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Laura Pacini,* Andrew D Jenks,* Simon Vyse,* Christopher P Wilding, Amani Arthur, Paul H Huang Division of Molecular Pathology, The Institute of Cancer Research, London, UK*These authors contributed equally to this workCorrespondence: Paul H HuangDivision of Molecular Pathology, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UKTel +44 207 153 5554Email paul.huang@icr.ac.ukAbstract: Insertion mutations in exon 20 (Ex20ins) of the epidermal growth factor receptor (EGFR) gene are the largest class of EGFR mutations in non-small cell lung cancer (NSCLC) for which there are currently no approved targeted therapies. NSCLC patients with these mutations do not respond to clinically approved EGFR tyrosine kinase inhibitors (TKIs) and have poor outcomes. A number of early phase clinical trials are currently underway to evaluate the efficacy of a new generation of TKIs that are capable of binding to and blocking Ex20ins. Although these agents have shown some clinical activity, patient responses have been restricted by dose-limiting toxicity or rapid acquisition of resistance after a short response. Here we review the current understanding of the mechanisms of resistance to these compounds, which include on-target EGFR secondary mutations, compensatory bypass pathway activation and acquisition of an EMT phenotype. Taking lessons from conventional EGFR inhibitor therapy in NSCLC, we also consider other potential sources of resistance including the presence of drug-tolerant persister cells. We will discuss therapeutic strategies which have the potential to overcome different forms of drug resistance. We conclude by evaluating recent technological developments in drug discovery such as PROTACs as a means to better tackle TKI resistance in NSCLC harbouring Ex20ins mutations.Keywords: EGFR, exon 20 insertions, poziotinib, lung cancer, drug resistance, PROTACs

Purpose: The landscape of extracellular matrix (ECM) alterations in soft tissue sarcomas (STS) remains poorly characterized. We aimed to investigate the tumor ECM and adhesion signaling networks present in STS and their clinical implications., Experimental Design: Proteomic and clinical data from 321 patients across 11 histological subtypes were analyzed to define ECM and integrin adhesion networks. Subgroup analysis was performed in leiomyosarcomas (LMS), dedifferentiated liposarcomas (DDLPS), and undifferentiated pleomorphic sarcomas (UPS)., Results: This analysis defined subtype-specific ECM profiles including enrichment of basement membrane proteins in LMS and ECM proteases in UPS. Across the cohort, we identified three distinct coregulated ECM networks which are associated with tumor malignancy grade and histological subtype. Comparative analysis of LMS cell line and patient proteomic data identified the lymphocyte cytosolic protein 1 cytoskeletal protein as a prognostic factor in LMS. Characterization of ECM network events in DDLPS revealed three subtypes with distinct oncogenic signaling pathways and survival outcomes. Evaluation of the DDLPS subtype with the poorest prognosis nominates ECM remodeling proteins as candidate antistromal therapeutic targets. Finally, we define a proteoglycan signature that is an independent prognostic factor for overall survival in DDLPS and UPS., Conclusions: STS comprise heterogeneous ECM signaling networks and matrix-specific features that have utility for risk stratification and therapy selection, which could in future guide precision medicine in these rare cancers., (©2024 The Authors; Published by the American Association for Cancer Research.)

Background: High-risk soft tissue sarcomas of the extremities and trunk wall (eSTS), as defined by the Sarculator nomogram, are more likely to benefit from (neo)adjuvant anthracycline-based therapy compared to low/intermediate-risk patients. The biology underpinning these differential treatment outcomes remain unknown., Methods: We analysed proteomic profiles and clinical outcomes of 123 eSTS patients. A Cox model for overall survival including the Sarculator was fitted to individual data to define four risk groups. A DNA replication protein signature-Sarcoma Proteomic Module 6 (SPM6) was evaluated for association with clinicopathological factors and risk groups. SPM6 was added as a covariate together with Sarculator in a multivariable Cox model to assess improvement in prognostic risk stratification., Results: DNA replication and cell cycle proteins were upregulated in high-risk versus very low-risk patients. Evaluation of the functional effects of CRISPR-Cas9 gene knockdown of proteins enriched in high-risk patients using the cancer cell line encyclopaedia database identified candidate drug targets. SPM6 was significantly associated with tumour malignancy grade (p = 1.6e-06), histology (p = 1.4e-05) and risk groups (p = 2.6e-06). Cox model analysis showed that SPM6 substantially contributed to a better calibration of the Sarculator nomogram (Index of Prediction Accuracy = 0.109 for Sarculator alone versus 0.165 for Sarculator + SPM6)., Conclusions: Risk stratification of patient with STS is defined by distinct biological pathways across a range of cancer hallmarks. Incorporation of SPM6 protein signature improves prognostic risk stratification of the Sarculator nomogram. This study highlights the utility of integrating protein signatures for the development of next-generation nomograms., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

Background: Adolescents and young adult (AYA) patients with soft tissue tumours including sarcomas are an underserved group with disparities in treatment outcomes., Methods: To define the molecular features between AYA and older adult (OA) patients, we analysed the proteomic profiles of a large cohort of soft tissue tumours across 10 histological subtypes (AYA n = 66, OA n = 243), and also analysed publicly available functional genomic data from soft tissue tumour cell lines (AYA n = 5, OA n = 8)., Results: Biological hallmarks analysis demonstrates that OA tumours are significantly enriched in MYC targets compared to AYA tumours. By comparing the patient-level proteomic data with functional genomic profiles from sarcoma cell lines, we show that the mRNA splicing pathway is an intrinsic vulnerability in cell lines from OA patients and that components of the spliceosome complex are independent prognostic factors for metastasis free survival in AYA patients., Conclusions: Our study highlights the importance of performing age-specific molecular profiling studies to identify risk stratification tools and targeted agents tailored for the clinical management of AYA patients., (© 2024. The Author(s).)

Soft tissue sarcomas (STS) are rare and diverse mesenchymal cancers with limited treatment options. Here we undertake comprehensive proteomic profiling of tumour specimens from 321 STS patients representing 11 histological subtypes. Within leiomyosarcomas, we identify three proteomic subtypes with distinct myogenesis and immune features, anatomical site distribution and survival outcomes. Characterisation of undifferentiated pleomorphic sarcomas and dedifferentiated liposarcomas with low infiltrating CD3 + T-lymphocyte levels nominates the complement cascade as a candidate immunotherapeutic target. Comparative analysis of proteomic and transcriptomic profiles highlights the proteomic-specific features for optimal risk stratification in angiosarcomas. Finally, we define functional signatures termed Sarcoma Proteomic Modules which transcend histological subtype classification and show that a vesicle transport protein signature is an independent prognostic factor for distant metastasis. Our study highlights the utility of proteomics for identifying molecular subgroups with implications for risk stratification and therapy selection and provides a rich resource for future sarcoma research., (© 2023. The Author(s).)

Intravenous leiomyomatosis (IVLM) is a rare benign smooth muscle tumour that is characterised by intravenous growth in the uterine and pelvic veins. Previous DNA copy number and transcriptomic studies have shown that IVLM harbors unique genomic and transcriptomic alterations when compared to uterine leiomyoma (uLM), which may account for their distinct clinical behaviour. Here we undertake the first comparative proteomic analysis of IVLM and other smooth muscle tumours (comprising uLM, soft tissue leiomyoma and benign metastasizing leiomyoma) utilising data-independent acquisition mass spectrometry. We show that, at the protein level, IVLM is defined by the unique co-regulated expression of splicing factors. In particular, IVLM is enriched in two clusters composed of co-regulated proteins from the hnRNP, LSm, SR and Sm classes of the spliceosome complex. One of these clusters (Cluster 3) is associated with key biological processes including nascent protein translocation and cell signalling by small GTPases. Taken together, our study provides evidence of co-regulated expression of splicing factors in IVLM compared to other smooth muscle tumours, which suggests a possible role for alternative splicing in the pathogenesis of IVLM.

Soft tissue sarcomas (STS) are a group of rare and heterogeneous cancers. While large-scale genomic and epigenomic profiling of STS have been undertaken, proteomic analysis has thus far been limited. Here we utilise sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) for proteomic profiling of formalin fixed paraffin embedded (FFPE) specimens from a cohort of STS patients (n = 36) across four histological subtypes (leiomyosarcoma, synovial sarcoma, undifferentiated pleomorphic sarcoma and dedifferentiated liposarcoma). We quantified 2951 proteins across all cases and show that there is a significant enrichment of gene sets associated with smooth muscle contraction in leiomyosarcoma, RNA splicing regulation in synovial sarcoma and leukocyte activation in undifferentiated pleomorphic sarcoma. We further identified a subgroup of STS cases that have a distinct expression profile in a panel of proteins, with worse survival outcomes when compared to the rest of the cohort. Our study highlights the value of comprehensive proteomic characterisation as a means to identify histotype-specific STS profiles that describe key biological pathways of clinical and therapeutic relevance; as well as for discovering new prognostic biomarkers in this group of rare and difficult-to-treat diseases., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Aims: COVID-19 represents one of the greatest global healthcare challenges in a generation. Orthopaedic departments within the UK have shifted care to manage trauma in ways that minimize exposure to COVID-19. As the incidence of COVID-19 decreases, we explore the impact and risk factors of COVID-19 on patient outcomes within our department., Methods: We retrospectively included all patients who underwent a trauma or urgent orthopaedic procedure from 23 March to 23 April 2020. Electronic records were reviewed for COVID-19 swab results and mortality, and patients were screened by telephone a minimum 14 days postoperatively for symptoms of COVID-19., Results: A total of 214 patients had orthopaedic surgical procedures, with 166 included for analysis. Patients undergoing procedures under general or spinal anaesthesia had a higher risk of contracting perioperative COVID-19 compared to regional/local anaesthesia (p = 0.0058 and p = 0.0007, respectively). In all, 15 patients (9%) had a perioperative diagnosis of COVID-19, 14 of whom had fragility fractures; six died within 30 days of their procedure (40%, 30-day mortality). For proximal femoral fractures, our 30-day mortality was 18.2%, compared to 7% in 2019., Conclusion: Based on our findings, patients undergoing procedures under regional or local anaesthesia have minimal risk of developing COVID-19 perioperatively. Those with multiple comorbidities and fragility fractures have a higher morbidity and mortality if they contract COVID-19 perioperatively; therefore, protective care pathways could go some way to mitigate the risk. Our 30-day mortality of proximal femoral fractures was 18.2% during the COVID-19 pandemic in comparison to the annual national average of 6.1% in 2018 and the University Hospital Coventry average of 7% for the same period in 2019, as reported in the National Hip Fracture Database. Patients undergoing procedures under general or spinal anaesthesia at the peak of the pandemic had a higher risk of contracting perioperative COVID-19 compared to regional block or local anaesthesia. We question whether young patients undergoing day-case procedures under regional block or local anaesthesia with minimal comorbidities require fourteen days self-isolation; instead, we advocate that compliance with personal protective equipment, a negative COVID-19 swab three days prior to surgery, and screening questionnaire may be sufficient.Cite this article: Bone Joint Open 2020;1-9:520-529., Competing Interests: ICMJE COI statement: The authors have no financial or competing interests to disclose.

Purpose of Review: Genetic aberrations resulting in tropomyosin receptor kinase (TRK) fusion proteins can drive oncogenesis and are postulated to occur in up to 1% of solid tumours. However, TRK fusions in adult sarcomas are rare and there is a significant challenge in identifying patients with sarcomas harbouring TRK fusions in the clinical setting. Despite a recent European Society of Medical Oncology consensus article regarding screening of tumours for TRK fusions, economical and practical limitations present a barrier to widespread screening of sarcomas., Recent Findings: Larotrectinib and entrectinib are pan-TRK inhibitors which have both received FDA approval for the management of solid tumours harbouring NTRK fusions. Initial results of a number of clinical trials have demonstrated promising efficacy and safety data, including dramatic and durable responses in patients with sarcomas. As such, TRK inhibitors represent a promising treatment option in a small cohort of adult sarcoma patients, where currently treatment options are limited. The emergence of acquired resistance is a concern associated with TRK inhibitor therapy and a number of second-generation agents targeting TRK kinase mutations driving acquired resistance have entered early-phase clinical trials., Summary: With the growing appreciation of the implications of TRK fusions, this review will summarize the emerging clinical trial data of TRK inhibitors in sarcomas. Although in their infancy, clinical trial results are encouraging, and as further results and analyses are released, we will have a greater understanding of their impact on clinical practice and the management of patients with sarcomas.

Sarcomas are a rare group of mesenchymal cancers comprising over 70 different histological subtypes. For the majority of these diseases, the molecular understanding of the basis of their initiation and progression remains unclear. As such, limited clinical progress in prognosis or therapeutic regimens have been made over the past few decades. Proteomics techniques are being increasingly utilised in the field of sarcoma research. Proteomic research efforts have thus far focused on histological subtype characterisation for the improvement of biological understanding, as well as for the identification of candidate diagnostic, predictive, and prognostic biomarkers for use in clinic. However, the field itself is in its infancy, and none of these proteomic research findings have been translated into the clinic. In this review, we provide a brief overview of the proteomic strategies that have been employed in sarcoma research. We evaluate key proteomic studies concerning several rare and ultra-rare sarcoma subtypes including, gastrointestinal stromal tumours, osteosarcoma, liposarcoma, leiomyosarcoma, malignant rhabdoid tumours, Ewing sarcoma, myxofibrosarcoma, and alveolar soft part sarcoma. Consequently, we illustrate how routine implementation of proteomics within sarcoma research, integration of proteomics with other molecular profiling data, and incorporation of proteomics into clinical trial studies has the potential to propel the biological and clinical understanding of this group of complex rare cancers moving forward., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction : Tyrosine kinases are key mediators of intracellular signaling cascades and aberrations in these proteins have been implicated in driving oncogenesis through the dysregulation of fundamental cellular processes including proliferation, migration, and apoptosis. As such, targeting these proteins with small molecule tyrosine kinase inhibitors (TKI) has led to significant advances in the treatment of a number of cancer types. Areas covered : Soft tissue sarcomas (STS) are a heterogeneous and challenging group of rare cancers to treat, but the approval of the TKI pazopanib for the treatment of advanced STS demonstrates that this class of drugs may have broad utility against a range of different sarcoma histological subtypes. Since the approval of pazopanib, a number of other TKIs have entered clinical trials to evaluate whether their activity in STS matches the promising results seen in other solid tumors. In this article, we review the emerging role of TKIs in the evolving landscape of sarcoma treatment. Expert opinion : As our biological understanding of response and resistance of STS to TKIs advances, we anticipate that patient management will move away from a 'one size fits all' paradigm toward personalized, multi-line, and patient-specific treatment regimens where patients are treated according to the underlying biology and genetics of their specific disease.

The characterisation and clinical relevance of tumour-infiltrating lymphocytes (TILs) in leiomyosarcoma (LMS), a subtype of soft tissue sarcoma that exhibits histological heterogeneity, is not established. The use of tissue microarrays (TMA) in studies that profile TIL burden is attractive but given the potential for intra-tumoural heterogeneity to introduce sampling errors, the adequacy of this approach is undetermined. In this study, we assessed the histological inter- and intra-tumoural heterogeneity in TIL burden within a retrospective cohort of primary LMS specimens. Using a virtual TMA approach, we also analysed the optimal number of TMA cores required to provide an accurate representation of TIL burden in a full tissue section. We establish that LMS have generally low and spatially homogenous TIL burdens, although a small proportion exhibit higher levels and more heterogeneous distribution of TILs. We show that a conventional and practical number (e.g. ≤3) of TMA cores is adequate for correct ordinal categorisation of tumours with high or low TIL burden, but that many more cores (≥11) are required to accurately estimate absolute TIL numbers. Our findings provide a benchmark for the design of future studies aiming to define the clinical relevance of the immune microenvironments of LMS and other sarcoma subtypes.

Background: Kneeling is an important activity of daily living, holding social, religious and occupational value. Following total knee replacement (TKR), many patients report they are unable to kneel or have been advised not to kneel., Methods: We observed 100 consecutive knee replacements in 79 patients attending outpatient clinic at a minimum 5 months post-TKR. The patients were asked to fill out a questionnaire detailing whether they were able to kneel prior to their knee replacement and whether they thought they were able to kneel since their knee replacement. The patients were then asked to kneel on a padded examination couch and then onto a pillow on the floor for 15 s. Degree of flexion achievable was also recorded., Results: Of the knees with patella resurfacing, 78.6% were able to kneel compared to only 45.6% knees with native patellae. Two-tailed Fisher's exact test showed this difference to be statistically significant ( p = 0.001). The χ 2 analysis showed that those patients with an achievable flexion of angle of greater than 100° were significantly more likely to be able to kneel than those with a flexion angle of less than 100° ( p = 0.0148). Comparing posterior cruciate ligament (PCL) retaining against PCL sacrificing implants, there was no statistically significant difference in kneeling ability ( p = 0.541)., Conclusion: Kneeling remains an important function in patients undergoing TKR, with patella resurfacing significantly improving the likelihood of a patient being able to kneel.

Background: In the unsalvageable, infected total knee arthroplasty, knee arthrodesis is one treatment option with lower reported reinfection rates compared with repeated 2-stage revision and improved function compared with amputation. One possible method for reducing incidence of recurrent infection treated by arthrodesis is the use of a silver-coated implant. We report our experience of silver-coated arthrodesis nails used for managing infected revision arthroplasty. We primarily assess the rate of reinfection and rate of amputation and report functional outcome measures., Methods: Retrospective analysis of all patients undergoing knee arthrodesis with a silver-coated arthrodesis nail between 2008 and 2014. Patient-reported data were recorded prearthrodesis and postarthrodesis (Oxford Knee Score and Short Form-36) as well as evidence of recurrent of infection, subsequent surgery, and the necessity for amputation., Results: Eight patients underwent arthrodesis using the silver-coated arthrodesis nail. Mean duration of follow-up was 16 months (5-35 months). At the point of follow-up, there were no amputations, deaths, or implant revisions. One case of recurrent infection was successfully treated with washout and debridement. The mean prearthrodesis and postarthrodesis Oxford Knee Score difference was +8.9 points (P = .086) with significantly improved pain (P = .019), night pain (P = .021), and ease of standing (P = .003)., Conclusion: Arthrodesis of the knee using a silver-coated intramedullary device is successful in eradicating infection and allowing limb conservation. Where infection does recur, this can be successfully treated with implant retention. The use of a silver-coated arthrodesis nail should be considered as an alternative to amputation for patients with a multiply revised and infected total knee arthroplasty., (Copyright © 2016 Elsevier Inc. All rights reserved.)