Translational Relevance Treatment paradigm for sarcomas has been unchanged for the past four decades with survival outcomes plateauing and patients with HR disease facing an abysmal prognosis. SARC028, trial testing pembrolizumab in STS, demonstrated responses in UPS and dedifferentiated liposarcomas. Alliance A091401, trial combining ipilimumab and nivolumab, showed responses in more histologies, but OS rates similar to standard chemotherapy. Here, we compare the immune TME of two molecularly distinct sarcomas: the genetically complex, ICR, UPS, and the genetically simple, fusion-driven, poorly ICR, RMS, to identify factors that may contribute to their immunotherapy responsiveness. These two subtypes represent the main genomic aberrancies observed in sarcomas. Results show both tumors dominated by TAMs. T-cells in UPS are diffusely distributed, while T-cells in RMS cluster with B-cells near perivascular beds forming TLS. Our findings suggest targeting the myeloid compartment and tumor angiogenesis could overcome the immunosuppressive niche sustained by TAMs and lead to potential therapeutic targets.

Remission durability following single-antigen targeted chimeric antigen receptor (CAR) T-cells is limited by antigen modulation, which may be overcome with combinatorial targeting. Building upon our experiences targeting CD19 and CD22 in B-cell acute lymphoblastic leukemia (B-ALL), we report on the experiences and limitations of a novel MSCV-CD19/CD22-4-1BB bivalent CAR T-cell (CD19.22.BBz). This phase I dose-escalation trial enrolled children and young adults (CAYA) with B-cell malignancies. Primary objectives included toxicity and dose-finding. Secondary objectives included response rates and relapse-free survival (RFS). Biologic correlatives, including CAR T-cell expansion and cytokine profiling, and laboratory investigations, were also analyzed. Twenty patients, ages 5.4-34.6 years, with B-ALL received CD19.22.BBz. The complete response (CR) rate was 60% (12/20) in the full cohort and 71.4% (10/14) in CAR-naïve patients. Ten (50%) developed cytokine release syndrome (CRS), with 3 (15%) having grade 3 CRS and only 1 experiencing any neurotoxicity (grade 3). The 6- and 12-month RFS in those achieving CR was 80.8% (95% CI: 42.4-94.9%) and 57.7% (95% CI: 22.1-81.9%), respectively. Limited CAR T-cell expansion and persistence of MSCV-CD19.22.BBz compared to EF1a-CD22.BBz prompted laboratory investigations comparing EF1a versus MSCV promoters, which did not reveal major differences. Limited CD22 targeting with CD19.22.BBz, as evaluated by ex vivo cytokine secretion and leukemia eradication in humanized mice, led to development of a novel bicistronic CD19.28z/CD22.BBz construct with enhanced cytokine production against CD22. With demonstrated safety and efficacy of CD19.22.BBz in a heavily pre-treated CAYA B-ALL cohort, further optimization of combinatorial antigen targeting serves to overcome identified limitations. (Clinicaltrials.gov NCT03448393)

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BackgroundCurrent therapy for osteosarcoma pulmonary metastases (PMs) is ineffective. The mechanisms that prevent successful immunotherapy in osteosarcoma are incompletely understood. We investigated the tumor microenvironment of metastatic osteosarcoma with the goal of harnessing the immune system as a therapeutic strategy.Methods66 osteosarcoma tissue specimens were analyzed by immunohistochemistry (IHC) and immune markers were digitally quantified. Tumor-infiltrating lymphocytes (TILs) from 25 specimens were profiled by functional cytometry. Comparative transcriptomic studies of distinct tumor-normal lung ‘PM interface’ and ‘PM interior’ regions from 16 PMs were performed. Clinical follow-up (median 24 months) was available from resection.ResultsIHC revealed a statistically significantly higher concentration of TILs expressing immune checkpoint and immunoregulatory molecules in PMs compared with primary bone tumors (including programmed cell death 1 (PD-1), programmed death ligand 1 (PD-L1), lymphocyte-activation gene 3 (LAG-3), T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), and indoleamine 2,3-dioxygenase (IDO1). Remarkably, these lymphocytes are excluded at the PM interface compared with PM interior. TILs from PMs exhibited significantly higher amounts of PD-1 and LAG-3 and functional cytokines including interferon-γ (IFNγ) by flow cytometry. Gene expression profiling further confirmed the presence of CD8 and CD4 lymphocytes concentrated at the PM interface, along with upregulation of immunoregulatory molecules and IFNγ-driven genes in the same region. We further discovered a strong alternatively activated macrophage signature throughout the entire PMs along with a polymorphonuclear myeloid-derived suppressor cell signature focused at the PM interface. Expression of PD-L1, LAG-3, and colony-stimulating factor 1 receptor (CSF1R) at the PM interface was associated with significantly worse progression-free survival (PFS), while gene sets indicative of productive T cell immune responses (CD8 T cells, T cell survival, and major histocompatibility complex class 1 expression) were associated with significantly improved PFS.ConclusionsOsteosarcoma PMs exhibit immune exclusion characterized by the accumulation of TILs at the PM interface. These TILs produce effector cytokines, suggesting their capability of activation and recognition of tumor antigens. Our findings suggest cooperative immunosuppressive mechanisms in osteosarcoma PMs including immune checkpoint molecule expression and the presence of immunosuppressive myeloid cells. We identify cellular and molecular signatures that are associated with patient outcomes, which could be exploited for successful immunotherapy.

Staphylococcus aureus is an important cause of morbidity among children with CHD. Infective endocarditis was common with S aureus bacteremia in this population; in addition, prolonged bacteremia, thrombocytopenia, and CRP >10 mg/dL may serve as diagnostic adjuncts for IE. qacA/B-positive isolates are associated with adverse clinical outcomes.

Staphylococcus aureus bacteremia without a localizing source in children is not well described. We identified patients with a positive blood culture for S. aureus from an 11-year surveillance study. Thirty-six cases of primary bacteremia were identified accounting for 5.7% of bacteremias. Most patients had comorbidities (86.1%), most commonly immunosuppression (33.3%).

Level II-prognostic, retrospective cohort comparison.

Chimeric antigen receptor (CAR) T-cells effectively eradicate medullary B-cell acute lymphoblastic leukemia (B-ALL) and can traffic to and clear central nervous system (CNS) involvement. CAR T-cell activity in non¬contral nervous system (CNS) extramedullary disease (EMD) has not been well-characterized. We systematically evaluated CAR T-cell kinetics, associated toxicities, and efficacy in B-ALL non-CNS EMD. We conducted a retrospective review of B-ALL patients with non-CNS EMD who were screened for/enrolled on one of three CAR trials at our institution (CD19, CD22, CD19/22). Non-CNS EMD was identified by histology or radiographic imaging at extramedullary sites excluding the cerebrospinal fluid and CNS parenchyma. Of approximately 180 patients with relapsed/refractory B-ALL screened across multiple early phase trials over an 8-year period, 38 (21.1%) presented with isolated non-CNS EMD (n=5) or combined medullary/non-CNS EMD (n=33) on FDG PET-CT imaging. A subset receiving CAR T-cells (18 infusions) obtained FDG PET-CT scans pre- and post-infusion to monitor response. At best response, 72.2% (13 of 18) of patients demonstrated a medullary MRD-negative complete remission and complete (CR, n=7) or partial (PR, n=6) non-CNS EMD response. Non-CNS EMD responses to CAR T-cells were delayed (n=3) and residual non-CNS EMD was substantial; rarely, discrepant responses (marrow without EMD response) were observed (n=2). Unique CAR-associated toxicities at non-CNS EMD sites were seen in select patients. CAR T-cells are active in B-ALL non-CNS EMD. Still, non-CNS EMD response to CAR T-cells may be delayed and sub-optimal, particularly with multifocal disease. Serial FDG PET-CT scans are necessary for identifying and monitoring non-CNS EMD.