Effect of radiotherapy on local recurrence, distant metastasis and overall survival in 1200 extremity soft tissue sarcoma patients. Retrospective analysis using IPTW-adjusted models.

BACKGROUND AND PURPOSE: Neoadjuvant (NRTX) and adjuvant radiotherapy (ARTX) reduce local recurrence (LR) risk in extremity soft tissue sarcoma (eSTS), yet their impact on distant metastasis (DM) and overall survival (OS) is less well defined. This study aimed at analysing the influence of NRTX/ARTX on all three endpoints using a retrospective, multicentre eSTS cohort. MATERIALS AND METHODS: 1200 patients (mean age: 60.7 ± 16.8 years; 44.4 % females) were retrospectively included, treated with limb sparing surgery and curative intent for localised, high grade (G2/3) eSTS. 194 (16.2 %), 790 (65.8 %), and 216 (18.0 %) patients had received NRTX, ARTX and no RTX, respectively. For the resulting three groups (no RTX vs. NRTX, no RTX vs. ARTX, NRTX vs. ARTX) Fine&Gray models for LR and DM, and Cox-regression models for OS were calculated, with IPTW-modelling adjusting for imbalances between groups. RESULTS: In the IPTW-adjusted analysis, NRTX was associated with lower LR-risk in comparison to no RTX (SHR [subhazard ratio]: 0.236; p = 0.003), whilst no impact on DM-risk (p = 0.576) or OS (p = 1.000) was found. IPTW-weighted analysis for no RTX vs. ARTX revealed a significant positive association between ARTX and lower LR-risk (SHR: 0.479, p = 0.003), but again no impact on DM-risk (p = 0.363) or OS (p = 0.534). IPTW-weighted model for NRTX vs. ARTX showed significantly lower LR-risk for NRTX (SHR for ARTX: 3.433; p = 0.003) but no difference regarding DM-risk (p = 1.000) or OS (p = 0.639). CONCLUSION: NRTX and ARTX are associated with lower LR-risk, but do not seem to affect DM-risk or OS. NRTX may be favoured over ARTX as our results indicate better local control rates.

Artificial Bone Graft Substitutes for Curettage of Benign and Low-Grade Malignant Bone Tumors: Clinical and Radiological Experience with Cerasorb.

Background: Artificial bone graft substitutes (ABGS) for curettage of bone tumors are becoming increasingly popular. The aim of this retrospective analysis was to determine the efficacy of the ABGS Cerasorb (Curasan-AG, Kleinostheim, Germany), a beta-tricalcium phosphate (beta-TCP), concerning resorption profile, bone healing, and remodeling after surgery and to evaluate potential complications. Methods: Forty-three patients suffering from benign and low-grade malignant bone tumors were treated with curettage and refilling of the bony cavity using the ABGS Cerasorb between 2018 and 2021 and included in the final analysis. Clinical follow-up exams with X-rays in two planes were performed 6 weeks, 3 months, 6 months, and 1 year after surgery. Results: After a mean follow-up period of 14.6 months, radiological consolidation following curettage was observed in all patients. Total resorption was observed in 16.3% of patients; in the other 83.7%, resorption was partial. In four patients, of whom two had a tumor in the distal femur and two in the humeral diaphysis, fractures occurred within 6 weeks after primary surgery. Conclusion: In conclusion, the beta-TCP Cerasorb seems to be a reliable bone graft substitute with low complication rates and is a suitable alternative to autologous bone grafts or allografts. Nonetheless, it shows a tendency of delayed resorption. Level of Evidence: III; retrospective cohort study.

Datasets for reporting of soft-tissue sarcoma: recommendations from the International Collaboration on Cancer Reporting (ICCR).

AIMS: Soft-tissue tumours are rare and both accurate diagnosis and proper treatment represent a global challenge. Current treatment guidelines also recommend review by specialised pathologists. Here we report on international consensus-based datasets for the pathology reporting of biopsy and resection specimens of soft-tissue sarcomas. The datasets were produced under the auspices of the International Collaboration on Cancer Reporting (ICCR), a global alliance of international pathology and cancer organisations. METHODS AND RESULTS: According to the ICCR's guidelines for dataset development, an international expert panel consisting of pathologists, a surgical oncologist, and a medical oncologist produced a set of core and noncore data items for biopsy and resection specimens based on a critical review and discussion of current evidence. All professionals involved were subspecialised soft-tissue sarcoma experts and affiliated with tertiary referral centres. Commentary was provided for each data item to explain the rationale for selecting it as a core or noncore element, its clinical relevance, and to highlight potential areas of disagreement or lack of evidence, in which case a consensus position was formulated. Following international public consultation, the documents were finalised and ratified, and the datasets, which included a synoptic reporting guide, were published on the ICCR website. CONCLUSION: These first international datasets for soft-tissue sarcomas are aimed to promote high-quality, standardised pathology reporting. Their adoption will improve consistency of reporting, facilitate multidisciplinary communication, and enhance comparability of data, all of which will help to improve patient's management.

Targeting epigenetic features in clear cell sarcomas based on patient-derived cell lines.

BACKGROUND: Clear cell sarcomas (CCSs) are translocated aggressive malignancies, most commonly affecting young adults with a high incidence of metastases and a poor prognosis. Research into the disease is more feasible when adequate models are available. By establishing CCS cell lines from a primary and metastatic lesion and isolating healthy fibroblasts from the same patient, the in vivo process is accurately reflected and aspects of clinical multistep carcinogenesis recapitulated. METHODS: Isolated tumor cells and normal healthy skin fibroblasts from the same patient were compared in terms of growth behavior and morphological characteristics using light and electron microscopy. Tumorigenicity potential was determined by soft agar colony formation assay and in vivo xenograft applications. While genetic differences between the two lineages were examined by copy number alternation profiles, nuclear magnetic resonance spectroscopy determined arginine methylation as epigenetic features. Potential anti-tumor effects of a protein arginine N-methyltransferase type I (PRMT1) inhibitor were elicited in 2D and 3D cell culture experiments using cell viability and apoptosis assays. Statistical significance was calculated by one-way ANOVA and unpaired t-test. RESULTS: The two established CCS cell lines named MUG Lucifer prim and MUG Lucifer met showed differences in morphology, genetic and epigenetic data, reflecting the respective original tissue. The detailed cell line characterization especially in regards to the epigenetic domain allows investigation of new innovative therapies. Based on the epigenetic data, a PRMT1 inhibitor was used to demonstrate the targeted antitumor effect; normal tissue cells isolated and immortalized from the same patient were not affected with the IC50 used. CONCLUSIONS: MUG Lucifer prim, MUG Lucifer met and isolated and immortalized fibroblasts from the same patient represent an ideal in vitro model to explore the biology of CCS. Based on this cell culture model, novel therapies could be tested in the form of PRMT1 inhibitors, which drive tumor cells into apoptosis, but show no effect on fibroblasts, further supporting their potential as promising treatment options in the combat against CCS. The data substantiate the importance of tailored therapies in the advanced metastatic stage of CCS.

Impact of allogeneic red blood cell transfusion on prognosis in soft tissue sarcoma patients. A single-centre study.

BACKGROUND: Perioperatively administered (leukocyte reduced) allogeneic red blood cell transfusions (lrRBCTs) may lead to transfusion-related immunomodulation and reduced overall survival (OS) in cancer patients. Herein, the effect of lrRBCT on local recurrence (LR), distant metastasis (DM), and OS in soft tissue sarcoma (STS) patients was analysed. METHODS: Retrospective study on 432 STS patients (mean age: 60.0 ± 17.8 years; 46.1% female), surgically treated at a tertiary tumour centre. Uni- and multivariate survival models were calculated to analyse impact of perioperative lrRBCTs on LR, DM, OS. RESULTS: Perioperatively, 75 patients (17.4%) had received lrRBCTs. Older patients, deep, large, lower limb STS rather required lrRBCTs (all p < 0.05). No significant association between lrRBCT administration and LR- (p = 0.582) or DM-risk (p = 0.084) was observed. LrRBCT was associated with worse OS in univariate analysis (HR: 2.222; p < 0.001), with statistical significance lost upon multivariate analysis (HR: 1.658; p = 0.059; including age, histology, size, grading, amputation, depth). Adding preoperative haemoglobin in subgroup of 220 patients with laboratory parameters revealed significant negative impact of low haemoglobin on OS (p = 0.014), whilst effect of lrRBCT was further diminished (p = 0.167). CONCLUSION: Unfavourable prognostic factors prevail in STS patients requiring lrRBCTs. Low haemoglobin levels rather than lrRBCT seem to reduce OS.

Implementation of Copy Number Variations-Based Diagnostics in Morphologically Challenging EWSR1/FUS::NFATC2 Neoplasms of the Bone and Soft Tissue.

In the last decade, new tumor entities have been described, including EWSR1/FUS::NFATC2-rearranged neoplasms of different biologic behavior. To gain further insights into the behavior of these tumors, we analyzed a spectrum of EWSR1/FUS::NFATC2-rearranged neoplasms and discuss their key diagnostic and molecular features in relation to their prognosis. We report five patients with EWSR1/FUS::NFATC2-rearranged neoplasms, including one simple bone cyst (SBC), two complex cystic bone lesions lacking morphological characteristics of SBC, and two sarcomas. In three cases, fluorescence in situ hybridization (FISH) and in all cases copy number variation (CNV) profiling and fusion analyses were performed. All patients were male, three cystic lesions occurred in children (aged 10, 14, and 17 years), and two sarcomas in adults (69 and 39 years). Fusion analysis revealed two FUS::NFATC2 rearrangements in two cystic lesions and three EWSR1::NFATC2 rearrangements in one complex cystic lesion and two sarcomas. EWSR1 FISH revealed tumor cells with break-apart signal without amplification in one complex cystic lesion and EWSR1 amplification in both sarcomas was documented. CNV analysis showed simple karyotypes in all cystic lesions, while more complex karyotypes were found in NFATC2-rearranged sarcomas. Our study supports and expands previously reported molecular findings of EWSR1/FUS::NFATC2-rearranged neoplasms. The study highlights the importance of combining radiology and morphologic features with molecular aberrations. The use of additional molecular methods, such as CNV and FISH in the routine diagnostic workup, can be crucial in providing a correct diagnosis and avoiding overtreatment.

In Model, In Vitro and In Vivo Killing Efficacy of Antitumor Peptide RDP22 on MUG-Mel2, a Patient Derived Cell Line of an Aggressive Melanoma Metastasis.

The host defense derived peptide was assessed in different model systems with increasing complexity employing the highly aggressive NRAS mutated melanoma metastases cell line MUG-Mel2. Amongst others, fluorescence microscopy and spectroscopy, as well as cell death studies were applied for liposomal, 2D and 3D in vitro models including tumor spheroids without or within skin models and in vivo mouse xenografts. Summarized, MUG-Mel2 cells were shown to significantly expose the negatively charged lipid phosphatidylserine on their plasma membranes, showing they are successfully targeted by RDP22. The peptide was able to induce cell death in MUG-Mel2 2D and 3D cultures, where it was able to kill tumor cells even inside the core of tumor spheroids or inside a melanoma organotypic model. In vitro studies indicated cell death by apoptosis upon peptide treatment with an LC50 of 8.5 µM and seven-fold specificity for the melanoma cell line MUG-Mel2 over normal dermal fibroblasts. In vivo studies in mice xenografts revealed effective tumor regression upon intratumoral peptide injection, indicated by the strong clearance of pigmented tumor cells and tremendous reduction in tumor size and proliferation, which was determined histologically. The peptide RDP22 has clearly shown high potential against the melanoma cell line MUG-Mel2 in vitro and in vivo.

Clinical implementation of plasma cell-free circulating tumor DNA quantification by digital droplet PCR for the monitoring of Ewing sarcoma in children and adolescents.

Background: Treatment stratification and response assessment in pediatric sarcomas has relied on imaging studies and surgical/histopathological evidence of vital tumor cells. Such studies and evidence collection processes often involve radiation and/or general anesthesia in children. Cell-free circulating tumor DNA (ctDNA) detection in blood plasma is one available method of so-called liquid biopsies that has been shown to correlate qualitatively and quantitatively with the existence of vital tumor cells in the body. Our clinical observational study focused on the utility and feasibility of ctDNA detection in pediatric Ewing sarcoma (EWS) as a marker of minimal residual disease (MRD). Patients and methods: We performed whole genome sequencing (WGS) to identify the exact breakpoints in tumors known to carry the EWS-FLI1 fusion gene. Patient-specific fusion breakpoints were tracked in peripheral blood plasma using digital droplet PCR (ddPCR) before, during, and after therapy in six children and young adults with EWS. Presence and levels of fusion breakpoints were correlated with clinical disease courses. Results: We show that the detection of ctDNA in the peripheral blood of EWS patients (i) is feasible in the clinical routine and (ii) allows for the longitudinal real-time monitoring of MRD activity in children and young adults. Although changing ctDNA levels correlated well with clinical outcome within patients, between patients, a high variability was observed (inter-individually). Conclusion: ctDNA detection by ddPCR is a highly sensitive, specific, feasible, and highly accurate method that can be applied in EWS for follow-up assessments as an additional surrogate parameter for clinical MRD monitoring and, potentially, also for treatment stratification in the near 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.

Quantification of increased MUC5AC expression in airway mucus of smoker using an automated image-based approach.

Microscopic analysis of mucus quantity and composition is crucial in research and diagnostics on muco-obstructive diseases. Currently used image-based methods are unable to extract concrete numeric values of mucosal proteins, especially on the expression of the key mucosal proteins MUC5AC and MUC5B. Since their levels increase under pathologic conditions such as extensive exposure to cigarette smoke, it is imperative to quantify them to improve treatment strategies of pulmonary diseases. This study presents a simple, image-based, and high-processing computational method that allows determining the ratio of MUC5AC and MUC5B within the overall airway mucus while providing information on their spatial distribution. The presented pipeline was optimized for automated downstream analysis using a combination of bright field and immunofluorescence imaging suitable for tracheal and bronchial tissue samples, and air-liquid interface (ALI) cell cultures. To validate our approach, we compared tracheal tissue and ALI cell cultures of isolated primary normal human bronchial epithelial cells derived from smokers and nonsmokers. Our data indicated 18-fold higher levels of MUC5AC in submucosal glands of smokers covering about 8% of mucosal areas compared to <1% in nonsmoking individuals, confirming results of previous studies. We further identified a subpopulation of nonsmokers with slightly elevated glandular MUC5AC levels suggesting moderate exposure to second-hand smoke or fine particulate air pollution. Overall, this study demonstrates a novel, user-friendly and freely available tool for digital pathology and the analysis of therapeutic interventions tested in ALI cell cultures.

Dilemma in the Treatment of a Central Giant Cell Granuloma.

Management of central giant cell granuloma (CGCG) presents a clinical challenge. While eradicating a lesion known for its high recurrence rate calls for radical surgical approaches, these cause significant esthetic and functional impairment. We present an eight-year-old boy suffering from an extraordinarily large CGCG expanding into the mandible and base of the mouth in the whole anterior region. Combined treatment with surgical intervention and corticosteroid application was successfully applied, and all six attached dental germs could be preserved. Different approaches for clinical management in pediatric cases are discussed.

Drug combination screening as a translational approach toward an improved drug therapy for chordoma.

PURPOSE: Drug screening programmes have revealed epidermal growth factor receptor inhibitors (EGFRis) as promising therapeutics for chordoma, an orphan malignant bone tumour, in the absence of a known genetic driver. Concurrently, the irreversible EGFRi afatinib (Giotrif®) is being evaluated in a multicentric Phase II trial. As tyrosine kinase inhibitor (TKI) monotherapies are invariably followed by resistance, we aimed to evaluate potential therapeutic combinations with EGFRis. METHODS: We screened 133 clinically approved anticancer drugs as single agents and in combination with two EGFRis (afatinib and erlotinib) in the clival chordoma cell line UM-Chor1. Synergistic combinations were analysed in a 7 × 7 matrix format. The most promising combination was further explored in clival (UM-Chor1, MUG-CC1) and sacral (MUG-Chor1, U-CH1) chordoma cell lines. Secretomes were analysed for receptor tyrosine kinase ligands (EGF, TGF-α, FGF-2 and VEGF-A) upon drug treatment. RESULTS: Drugs that were active as single agents (n = 45) included TKIs, HDAC and proteasome inhibitors, and cytostatic drugs. Six combinations were analysed in a matrix format: n = 4 resulted in a significantly increased cell killing (crizotinib, dabrafenib, panobinostat and doxorubicin), and n = 2 exhibited no or negligible effects (regorafenib, venetoclax). Clival chordoma cell lines were more responsive to combined EGFR-MET inhibition. EGFR-MET cross-talk (e.g. via TGF-α secretion) likely accounts for the synergistic effects of EGFR-MET inhibition. CONCLUSION: Our screen revealed promising combinations with EGFRis, such as the ALK/MET-inhibitor crizotinib, the HDAC-inhibitor panobinostat or the topoisomerase-II-inhibitor doxorubicin, which are part of standard chemotherapy regimens for various bone and soft-tissue sarcomas.

Molecular profiling of soft-tissue sarcomas with FoundationOne® Heme identifies potential targets for sarcoma therapy: a single-centre experience.

BACKGROUND: Molecular diagnosis has become an established tool in the characterisation of adult soft-tissue sarcomas (STS). FoundationOne® Heme analyses somatic gene alterations in sarcomas via DNA and RNA-hotspot sequencing of tumour-associated genes. METHODS: We evaluated FoundationOne® Heme testing in 81 localised STS including 35 translocation-associated and 46 complex-karyotyped cases from a single institution. RESULTS: Although FoundationOne® Heme achieved broad patient coverage and identified at least five genetic alterations in each sample, the sensitivity for fusion detection was rather low, at 42.4%. Nevertheless, potential targets for STS treatment were detected using the FoundationOne® Heme assay: complex-karyotyped sarcomas frequently displayed copy-number alterations of common tumour-suppressor genes, particularly deletions in TP53, NF1, ATRX, and CDKN2A. A subset of myxofibrosarcomas (MFS) was amplified for HGF (n = 3) and MET (n = 1). PIK3CA was mutated in 7/15 cases of myxoid liposarcoma (MLS; 46.7%). Epigenetic regulators (e.g. MLL2 and MLL3) were frequently mutated. CONCLUSIONS: In summary, FoundationOne® Heme detected a broad range of genetic alterations and potential therapeutic targets in STS (e.g. HGF/MET in a subset of MFS, or PIK3CA in MLS). The assay's sensitivity for fusion detection was low in our sample and needs to be re-evaluated in a larger cohort.

YAP1-TFE3 gene fusion variant in clear cell stromal tumour of lung: report of two cases in support of a distinct entity.

AIMS: Clear cell (haemangioblastoma-like) stromal tumour of the lung is a newly described, rare pulmonary neoplasm. Recurrent YAP1-TFE3 gene fusions have recently been reported in three cases. We describe two additional cases and confirm the characteristic YAP1-TFE3 gene fusion. METHODS AND RESULTS: Two mesenchymal tumours of lung were identified from our soft tissue pathology consultation services and RNA sequencing was performed. Both cases were in male patients, aged 35 and 77 years. Both presented as solitary lung nodules measuring 3.9 and 7.5 cm in greatest dimension. Histopathologically, the tumours were composed of epithelioid to plump spindle cells arranged in packets and solid sheets. The cells showed fusiform to ovoid nuclei with open chromatin, variably prominent nucleoli and scant to moderate, clear to eosinophilic cytoplasm. Cytological atypia and significant mitotic activity were minimal. None of the tumours expressed lineage-specific immunophenotypical markers. Both cases were diffusely positive for nuclear TFE3. Unlike YAP1-TFE3-fused epithelioid haemangioendothelioma, for which the fusion breakpoint occurs in YAP1 exon 1 and TFE3 exons 4 or 6, the fusion breakpoints of these tumours were located in YAP1 exon 4 and TFE3 exon 7. Following complete surgical resection, neither of the tumours has recurred or metastasised (follow-up period 6-7 months). CONCLUSIONS: We validate the presence of YAP1-TFE3 gene fusion in a unique primary mesenchymal tumour of lung, adding additional support for clear cell stromal tumour of the lung as a distinct entity.

The clinicopathologic spectrum and genomic landscape of de-/trans-differentiated melanoma.

Dedifferentiation and transdifferentiation are rare and only poorly understood phenomena in cutaneous melanoma. To study this disease more comprehensively we have retrieved 11 primary cutaneous melanomas from our pathology archives showing biphasic features characterized by a conventional melanoma and additional areas of de-/trans-differentiation as defined by a lack of immunohistochemical expression of all conventional melanocytic markers (S-100 protein, SOX10, Melan-A, and HMB-45). The clinical, histologic, and immunohistochemical findings were recorded and follow-up was obtained. The patients were mostly elderly (median: 81 years; range: 42-86 years) without significant gender predilection, and the sun-exposed skin of the head and neck area was most commonly affected. The tumors were deeply invasive with a mean depth of 7 mm (range: 4-80 mm). The dedifferentiated component showed atypical fibroxanthoma-like features in the majority of cases (7), while additional rhabdomyosarcomatous and epithelial transdifferentiation was noted histologically and/or immunohistochemically in two tumors each. The background conventional melanoma component was of desmoplastic (4), superficial spreading (3), nodular (2), lentigo maligna (1), or spindle cell (1) types. For the seven patients with available follow-up data (median follow-up period of 25 months; range: 8-36 months), two died from their disease, and three developed metastases. Next-generation sequencing of the cohort revealed somatic mutations of established melanoma drivers including mainly NF1 mutations (5) in the conventional component, which was also detected in the corresponding de-/trans-differentiated component. In summary, the diagnosis of primary cutaneous de-/trans-differentiated melanoma is challenging and depends on the morphologic identification of conventional melanoma. Molecular analysis is diagnostically helpful as the mutated gene profile is shared between the conventional and de-/trans-differentiated components. Importantly, de-/trans-differentiation does not appear to confer a more aggressive behavior.

Higher cMET dependence of sacral compared to clival chordoma cells: contributing to a better understanding of cMET in chordoma.

Chordomas are rare slow growing, malignant bone tumors of the axial skeleton with no approved medical treatment. As the majority of chordomas express cMET and its ligand, HGF, and crosstalks between EGFR and MET-signaling exist, we aimed to explore cMET activity in chordoma cell lines and clinical samples. We investigated nine chordoma patients and four chordoma cell lines for cMET expression. Two clival and two sacral chordoma cell lines were tested for chromosomal abnormalities of the MET gene locus; we studied the influence of HGF on the autocrine secretion and migration behavior, as well as protein expression and phosphorylation. Two MET/ALK inhibitors were investigated for their effects on cell viability, cell cycle, cyclin alterations, apoptosis, and downstream signaling pathways. Moderate and strong expression of membrane and cytoplasmic cMET in chordoma patients and cell lines used, as well as concentration-dependent increase in phospho cMET expression after HGF stimulation in all four chordoma cell lines was shown. U-CH2, MUG-Chor1, and UM-Chor1 are polysomic for MET. Chordoma cell lines secreted EGF, VEGF, IL-6, and MMP9 upon HGF-stimulation. Sacral cell lines showed a distinct HGF-induced migration. Both inhibitors dose-dependently inhibited cell growth, induce apoptosis and cell-cycle arrest, and suppress downstream pathways. Heterogeneous responses obtained in our in vitro setting indicate that cMET inhibitors alone or in combination with other drugs might particularly benefit patients with sacral chordomas.

T-regulatory cells predict clinical outcome in soft tissue sarcoma patients: a clinico-pathological study.

BACKGROUND: Soft tissue sarcomas (STS) are generally considered non-immunogenic, although specific subtypes respond to immunotherapy. Antitumour response within the tumour microenvironment relies on a balance between inhibitory and activating signals for tumour-infiltrating lymphocytes (TILs). This study analysed TILs and immune checkpoint molecules in STS, and assessed their prognostic impact regarding local recurrence (LR), distant metastasis (DM), and overall survival (OS). METHODS: One-hundred and ninety-two surgically treated STS patients (median age: 63.5 years; 103 males [53.6%]) were retrospectively included. Tissue microarrays were constructed, immunohistochemistry for PD-1, PD-L1, FOXP3, CD3, CD4, and CD8 performed, and staining assessed with multispectral imaging. TIL phenotype abundance and immune checkpoint markers were correlated with clinical and outcome parameters (LR, DM, and OS). RESULTS: Significant differences between histology and all immune checkpoint markers except for FOXP3+ and CD3-PD-L1+ cell subpopulations were found. Higher levels of PD-L1, PD-1, and any TIL phenotype were found in myxofibrosarcoma as compared to leiomyosarcoma (all p < 0.05). The presence of regulatory T cells (Tregs) was associated with increased LR risk (p = 0.006), irrespective of margins. Other TILs or immune checkpoint markers had no significant impact on outcome parameters. CONCLUSIONS: TIL and immune checkpoint marker levels are most abundant in myxofibrosarcoma. High Treg levels are independently associated with increased LR risk, irrespective of margins.

Applicability of pan-TRK immunohistochemistry for identification of NTRK fusions in lung carcinoma.

In the last two decades, various therapies have been introduced for lung carcinoma patients, including tyrosine-kinase inhibitors for different mutations. While some of them are specific to specific tumor types, others, like NTRK1-3 fusions, are found in various solid tumors. The occurrence of an NTRK1,2 or 3 fusion acts as a biomarker for efficient treatment with NTRK inhibitors, irrespectively of the tumor type. However, the occurrence of the NTRK1-3 fusions in lung carcinomas is extremely rare. We performed a retrospective analysis to evaluate the applicability of immunohistochemistry with the pan-TRK antibody in the detection of NTRK fusions in lung carcinomas. The study cohort included 176 adenocarcinomas (AC), 161 squamous cell carcinomas (SCC), 31 large-cell neuroendocrine carcinomas (LCNEC), and 19 small cell lung carcinomas (SCLC). Immunohistochemistry (IHC) was performed using the pan-TRK antibody (clone EPR17341, Ventana) on tissue microarrays, while confirmation for all positive cases was done using RNA-based Archer FusionPlex MUG Lung Panel. On IHC staining, 12/387 samples (3.1%) demonstrated a positive reaction. Ten SCC cases (10/161, 6.2%), and two LCNEC cases (2/31, 6.5%) were positive. Positive cases demonstrated heterogeneous staining of tumor cells, mostly membranous with some cytoplasmic and in one case nuclear pattern. RNA-based sequencing did not demonstrate any NTRK1-3 fusion in our patients' collective. Our study demonstrates that pan-TRK expression in lung carcinoma is very low across different histologic types. NTRK1-3 fusions using an RNA-based sequencing approached could not be detected. This stresses the importance of confirmation of immunohistochemistry results by molecular methods.

Comparison of the 7th and 8th version of the AJCC classification system for soft tissue sarcomas of extremities and trunk in patients with localised, intermediate or high-grade disease treated at European tertiary sarcoma centres.

BACKGROUND: The updated 8th version of the AJCC-staging system for soft tissue sarcomas (STS) has been criticised for omitting tumour depth as category-defining variable and eventually not improving prognostic accuracy in comparison to the 7th version. This study aimed at investigating the prognostic accuracy of both AJCC-versions in STS-patients treated at European tertiary sarcoma centres. METHODS: 1032 patients (mean age: 60.7 ± 16.3 years; 46.0% [n = 475] females; median follow-up: 38.6 months), treated at five tertiary sarcoma centres for localised, intermediate or high-grade STS of extremities and trunk were retrospectively included. Uni- and multivariate Cox-regression models and Harrell's C-indices were calculated to analyse prognostic factors for overall survival (OS) and assess prognostic accuracy. RESULTS: In univariate analysis, prognostic accuracy for OS was comparable for both AJCC-versions (C-index: 0.620 [8th] vs. 0.614 [7th]). By adding margins, age, gender, and histology to the multivariate models, prognostic accuracy of both versions could be likewise improved (C-index: 0.714 [8th] vs. 0.705 [7th]). Moreover, tumour depth did not significantly contribute to prognostic accuracy of the 8th version's multivariate model (C-index for both models: 0.714). Stratification into four main T-stages based on tumour size only, as implemented in the 8th version, significantly improved prognostic accuracy between each category. However, T-stages as defined in the 7th version had poorer discriminatory power (C-index: 0.625 [8th] vs. 0.582 [7th]). CONCLUSION: Both AJCC-versions perform equally well regarding prognostic accuracy. Yet, simplification of the 8 th version by omitting tumour depth as T-stage-defining parameter, whilst emphasizing the importance of tumour size, should be considered advantageous.