples and ctDNA quantified therein. Identification of breakpoints is carried out using FACTERA. Bioinformatic analysis is performed using samtools, picard, fgbio, and the MIRACUM Pipeline. MPMRI: Combination of conventional MRI sequences with diffusion-weighted imaging, intravoxel-incoherent motion, and dynamic contrast enhancement. Multiparametric MRI is performed before, during, and after NRT. We aim to correlate mpMRI data with the resected specimen's macroscopical, histological, and immunohistochemical findings. RESULTS: Preliminary data support the notion that quantification of ctDNA in combination with tumor mass characterization through co-registration of mpMRI and histopathology can predict NRT response of STS. CLINICAL RELEVANCE: The methods presented in this prospective study are necessary to assess therapy response in heterogeneous tumors and lay the foundation of future patient- and tumor-specific therapy concepts. These methods can be applied to various tumor entities. Thus, the participation and support of a wider group of oncologic surgeons are needed to validate these findings on a larger patient cohort.

Individualized Mini-Panel Sequencing of ctDNA Allows Tumor Monitoring in Complex Karyotype Sarcomas.

Soft tissue sarcomas (STS) are rare tumors of mesenchymal origin with high mortality. After curative resection, about one third of patients suffer from distant metastases. Tumor follow-up only covers a portion of recurrences and is associated with high cost and radiation burden. For metastasized STS, only limited inferences can be drawn from imaging data regarding therapy response. To date there are no established and evidence-based diagnostic biomarkers for STS due to their rarity and diversity. In a proof-of-concept study, circulating tumor DNA (ctDNA) was quantified in (n = 25) plasma samples obtained from (n = 3) patients with complex karyotype STS collected over three years. Genotyping of tumor tissue was performed by exome sequencing. Patient-individual mini-panels for targeted next-generation sequencing were designed encompassing up to 30 mutated regions of interest. Circulating free DNA (cfDNA) was purified from plasma and ctDNA quantified therein. ctDNA values were correlated with clinical parameters. ctDNA concentrations correlated with the tumor burden. In case of full remission, no ctDNA was detectable. Patients with a recurrence at a later stage showed low levels of ctDNA during clinical remission, indicating minimal residual disease. In active disease (primary tumor or metastatic disease), ctDNA was highly elevated. We observed direct response to treatment, with a ctDNA decline after tumor resections, radiotherapy, and chemotherapy. Quantification of ctDNA allows for the early detection of recurrence or metastases and can be used to monitor treatment response in STS. Therapeutic decisions can be made earlier, such as the continuation of a targeted adjuvant therapy or the implementation of extended imaging to detect recurrences. In metastatic disease, therapy can be adjusted promptly in case of no response. These advantages may lead to a survival benefit for patients in the future.

Genotyping of Circulating Free DNA Enables Monitoring of Tumor Dynamics in Synovial Sarcomas.

BACKGROUND: Synovial sarcoma (SS) is a malignant soft tissue tumor of mesenchymal origin that frequently occurs in young adults. Translocation of the SYT gene on chromosome 18 to the SSX genes on chromosome X leads to the formation of oncogenic fusion genes, which lead to initiation and proliferation of tumor cells. The detection and quantification of circulating tumor DNA (ctDNA) can serve as a non-invasive method for diagnostics of local or distant tumor recurrence, which could improve survival rates due to early detection. METHODS: We developed a subtype-specific targeted next-generation sequencing (NGS) approach specifically targeting SS t(X;18)(p11;q11), which fuses SS18 (SYT) in chromosome 18 to SSX1 or SSX2 in chromosome x, and recurrent point mutations. In addition, patient-specific panels were designed from tumor exome sequencing. Both approaches were used to quantify ctDNA in patients' plasma. RESULTS: The subtype-specific assay allowed detection of somatic mutations from 25/25 tumors with a mean of 1.68 targetable mutations. The minimal limit of detection was determined at a variant allele frequency of 0.05%. Analysis of 29 plasma samples from 15 tumor patients identified breakpoint ctDNA in 6 patients (sensitivity: 40%, specificity 100%). The addition of more mutations further increased assay sensitivity. Quantification of ctDNA in plasma samples (n = 11) from one patient collected over 3 years, with a patient-specific panel based on tumor exome sequencing, correlated with the clinical course, response to treatment and tumor volume. CONCLUSIONS: Targeted NGS allows for highly sensitive tumor profiling and non-invasive detection of ctDNA in SS patients, enabling non-invasive monitoring of tumor dynamics.

Deciphering the regulatory landscape of fetal and adult γδ T-cell development at single-cell resolution.

γδ T cells with distinct properties develop in the embryonic and adult thymus and have been identified as critical players in a broad range of infections, antitumor surveillance, autoimmune diseases, and tissue homeostasis. Despite their potential value for immunotherapy, differentiation of γδ T cells in the thymus is incompletely understood. Here, we establish a high-resolution map of γδ T-cell differentiation from the fetal and adult thymus using single-cell RNA sequencing. We reveal novel sub-types of immature and mature γδ T cells and identify an unpolarized thymic population which is expanded in the blood and lymph nodes. Our detailed comparative analysis reveals remarkable similarities between the gene networks active during fetal and adult γδ T-cell differentiation. By performing a combined single-cell analysis of Sox13, Maf, and Rorc knockout mice, we demonstrate sequential activation of these factors during IL-17-producing γδ T-cell (γδT17) differentiation. These findings substantially expand our understanding of γδ T-cell ontogeny in fetal and adult life. Our experimental and computational strategy provides a blueprint for comparing immune cell differentiation across developmental stages.