Intratumoral NKp46+ natural killer cells are spatially distanced from T and MHC-I+ cells with prognostic implications in soft tissue sarcoma.

Introduction: Soft tissue sarcomas (STS) are rare, heterogenous malignancies with an unmet need for novel immunotherapies. Tumor infiltrating lymphocytes (TILs) have been linked with favorable outcomes in STS patients, though the contribution of natural killer (NK) cells and spatial relationships of TILs with MHC-I expressing cells lacks detailed characterization. Experimental design: Using archived and prospectively collected specimens, we evaluated intratumoral NK cells by immunohistochemistry (IHC), flow cytometry, and immunofluorescence (IF). We assessed spatial localization of NK and T cells by multiplex IF, analyzing the effects of MHC-I expression status on NK and T cell clustering. Results: Both intratumoral NKp46 and CD56dim expression were associated with significantly improved overall survival (P=0.05), while higher infiltrates of CD56bright NK cells predicted a worse prognosis (P=0.05). The presence of intratumoral NK cells was inversely proportional to CD3+ T cells. Spatial analyses showed NK cells preferentially clustering close to other NK cells with sparse CD3+ T and CD8+ T cells in range (P<0.0001). Additionally, CD3+ T and CD8+ T cells showed significantly greater co-localization with MHC-I+ cells, compared to NK cells (P<0.0001). After neoadjuvant radiotherapy, there was greater CD8 clustering, while after neoadjuvant chemotherapy, there was overall lower TIL clustering. Conclusion: Intratumoral NK cells are prognostic in STS and localize closer to MHC-I- cells than T cells. Although both NK and T cells are associated with improved survival in STS, their differential distribution in the TME based on MHC-I expression status may serve as a biomarker for improved immunotherapy treatment selection.

circCsnk1g3- and circAnkib1-regulated interferon responses in sarcoma promote tumorigenesis by shaping the immune microenvironment.

Exonic circular RNAs (circRNAs) produce predominantly non-coding RNA species that have been recently profiled in many tumors. However, their functional contribution to cancer progression is still poorly understood. Here, we identify the circRNAs expressed in soft tissue sarcoma cells and explore how the circRNAs regulate sarcoma growth in vivo. We show that circCsnk1g3 and circAnkib1 promote tumor growth by shaping a pro-tumorigenic microenvironment, possibly due to their capabilities to regulate tumor-promoting elements extrinsic to the tumor cells. Accordingly, circCsnk1g3 and circAnkib1 can control the expression of interferon-related genes and pro-inflammatory factors in the sarcoma cells, thus directing immune cell recruitment into the tumor mass, and hence their activation. Mechanistically, circRNAs may repress pro-inflammatory elements by buffering activation of the pathways mediated by RIG-I, the cytosolic viral RNA sensor. The current findings suggest that the targeting of specific circRNAs could augment the efficacy of tumor and immune response to mainstay therapies.

Immune-Desert Tumor Microenvironment in Thoracic SMARCA4-Deficient Undifferentiated Tumors with Limited Efficacy of Immune Checkpoint Inhibitors.

BACKGROUND: Thoracic SMARCA4-deficient undifferentiated tumors (SMARCA4-UT) are aggressive neoplasms. Data linking BAF alterations with tumor microenvironment (TME) and efficacy of immune checkpoint inhibitors (ICI) are contradictory. The TME of SMARCA4-UT and their response to ICI are unknown. MATERIALS AND METHODS: Patients diagnosed with SMARCA4-UT in our institution were included. Immunostainings for tertiary lymphoid structures (TLS), immune cell markers, and checkpoints were assessed. Validation was performed using an independent transcriptome dataset including SMARCA4-UT, non-small cell lung cancers (NSCLC) with/without SMARCA4 mutations, and unclassified thoracic sarcomas (UTS). CXCL9 and PD-L1 expressions were assessed in NSCLC and thoracic fibroblast cell lines, with/without SMARCA4 knockdown, treated with/without interferon gamma. RESULTS: Nine patients were identified. All samples but one showed no TLS, consistent with an immune desert TME phenotype. Four patients received ICI as part of their treatment, but the only one who responded, had a tumor with a TLS and immune-rich TME. Unsupervised clustering of the validation cohort using immune cell scores identified 2 clusters associated with cell ontogeny and immunity (cluster 1 enriched for NSCLC independently of SMARCA4 status (n = 9/10; P = .001); cluster 2 enriched for SMARCA4-UT (n = 11/12; P = .005) and UTS (n = 5/5; P = .0005). SMARCA4 loss-of-function experiments revealed interferon-induced upregulation of CXCL9 and PD-L1 expression in the NSCLC cell line with no effect on the thoracic fibroblast cell line. CONCLUSION: SMARCA4-UT mainly have an immune desert TME with limited efficacy to ICI. TME of SMARCA4-driven tumors varies according to the cell of origin questioning the interplay between BAF alterations, cell ontogeny and immunity.

HMGB1 Translocation is Associated with Tumor-Associated Myeloid Cells and Involved in the Progression of Fibroblastic Sarcoma.

The morphological variability and genetic complexity of fibroblastic sarcoma makes its diagnosis and treatment a challenge. High-mobility group box 1 protein (HMGB1), which functions as a DNA chaperone and a prototypical damage-associated molecular pattern, plays a paradoxical role in cancer. However, the expression pattern and role of HMGB1 in fibroblastic sarcomas is ill defined. By immunostaining of 95 tissue microarray cores of fibroblastic sarcomas, HMGB1 was found to be expressed in most tumor tissues. Nuclear HMGB1 translocation to cytoplasm was observed both in tumor cells and vascular endothelial cells. A visible number of tumor-associated myeloid cells including CD68+ and CD163+ macrophages and CD33+ myeloid cells were also detected in most tumor tissues. HMGB1 translocation was not only associated with CD68, CD163, and CD33 density, but also with disease progression. These results imply that HMGB1, an important regulator of the tumor microenvironment, is associated with tumor-associated myeloid cells and involved in the progression of fibroblastic sarcomas; HMGB1 may serve as a promising prognostic biomarker and a potential therapeutic target for fibroblastic sarcoma.

Clinical and immunological characteristics of sarcomas patients with clonogenic tumors.

Tumorigenesis is related to the generation of heterogeneous tumor cell population, which is the result of genetic and epigenetic alterations followed by clonal selections and subsequent expansion. In basic studies genetic, histological and morphological diversity of different clones within a patient's neoplasm and specifics of their interrelation with patient's immune system are investigated mostly on the models of tumors of epithelial origin. Mesenchymal tumors such as soft tissue and bone-derived sarcomas (STBS) have been poorly studied in this regard. The molecular genetic methods used to examine intratumoral heterogeneity do not currently provide insight into which portion of the identified subclones are able to grow autonomously. Limiting dilution cloning demonstrates the existence of self-regulating tumor cells in the population and can serve as an independent prognostic predictor of poor prognosis. Intratumoral heterogeneity results not only in differences in growth dynamics, gene expression, and phenotypic markers, but also in the resistance to treatment, especially immunotherapy, thus causing tumor eluding immune escape. The changes that accompany this process can be affected by the cellular immune system, resulting in an imbalance between populations. The variations in the population composition of immune system cells are now widely debated as a predictor of response to immunotherapy, which is of obvious interest for sarcomas, where the effectiveness of chemotherapy is low and the prognosis is unfavorable, especially in case of metastatic disease development. The search for new predictive markers of disease prognosis and treatment efficacy is an important task, to which this study is focused. Our results demonstrate that clonogenic tumor characteristics such as clonogenic potential is independent predictor of unfavorable prognosis in cases of cancer and correlate with the clinical characteristics of the tumor such as overall survival (OS) and progression free survival (PFS). It was found that patients with clonogenic sarcomas had a lower content of activated cytotoxic T-lymphocytes (CTL) with the CD3+CD8+HLA-DR+ phenotype and an increased number of natural NK killers (p < 0.05) compared to nonclonogenic tumors. In addition, according to our data, a high neutrophil to lymphocyte ratio (NLR), a low value of major T-lymphocyte populations, and a higher number of natural killer cells (NK) in the blood can be negative prognostic factors for the immunotherapy of this disease.

Depth of tumor implantation affects response to in situ vaccination in a syngeneic murine melanoma model.

An important component of research using animal models is ensuring rigor and reproducibility. This study was prompted after two experimenters performing virtually identical studies obtained different results when syngeneic B78 murine melanoma cells were implanted into the skin overlying the flank and treated with an in situ vaccine (ISV) immunotherapy. Although both experimenters thought they were using identical technique, we determined that one was implanting the tumors intradermally (ID) and the other was implanting them subcutaneously (SC). Though the baseline in vivo immunogenicity of tumors can depend on depth of their implantation, the response to immunotherapy as a function of tumor depth, particularly in immunologically 'cold' tumors, has not been well studied. The goal of this study was to evaluate the difference in growth kinetics and response to immunotherapy between identically sized melanoma tumors following ID versus SC implantation. We injected C57BL/6 mice with syngeneic B78 melanoma cells either ID or SC in the flank. When tumors reached 190-230 mm3, they were grouped into a 'wave' and treated with our previously published ISV regimen (12 Gy local external beam radiation and intratumoral hu14.18-IL2 immunocytokine). Physical examination demonstrated that ID-implanted tumors were mobile on palpation, while SC-implanted tumors became fixed to the underlying fascia. Histologic examination identified a critical fascial layer, the panniculus carnosus, which separated ID and SC tumors. SC tumors reached the target tumor volume significantly faster compared with ID tumors. Most ID tumors exhibited either partial or complete response to this immunotherapy, whereas most SC tumors did not. Further, the 'mobile' or 'fixed' phenotype of tumors predicted response to therapy, regardless of intended implantation depth. These findings were then extended to additional immunotherapy regimens in four separate tumor models. These data indicate that the physical 'fixed' versus 'mobile' characterization of the tumors may be one simple method of ensuring homogeneity among implanted tumors prior to initiation of treatment. Overall, this short report demonstrates that small differences in depth of tumor implantation can translate to differences in response to immunotherapy, and proposes a simple physical examination technique to ensure consistent tumor depth when conducting implantable tumor immunotherapy experiments.

Dose escalation and expansion phase I studies with the tumour-targeting antibody-tumour necrosis factor fusion protein L19TNF plus doxorubicin in patients with advanced tumours, including sarcomas.

BACKGROUND: L19TNF is a recombinant fusion protein composed of a human antibody fragment and human tumour necrosis factor. L19TNF targets the EDB domain of oncofetal fibronectin highly expressed in tumour vasculature and induces tumour remission in mouse tumours. We summarise two phase I trials testing a combination of L19TNF with doxorubicin in patients with solid tumours, particularly soft tissue sarcomas (STS). PATIENTS AND METHODS: The first study, an open-label, dose-escalation and expansion phase I study of L19TNF plus doxorubicin, enrolled 27 patients. Three cohorts (10.4-17 µg/kg L19TNF) of patients received L19TNF intravenously at days 1, 3, and 5 and doxorubicin (75 mg/m2, then 60 mg/m2) on day 1 every 3 weeks. The expansion cohort enrolled patients with STS. The second study tried to re-escalate the doxorubicin dose to 75 mg/m2 with 13 µg/kg L19TNF. Among primary objectives was the establishment of a recommended dose (RD). RESULTS: The combination was safely applicable. Dose-limiting toxicity occurred either at 17 µg/kg L19TNF or at 75 mg/m2 doxorubicin. RD is 13 µg/kg L19TNF plus 60 mg/m2 doxorubicin. In 15 STS patients of the extension cohort evaluable for efficacy, antitumour activity was observed with complete remission in 1, partial remission in 1 and minor tumour shrinkage in 7 patients. The median overall survival for this heavily pretreated cohort was 14.9 months. CONCLUSION: L19TNF can be safely applied in combination with doxorubicin and induces encouraging tumour remissions in patients with soft tissue sarcomas.

Tumor DNA methylation profiles correlate with response to anti-PD-1 immune checkpoint inhibitor monotherapy in sarcoma patients.

BACKGROUND: Some sarcomas respond to immune checkpoint inhibition, but predictive biomarkers are unknown. We analyzed tumor DNA methylation profiles in relation to immunological parameters and response to anti-programmed cell death 1 (anti-PD-1) immune checkpoint inhibitor (ICI) therapy in patients with sarcoma. PATIENTS AND METHODS: We retrospectively identified adult patients who had received anti-PD-1 ICI therapy for recurrent sarcoma in two independent centers. We performed (1) blinded radiological response evaluation according to immune response evaluation criteria in solid tumors (iRECIST) ; (2) tumor DNA methylation profiling of >850,000 probes using Infinium MethylationEPIC microarrays; (3) analysis of tumor-infiltrating immune cell subsets (CD3, CD8, CD45RO, FOXP3) and intratumoral expression of immune checkpoint molecules (PD-L1, PD-1, LAG-3) using immunohistochemistry; and (4) evaluation of blood-based systemic inflammation scores (neutrophil-to-lymphocyte ratio, leucocyte-to-lymphocyte ratio, monocyte-to-lymphocyte ratio, platelet-to-lymphocyte ratio). Response to anti-PD-1 ICI therapy was bioinformatically and statistically correlated with DNA methylation profiles and immunological data. RESULTS: 35 patients (median age of 50 (23-81) years; 18 females, 17 males; 27 soft tissue sarcomas; 8 osteosarcomas) were included in this study. The objective response rate to anti-PD-1 ICI therapy was 22.9% with complete responses in 3 out of 35 and partial responses in 5 out of 35 patients. Adjustment of DNA methylation data for tumor-infiltrating immune cells resulted in identification of methylation differences between responders and non-responders to anti-PD-1 ICI. 2453 differentially methylated CpG sites (DMPs; 2043 with decreased and 410 with increased methylation) were identified. Clustering of sarcoma samples based on these DMPs revealed two main clusters: methylation cluster 1 (MC1) consisted of 73% responders and methylation cluster 2 (MC2) contained only non-responders to anti-PD-1 ICI. Median progression-free survival from anti-PD-1 therapy start of MC1 and MC2 patients was 16.5 and 1.9 months, respectively (p=0.001). Median overall survival of these patients was 34.4 and 8.0 months, respectively (p=0.029). The most prominent DNA methylation differences were found in pathways implicated in Rap1 signaling, focal adhesion, adherens junction Phosphoinositide 3-kinase (PI3K)-Akt signaling and extracellular matrix (ECM)-receptor interaction. CONCLUSIONS: Our data demonstrate that tumor DNA methylation profiles may serve as a predictive marker for response to anti-PD-1 ICI therapy in sarcoma.

Perioperative chemotherapy and regional hyperthermia for high-risk adult-type soft tissue sarcomas.

A group of patients with adult-type soft tissue sarcoma is at high risk of local recurrence and distant metastases. Age, tumour site, histological subtype, tumour size and grade have been identified as the most important independent adverse prognostic factors. Macroscopically complete tumour resection is considered as the mainstay of treatment with the addition of preoperative or postoperative radiotherapy for extremity or trunk localisation. Retroperitoneal localisation requires compartmental resection and is associated with a worse prognosis. Here, radiotherapy is of no proven value. Perioperative chemotherapy is considered to treat micrometastatic disease not detectable at the time of diagnosis. The neoadjuvant application gives the risk of distant metastasis the greatest importance as therapy is carried out at the earliest possible time, whereas adjuvant chemotherapy is delayed by surgery and the necessary wound healing. With reported response rates up to 30%, both the operability may be improved and the risk of intraoperative tumour cell dissemination may be reduced, resulting also in reduced local relapse rates. However, the potential risk of early tumour progression may counteract this benefit. Optimised strategies with multimodality approaches including chemotherapy, regional hyperthermia (RHT) and immunotherapeutic agents have been shown to improve survival in high-risk patients. Here, we focus on the data from available randomised studies investigating the use of perioperative chemotherapy in patients with high-risk adult-type soft tissue sarcoma, including the use of RHT for local enhancement of chemotherapy effect and immune induction.

Intraarticular Inflammatory Myofibroblastic Tumor of the Left Knee With ALK-CARS Fusion Detected With Archer Fusionplex Sarcoma NGS Panel: Case Report and Literature Review.

Inflammatory myofibroblastic tumor (IMT) is a lesion of intermediate biological potential with local recurrences and rare metastases found in multiple anatomical locations. We present a case of a pure intraarticular IMT of the knee, a location that has not been previously documented, with genetic confirmation of ALK-CARS fusion detected with next-generation sequencing. A 20-year-old healthy male was admitted to the orthopedic oncology department due to several months of pain and restriction in movement of his left knee. On magnetic resonance imaging, multiple intraarticular nodular lesions were seen. The patient underwent 2 synovectomies within the course of 1 year. The initial biopsy was interpreted as nodular fasciitis. The second biopsy revealed exuberant tissue displaying compact fascicles of spindle cells intermixed with myxoid areas in a background of inflammatory cells, highly suggestive for IMT. Due to the unusual intraarticular location, equivocal ALK immunostaining and the differential diagnosis with nodular fasciitis, we performed targeted next-generation sequencing using Archer FusionPlex Sarcoma panel, which can identify multiple fusions in a single assay. An ALK-CARS fusion was found, supporting the diagnosis of IMT. This report emphasizes the added value of broad molecular analysis in cases with unusual clinical presentation, equivocal immunohistochemistry, and a wide differential diagnosis.

Novel therapeutic options for alveolar soft part sarcoma: antiangiogenic therapy, immunotherapy and beyond.

PURPOSE OF REVIEW: Alveolar soft part sarcoma (ASPS) represent 0.5% of sarcomas, defining a rarest among rare malignancies. It affects young adults, displaying slow-growing mass of the thigh, head and neck, and trunk. Although quite indolent, a majority of cases displays an advanced disease with lung bone or central nervous system metastasis. Complete surgery is the cornerstone of localized ASPS, and advanced diseases poorly respond to chemotherapy. Here discuss recent progress in molecular characterization of ASPS and future prospects of therapeutic approaches. RECENT FINDINGS: ASPS is characterized by a specific oncogenic translocation ASPSCR1-TFE3 that induce hepatocyte growth factor receptor (MET) overexpression, angiogenesis, and immunosuppression in the tumor microenvironment. These specific biological features have encouraged the successful exploration of MET inhibitors, antiangiogenic drugs, and immunotherapy. We reviewed the main tracks of ASPS biology and recent insights from targeted therapies is ASPS mainly driven tyrosine kinase inhibitors (especially antiangiogenics), immune-checkpoint inhibitors, and their combinations. SUMMARY: Overall, antiangiogenics and anti Programmed cell death 1/Programmed cell death ligand 1 therapies showed a significant activity in ASPS that warrants additional investigation through randomized trials to validate those results and through ancillary biological studies to better understand resistance mechanisms and biomarkers of response.

Tumor mutation burden and checkpoint immunotherapy markers in primary and metastatic synovial sarcoma.

Synovial sarcoma (SS) is a soft-tissue malignancy that most often affects patients aged between 15 and 40 years, and the prognosis for patients with metastatic disease is generally poor. This study was performed to evaluate checkpoint blockade immunotherapy markers in SS, including tumor mutational burden (TMB), DNA mismatch repair (MMR) status, and PDL-1 (programmed cell death ligand 1), PD1 (programmed cell death 1), and CD8 expression by normal-tumor paired whole-exome sequencing (WES) and immunohistochemistry (IHC). Outcomes evaluated included event-free and overall survival. Twenty one (21) FISH (Fluorescence In Situ Hybridization)-confirmed SS cases (11 F, 10 M) were studied, with age ranging from 8 to 89 years at diagnosis and follow-up ranging from 1 to 16 years. TMB (n = 16) ranged from 0.83 to 212/Mb (median, 1.7). Only one case showed a high TMB of 212/Mb and missense variants of MMR genes in the primary tumor, while the other 15 cases had a low TMB of less than 5/Mb. IHC was performed on all 21 tumor samples for PD-L1, PD1, CD8, and MMR proteins. PD-L1 membranous staining was detected in 3 of 21 cases (14.3%), ranging from 1 to 5% for tumor proportion score and 1-10 for combined positive score. PD1 was detected in 15 of 21 cases (71.4%), ranging from 1 to 25/HPF (high power field) (median, 2). CD8 stain was seen in all cases, ranging from 2 to 60/HPF (median, 5). PD1 staining results correlated with CD8 staining results (P < 0.0001). No correlation of TMB or IHC markers was found with survival. No fixed pattern of TMB or IHCs between primary and metastatic tumors was observed; there was no correlation between TMB or IHCs and age, location, or diagnosis subtype. All of the cases tested showed retained expression of MMR proteins. The results show that for SS, a tumor with strong driver translocation, most cases have a low TMB, but occasionally a high TMB may be present, as observed in 1 of the 16 (6.25%) cases. The demonstration of a subgroup of SS cases with high TMB might explain the 10% response rate to checkpoint immunotherapy observed in clinical trials in patients with SS.

TEG011 persistence averts extramedullary tumor growth without exerting off-target toxicity against healthy tissues in a humanized HLA-A*24:02 transgenic mice.

γδT cells play an important role in cancer immunosurveillance and are able to distinguish malignant cells from their healthy counterparts via their γδTCR. This characteristic makes γδT cells an attractive candidate for therapeutic application in cancer immunotherapy. Previously, we have identified a novel CD8α-dependent tumor-specific allo-HLA-A*24:02-restricted Vγ5Vδ1TCR with potential therapeutic value when used to engineer αßT cells from HLA-A*24:02 harboring individuals. αßT cells engineered to express this defined Vγ5Vδ1TCR (TEG011) have been suggested to recognize spatial changes in HLA-A*24:02 present selectively on tumor cells but not their healthy counterparts. However, in vivo efficacy and toxicity studies of TEG011 are still limited. Therefore, we extend the efficacy and toxicity studies as well as the dynamics of TEG011 in vivo in a humanized HLA-A*24:02 transgenic NSG (NSG-A24:02) mouse model to allow the preparation of a first-in-men clinical safety package for adoptive transfer of TEG011. Mice treated with TEG011 did not exhibit any graft-versus-host disease-like symptoms and extensive analysis of pathologic changes in NSG-A24:02 mice did not show any off-target toxicity of TEG011. However, loss of persistence of TEG011 in tumor-bearing mice was associated with the outgrowth of extramedullary tumor masses as also observed for mock-treated mice. In conclusion, TEG011 is well tolerated without harming HLA-A*24:02+ expressing healthy tissues, and TEG011 persistence seems to be crucial for long-term tumor control in vivo.

Correlative Analyses of the SARC028 Trial Reveal an Association Between Sarcoma-Associated Immune Infiltrate and Response to Pembrolizumab.

PURPOSE: We recently reported a 17.5% objective RECIST 1.1 response rate in a phase II study of pembrolizumab in patients with advanced sarcoma (SARC028). The majority of responses occurred in undifferentiated pleomorphic sarcoma (UPS) and dedifferentiated liposarcoma (DDLPS). We sought to determine whether we can identify immune features that correlate with clinical outcomes from tumor tissues obtained pre- and on-treatment. PATIENTS AND METHODS: Pretreatment (n = 78) and 8-week on-treatment (n = 68) tumor biopsies were stained for PD-L1 and multiplex immunofluorescence panels. The density of positive cells was quantified to determine associations with anti-PD-1 response. RESULTS: Patients that responded to pembrolizumab were more likely to have higher densities of activated T cells (CD8+ CD3+ PD-1+) and increased percentage of tumor-associated macrophages (TAM) expressing PD-L1 pre-treatment compared with non-responders. Pre-treatment tumors from responders also exhibited higher densities of effector memory cytotoxic T cells and regulatory T cells compared with non-responders. In addition, higher density of cytotoxic tumor-infiltrating T cells at baseline correlated with a better progression-free survival (PFS). CONCLUSIONS: We show that quantitative assessments of CD8+ CD3+ PD-1+ T cells, percentage of TAMs expressing PD-L1, and other T-cell densities correlate with sarcoma response to pembrolizumab and improved PFS. Our findings support that multiple cell types present at the start of treatment may enhance tumor regression following anti-PD-1 therapy in specific advanced sarcomas. Efforts to confirm the activity of pembrolizumab in an expansion cohort of patients with UPS/DDLPS are underway.

Arteriovenous graft in kidney transplant patients: Lookout for the rare but fearsome angiosarcoma.

INTRODUCTION: Angiosarcomas are rare tumors, comprising less than 1% of all sarcomas. However, they portend a poor prognosis, as they tend to metastasize early, being of uttermost importance a prompt diagnosis and treatment. CASE DESCRIPTION: We present the case of a 55-year-old female with history of kidney transplantation, immunosuppressed with tacrolimus, prednisolone, and mofetil mycophenolate. Fifteen years after the transplant, she developed an ulcerated lesion on the site of a nonfunctioning arteriovenous graft, which was excised. Histology was compatible with a high grade angiosarcoma that invaded the margins, and immunosuppression was switched to everolimus. Staging imaging exams revealed lymph node, muscle, and lung metastases. Shortly after, nodular lesions appeared compatible with local recurrence of the disease, and the patient showed severe deterioration of her clinical condition, being proposed for palliative chemotherapy. However, the disease showed an explosive progression and the patient died before starting the treatment. CONCLUSION: This case emphasizes the importance of including inspection of the vascular access (functioning or not) in regular post-transplant consultation and value any alterations in the attempt of a timely diagnosis. Although rare, angiosarcoma is an important entity that should be considered in the differential diagnosis of soft tissue masses arising from a vascular access, especially in immunocompromised patients. Aggressive treatment should be offered whenever possible.

The adequacy of tissue microarrays in the assessment of inter- and intra-tumoural heterogeneity of infiltrating lymphocyte burden in leiomyosarcoma.

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.

Expression of lymphocyte immunoregulatory biomarkers in bone and soft-tissue sarcomas.

Despite advances in our understanding of the underlying molecular drivers of sarcomas, few treatments are available with proven benefit for advanced metastatic sarcomas. Immunotherapy has value in this setting for some types of cancers, but sarcomas, with their multiplicity of rare types, have not been characterized in detail for their expression of targetable immune biomarkers. This study provides the most systematic evaluation to date of tumor-infiltrating lymphocytes and immune checkpoint biomarker expression in sarcomas. We examined by morphology and immunohistochemistry 1072 sarcoma specimens representing 22 types, in addition to 236 benign bone and soft-tissue tumors. Genomically-complex sarcoma types-those driven by mutations and/or copy-number alterations-had much higher numbers of tumor-infiltrating lymphocytes than translocation-associated sarcomas. Prior exposure to radiotherapy was associated with increased immune infiltrates. Higher lymphocytic infiltration was associated with better overall survival among the non-translocation-associated sarcomas. Expression of PD-1 and CD56 were associated with worse overall survival. LAG-3 and TIM-3, two emerging immune checkpoints, were frequently expressed in most sarcoma types. Indeed, most cases positive for PD-(L)1 coexpressed one or both of these novel biomarkers, providing a potential rationale in support for trials targeting LAG-3 and/or TIM-3 in conjunction with PD-1 inhibition.

Ectopic hamartomatous thymoma in an immunocompromised male.

Ectopic hamartomatous thymoma (EHT) is a rare benign neoplasm classically occurring in the lower neck of adult males. Here we present a case of EHT occurring in a 43-year-old immunocompromised male and a brief review of existing literature. The patient presented with a palpable mass overlying the left clavicle which, on imaging, showed a solitary nodule possibly eroding the cortical bone. A biopsy predominantly showed spindle cells that were immunopositive for keratin AE1/AE3 as well as weakly positive for CD99, SMA, and CD34. A diagnosis of synovial sarcoma was favored; at which point surgical resection was performed. The resected mass was well-demarcated with a tan-yellow cut surface. Microscopically, the lesion was composed of a mixture of spindle cells, glands, and mature adipose tissue. The spindle cells were plump with bland nuclei, and the epithelial component showed morphology similar to glands of salivary or breast tissue with a bilayered appearance (luminal and basal). No pleomorphism, mitotic figures, or necrosis was present. Immunohistochemical stains were performed and showed the spindle cells to express a myoepithelial phenotype (cytokeratin AE1/AE3, p63, calponin positive). The glands showed SMA and p63 positivity in the basal cells (similar to salivary gland and breast). Overall, given the clinical context, histomorphologic, and immunohistochemical profile, a diagnosis of EHT was made. At 12 months of follow-up there was no evidence of recurrence.

An immunosuppressive macrophage profile attenuates the prognostic impact of CD20-positive B cells in human soft tissue sarcoma.

BACKGROUND: Immune cells can regulate disease progression and response to treatment in multiple tumor types, but their activities in human soft tissue sarcoma are poorly characterized. METHODS: Marker-defined immune cell subsets were characterized from a tumor microenvironmental perspective in two independent cohorts of human soft tissue sarcoma by multiplex IHC, quantitative PCR and/or bioinformatics. RESULTS: B cell profiling revealed a prognostic role for CD20 protein (cohort 1, 33 patients) and MS4A1 gene expression (cohort 2, 265 patients). Multiplex IHC and gene correlation analysis supported a role in antigen presentation, immune cell differentiation and T cell activation. The prognostic role of MS4A1 expressing B cells was only observed in an IL10low, PTGS2low or CD163low tumor microenvironment according to the transcriptomic data. IL10 levels consistently correlated with the M2-like macrophage marker CD163, which also defined the majority of macrophages. A polarization of these cells toward a pro-tumoral phenotype was further supported by lack of correlation between CD163 and M1 markers like NOS2, as well as by low abundance of CD80 positive cells in tissue. CONCLUSIONS: Analysis of CD20/MS4A1 expression in soft tissue sarcoma merits further attention as a promising candidate prognostic tool for survival, but not in patients with a pronounced immunosuppressive tumor microenvironment. Macrophages are ubiquitous and polarized toward a protumoral phenotype. This provides a rationale for further studies on B cell function and immunotherapy targeting M2-polarized macrophages.