Impact of Consolidative Radiation on Overall and Progression-Free Survival in Pediatric, Adolescent, and Young Adult Metastatic Bone and Soft Tissue Sarcoma.

PURPOSE: To determine the association between consolidative radiation (RT) and survival in children, adolescents, and young adults with metastatic sarcoma. METHODS AND MATERIALS: Eligibility criteria included patients aged ≤39 years with newly diagnosed metastatic bone or soft tissue sarcoma who completed local control of the primary tumor without disease progression. Consolidative RT was defined as RT to all known sites of metastatic disease. The Kaplan-Meier method was used to estimate overall survival (OS) and progression-free survival (PFS). The least absolute shrinkage and selection operator Cox provided adjusted estimates. To account for immortal time bias, consolidative RT was used as a time-varying covariate in a time dependent Cox model. Distant failure was estimated using the Fine-Gray model. RESULTS: Patients (n = 85) had a median age at diagnosis of 14.8 years. Most common histology was Ewing Sarcoma (45.9%) followed by rhabdomyosarcoma (40.0%). Receipt of consolidative RT was associated with Ewing Sarcoma (P < .001) and local control modality as those who underwent local control with surgery and RT compared with surgery alone were more likely to be treated with consolidative RT (P = .034). Consolidative RT was independently associated with improved OS (hazard ratio [HR], 0.41; 95% CI, 0.17-0.98; P = .045) and improved PFS (HR, 0.37; 95% CI, 0.16-0.88; P = .024) after adjusting for confounding variables and immortal time bias. Patients treated with consolidative RT also experienced a lower risk of distant failure (HR, 0.33; 95% CI, 0.17-0.64; P = .001). In an independent data set of patients with metachronous progression (n = 36), consolidative RT remained independently associated with improved OS. CONCLUSIONS: Consolidative RT was independently associated with improved OS and PFS and decreased risk of distant failure in child, adolescent, and young adult patients with metastatic sarcoma. Future work should evaluate biomarkers to optimize patient selection, timing, and dose for consolidative RT.

Atezolizumab for Advanced Alveolar Soft Part Sarcoma.

BACKGROUND: Alveolar soft part sarcoma (ASPS) is a rare soft-tissue sarcoma with a poor prognosis and no established therapy. Recently, encouraging responses to immune checkpoint inhibitors have been reported. METHODS: We conducted an investigator-initiated, multicenter, single-group, phase 2 study of the anti-programmed death ligand 1 (PD-L1) agent atezolizumab in adult and pediatric patients with advanced ASPS. Atezolizumab was administered intravenously at a dose of 1200 mg (in patients ≥18 years of age) or 15 mg per kilogram of body weight with a 1200-mg cap (in patients <18 years of age) once every 21 days. Study end points included objective response, duration of response, and progression-free survival according to Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1, as well as pharmacodynamic biomarkers of multistep drug action. RESULTS: A total of 52 patients were evaluated. An objective response was observed in 19 of 52 patients (37%), with 1 complete response and 18 partial responses. The median time to response was 3.6 months (range, 2.1 to 19.1), the median duration of response was 24.7 months (range, 4.1 to 55.8), and the median progression-free survival was 20.8 months. Seven patients took a treatment break after 2 years of treatment, and their responses were maintained through the data-cutoff date. No treatment-related grade 4 or 5 adverse events were recorded. Responses were noted despite variable baseline expression of programmed death 1 and PD-L1. CONCLUSIONS: Atezolizumab was effective at inducing sustained responses in approximately one third of patients with advanced ASPS. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT03141684.).

Gut colonization with an obesity-associated enteropathogenic microbe modulates the premetastatic niches to promote breast cancer lung and liver metastasis.

Introduction: Obesity, an independent risk factor for breast cancer growth and metastatic progression, is also closely intertwined with gut dysbiosis; and both obese state and dysbiosis promote each other. Enteric abundance of Bacteroides fragilis is strongly linked with obesity, and we recently discovered the presence of B. fragilis in malignant breast cancer. Given that enterotoxigenic B. fragilis or ETBF, which secretes B. fragilis toxin (BFT), has been identified as a procarcinogenic microbe in breast cancer, it is necessary to examine its impact on distant metastasis and underlying systemic and localized alterations promoting metastatic progression of breast cancer. Methods: We used syngeneic mammary intraductal (MIND) model harboring gut colonization with ETBF to query distant metastasis of breast cancer cells. Alterations in the immune network and cytokines/chemokines in the tumor microenvironment and distant metastatic sites were examined using flow cytometry, immunohistochemistry, and multiplex arrays. Results: ETBF infection initiates a systemic inflammation aiding in the establishment of the premetastatic niche formation in vital organs via increased proinflammatory and protumorigenic cytokines like IL17A, IL17E, IL27p28, IL17A/F, IL6, and IL10 in addition to creating a prometastatic immunosuppressive environment in the liver and lungs rich in myeloid cells, macrophages, and T regulatory cells. It induces remodeling of the tumor microenvironment via immune cell and stroma infiltration, increased vasculogenesis, and an EMT-like response, thereby encouraging early metastatic dissemination ready to colonize the conducive environment in liver and lungs of the breast tumor-bearing mice. Discussion: In this study, we show that enteric ETBF infection concomitantly induces systemic inflammation, reshapes the tumor immune microenvironment, and creates conducive metastatic niches to potentiate early dissemination and seeding of metastases to liver and lung tissues in agreement with the "seed and soil hypothesis." Our results also support the ETBF-induced "parallel model" of metastasis that advocates for an early dissemination of tumor cells that form metastatic lesions independent of the primary tumor load.

Complex/cryptic EWSR1::FLI1/ERG Gene Fusions and 1q Jumping Translocation in Pediatric Ewing Sarcomas.

Ewing sarcomas (ES) are rare small round cell sarcomas often affecting children and characterized by gene fusions involving one member of the FET family of genes (usually EWSR1) and a member of the ETS family of transcription factors (usually FLI1 or ERG). The detection of EWSR1 rearrangements has important diagnostic value. Here, we conducted a retrospective review of 218 consecutive pediatric ES at diagnosis and found eight patients having data from chromosome analysis, FISH/microarray, and gene-fusion assay. Three of these eight ES had novel complex/cryptic EWSR1 rearrangements/fusions by chromosome analysis. One case had a t(9;11;22)(q22;q24;q12) three-way translocation involving EWSR1::FLI1 fusion and 1q jumping translocation. Two cases had cryptic EWSR1 rearrangements/fusions, including one case with a cryptic t(4;11;22)(q35;q24;q12) three-way translocation involving EWSR1::FLI1 fusion, and the other had a cryptic EWSR1::ERG rearrangement/fusion on an abnormal chromosome 22. All patients in this study had various aneuploidies with a gain of chromosome 8 (75%), the most common, followed by a gain of chromosomes 20 (50%) and 4 (37.5%), respectively. Recognition of complex and/or cryptic EWSR1 gene rearrangements/fusions and other chromosome abnormalities (such as jumping translocation and aneuploidies) using a combination of various genetic methods is important for accurate diagnosis, prognosis, and treatment outcomes of pediatric ES.

Adoptive Cellular Therapy for Pediatric Solid Tumors: Beyond Chimeric Antigen Receptor-T Cell Therapy.

ABSTRACT: Children and adolescents with high-risk (metastatic and relapsed) solid tumors have poor outcomes despite intensive multimodal therapy, and there is a pressing need for novel therapeutic strategies. Adoptive cellular therapy (ACT) has demonstrated activity in multiple adult cancer types, and opportunity exists to expand the use of this therapy in children. Employment of immunotherapy in the pediatric population has realized only modest overall clinical trial results, with success thus far restricted mainly to antibody-based therapies and chimeric antigen receptor T-cell therapies for lymphoid malignancy. As we improve our understanding of the orchestrated cellular and molecular mechanisms involved in ACT, this will provide biologic insight and improved ACT strategies for pediatric malignancies. This review focuses on ACT strategies outside of chimeric antigen receptor T-cell therapy, including completed and ongoing clinical trials, and highlights promising preclinical data in tumor-infiltrating lymphocytes that enhance the clinical efficacy of ACT for high-risk pediatric solid tumors.

Exceptional response to the ALK and ROS1 inhibitor lorlatinib and subsequent mechanism of resistance in relapsed ALK F1174L-mutated neuroblastoma.

Treatment of high-risk neuroblastoma typically incorporates multiagent chemotherapy, surgery, radiation therapy, autologous stem cell transplantation, immunotherapy, and differentiation therapy. The discovery of activating mutations in ALK receptor tyrosine kinase (ALK) in ∼8% of neuroblastomas opens the possibility of further improving outcomes for this subset of patients with the addition of ALK inhibitors. ALK inhibitors have shown efficacy in tumors such as non-small-cell lung cancer and anaplastic large cell lymphoma in which wild-type ALK overexpression is driven by translocation events. In contrast, ALK mutations driving neuroblastomas are missense mutations in the tyrosine kinase domain yielding constitutive activation and differing sensitivity to available ALK inhibitors. We describe a case of a patient with relapsed, refractory, metastatic ALK F1174L-mutated neuroblastoma who showed no response to the first-generation ALK inhibitor crizotinib but had a subsequent complete response to the ALK/ROS1 inhibitor lorlatinib. The patient's disease relapsed after 13 mo of treatment. Sequencing of cell-free DNA at the time of relapse pointed toward a potential mechanism of acquired lorlatinib resistance: amplification of CDK4 and FGFR1 and a NRAS Q61K mutation. We review the literature regarding differing sensitivity of ALK mutations found in neuroblastoma to current FDA-approved ALK inhibitors and known pathways of acquired resistance. Our report adds to the literature of important correlations between neuroblastoma ALK mutation status and clinical responsiveness to ALK inhibitors. It also highlights the importance of understanding acquired mechanisms of resistance.

Predictors of Recurrence and Patterns of Initial Failure in Localized Ewing Sarcoma: A Contemporary 20-Year Experience.

BACKGROUND: The majority of patients with localized Ewing sarcoma will remain disease-free long term, but for those who suffer recurrence, successful treatment remains a challenge. Identification of clinicopathologic factors predictive of recurrence could suggest areas for treatment optimization. We sought to describe our experience regarding predictors of recurrence and patterns of first failure in patients receiving modern systemic therapy for nonmetastatic Ewing sarcoma. METHODS: The medical records of pediatric and adult patients treated for localized Ewing sarcoma between 1999 and 2019 at Johns Hopkins Hospital were retrospectively analyzed. Local control was surgery, radiotherapy, or both. Recurrence-free survival (RFS) was calculated using the Kaplan-Meier method. Univariable and multivariable Cox proportional-hazards modeling was performed to obtain hazard ratios (HR) for recurrence. RESULTS: In 94 patients with initially localized disease, there were 21 recurrences: 4 local, 14 distant, and 3 combined. 5-year and 10-year RFS were 75.6% and 70.5%, respectively. On multivariable analysis including age at diagnosis and tumor size, <95% tumor necrosis following neoadjuvant chemotherapy (NAC; HR 14.3, p = 0.028) and radiological tumor size change during NAC (HR 1.04 per 1% decrease in size change, p = 0.032) were independent predictors of recurrence. Among patients experiencing distant recurrence, pulmonary metastases were present in 82% and were the only identifiable site of disease in 53%. CONCLUSIONS: Poor pathologic or radiologic response to NAC is predictive of recurrence in patients with localized Ewing sarcoma. Suboptimal tumor size reduction following chemotherapy provides a means to risk-stratify patients who do not undergo definitive resection. Isolated pulmonary recurrence was a common event.

Moving Toward the Ideal Autologous Adoptive T-cell Therapy for Cancer.

Despite being one of the earliest immunotherapies to prove that the immune system can effectively recognize and eradicate cancer, autologous adoptive T-cell therapies remain largely limited to academic centers and research trials. The highly individualized protocols and the heterogeneous nature of the expanded T-cell products hinder effectiveness, commercial development, and regulatory approvals. The report by Li and colleagues details a novel method of generating cancer-specific autologous T cells from patients receiving anti-PD-1 checkpoint blockade immunotherapy. Their method achieved promising results in four initial patients treated in a pilot study. While further studies are required to characterize the autologous T-cell products generated and their effectiveness in larger cohorts of patients, the protocol they describe addresses several of the roadblocks that have prevented more wide-spread use of autologous adoptive T-cell therapy.See related article by Li et al., p. 2184.

Neoantigen-based EpiGVAX vaccine initiates antitumor immunity in colorectal cancer.

Metastatic colorectal cancer (CRC) is poorly immunogenic, with limited neoantigens that can be targeted by cancer vaccine. Previous approaches to upregulate neoantigen have had limited success. In this study, we investigated the role of a DNA methyltransferase inhibitor (DNMTi), 5-aza-2'-deoxycytidine (DAC), in inducing cancer testis antigen (CTA) expression and evaluated the antitumor efficacy of a combinatorial approach with an epigenetically regulated cancer vaccine EpiGVAX and DAC. A murine model of metastatic CRC treated with combination therapy with an irradiated whole-cell CRC vaccine (GVAX) and DAC was used to assess the antitumor efficacy. DAC significantly induced expression of CTAs in CRC, including a new CTA Tra-P1A with a known neoepitope, P1A. Epigenetically modified EpiGVAX with DAC improved survival outcomes of GVAX. Using the epigenetically regulated antigen Tra-P1A as an example, our study suggests that the improved efficacy of EpiGVAX with DAC may due in part to the enhanced antigen-specific antitumor immune responses. This study shows that epigenetic therapy with DNMTi can not only induce new CTA expression but may also sensitize tumor cells for immunotherapy. Neoantigen-based EpiGVAX combined with DAC can improve the antitumor efficacy of GVAX by inducing antigen-specific antitumor T cell responses to epigenetically regulated proteins.

The Immunosuppressive Niche of Soft-Tissue Sarcomas is Sustained by Tumor-Associated Macrophages and Characterized by Intratumoral Tertiary Lymphoid Structures.

PURPOSE: Clinical trials with immune checkpoint inhibition in sarcomas have demonstrated minimal response. Here, we interrogated the tumor microenvironment (TME) of two contrasting soft-tissue sarcomas (STS), rhabdomyosarcomas and undifferentiated pleomorphic sarcomas (UPS), with differing genetic underpinnings and responses to immune checkpoint inhibition to understand the mechanisms that lead to response. EXPERIMENTAL DESIGN: Utilizing fresh and formalin-fixed, paraffin-embedded tissue from patients diagnosed with UPS and rhabdomyosarcomas, we dissected the TME by using IHC, flow cytometry, and comparative transcriptomic studies. RESULTS: Our results demonstrated both STS subtypes to be dominated by tumor-associated macrophages and infiltrated with immune cells that localized near the tumor vasculature. Both subtypes had similar T-cell densities, however, their in situ distribution diverged. UPS specimens demonstrated diffuse intratumoral infiltration of T cells, while rhabdomyosarcomas samples revealed intratumoral T cells that clustered with B cells near perivascular beds, forming tertiary lymphoid structures (TLS). T cells in UPS specimens were comprised of abundant CD8+ T cells exhibiting high PD-1 expression, which might represent the tumor reactive repertoire. In rhabdomyosarcomas, T cells were limited to TLS, but expressed immune checkpoints and immunomodulatory molecules which, if appropriately targeted, could help unleash T cells into the rest of the tumor tissue. CONCLUSIONS: Our work in STS revealed an immunosuppressive TME dominated by myeloid cells, which may be overcome with activation of T cells that traffic into the tumor. In rhabdomyosarcomas, targeting T cells found within TLS may be key to achieve antitumor response.

T-cell receptor signal strength and epigenetic control of Bim predict memory CD8+ T-cell fate.

Most effector CD8+ T cells die, while some persist and become either "effector" (TEM) or "central" (TCM) memory T cells. Paradoxically, effector CD8+ T cells with greater memory potential have higher levels of the pro-apoptotic molecule Bim. Here, we report, using a novel Bim-mCherry knock-in mouse, that cells with high levels of Bim preferentially develop into TCM cells. Bim levels remained stable and were regulated by DNA methylation at the Bim promoter. Notably, high levels of Bcl-2 were required for Bimhi cells to survive. Using Nur77-GFP mice as an indicator of TCR signal strength, Nur77 levels correlated with Bim expression and Nur77hi cells also selectively developed into TCM cells. Altogether, these data show that Bim levels and TCR signal strength are predictive of TEM- vs. TCM-cell fate. Further, given the many other biologic functions of Bim, these mice will have broad utility beyond CD8+ T-cell fate.

De novo DNA methylation by DNA methyltransferase 3a controls early effector CD8+ T-cell fate decisions following activation.

DNMT3a is a de novo DNA methyltransferase expressed robustly after T-cell activation that regulates plasticity of CD4(+) T-cell cytokine expression. Here we show that DNMT3a is critical for directing early CD8(+) T-cell effector and memory fate decisions. Whereas effector function of DNMT3a knockout T cells is normal, they develop more memory precursor and fewer terminal effector cells in a T-cell intrinsic manner compared with wild-type animals. Rather than increasing plasticity of differentiated effector CD8(+) T cells, loss of DNMT3a biases differentiation of early effector cells into memory precursor cells. This is attributed in part to ineffective repression of Tcf1 expression in knockout T cells, as DNMT3a localizes to the Tcf7 promoter and catalyzes its de novo methylation in early effector WT CD8(+) T cells. These data identify DNMT3a as a crucial regulator of CD8(+) early effector cell differentiation and effector versus memory fate decisions.

De novo DNA methylation is required to restrict T helper lineage plasticity.

Naïve CD4+ T cells are highly plastic and can differentiate into discrete lineages with unique functions during an immune response. Once differentiated, helper T cells maintain a stable transcriptional memory of their initial lineage choice and resist redifferentiation. During embryogenesis, de novo DNA methylation operates on the hypomethylated genome of the blastocyst to achieve tissue-specific patterns of gene expression. Similarly, the ifnγ promoter is hypomethylated in naïve T cells, but Th2, Th17, and iTreg differentiation is accompanied by substantial de novo DNA methylation at this locus. To determine whether de novo DNA methylation is required to restrict T helper lineage plasticity, we used mice with T cell-specific deletion of the methyltransferase DNMT3a. Induction of lineage-specific cytokines occurred normally in the absence of DNMT3a, however, DNMT3a-deficient Th2, Th17, and iTreg completely failed to methylate the ifnγ promoter. This was accompanied by an increase in the transcriptionally permissive trimethyl H3K4 mark, and a reduction in inhibitory H3K27 methylation at the ifnγ locus. Failed de novo methylation resulted in failed silencing of the ifnγ gene, as DNMT3a-deficient Th2, Th17, and iTreg cells produced significant levels of IFNγ following restimulation in the presence of IL-12. Therefore, DNMT3a-mediated DNA methylation restricts T helper plasticity by establishing an epigenetically silent chromatin structure at regulatory regions of the ifnγ gene.

CD8⁺ Foxp3⁺ tumor infiltrating lymphocytes accumulate in the context of an effective anti-tumor response.

The composition of tumor infiltrating lymphocytes (TIL) is heterogeneous. In addition, the ratio of various subpopulations in the tumor microenvironment is highly dependent on the nature of the host's immune response. Here, we characterize Foxp3-expressing CD8(+) T cells in the tumor that demonstrate effector function and accumulate in the context of an effective anti-tumor response. CD8(+) Foxp3(+) T cells are induced in TIL in regressing tumors of FVB/N mice treated with a GM-CSF secreting HER-2/neu targeted whole cell vaccine. Foxp3 expression in tumor antigen-specific CD8 T cells is restricted to the tumor microenvironment and influenced by cues in the tumor. Interestingly, Foxp3(+) and Foxp3(-) CD8(+) T cells have similar IFN-γ production and antigen-specific degranulation after stimulation with RNEU(420-429) , the immunodominant HER-2/neu (neu) epitope in this model. Adoptive transfer studies, using RNEU((420-429)) -specific effector T cells into neu-N mice (a model that results in immune tolerance to neu), confirm that CD8(+) Foxp3(+) T cells are present in tumors only if there is an existing pool of tumor-rejecting effector T cells. CD8(+) Foxp3(+) TILs mark the presence of tumor-rejecting antigen-specific T cells and their accumulation serves as a marker for an effective T cell response.

OX40 costimulation synergizes with GM-CSF whole-cell vaccination to overcome established CD8+ T cell tolerance to an endogenous tumor antigen.

T cell costimulation via OX40 is known to increase CD4+ T cell expansion and effector function and enhances the development of T cell memory. OX40 costimulation can also prevent, and even reverse, CD4+ T cell anergy. However, the role of OX40 in CD8+ T cell function is less well defined, particularly in the setting of immune tolerance. To determine the effects of OX40 costimulation on the induction of the host CD8+ T cell repertoire to an endogenous tumor Ag, we examined the fate of CD8+ T cells specific for the immunodominant rat HER-2/neu epitope, RNEU420-429, in FVB MMTV-neu (neu-N) mice, which express rat HER-2/neu protein in a predominantly mammary-restricted fashion. We show that the RNEU420-429-specific T cell repertoire in neu-N mice expands transiently after vaccination with a neu-targeted GM-CSF-secreting whole-cell vaccine, but quickly declines to an undetectable level. However, OX40 costimulation, when combined with GM-CSF-secreting tumor-targeted vaccination, can break established CD8+ T cell tolerance in vivo by enhancing the expansion, and prolonging the survival and effector function of CD8+ T cells specific for RNEU420-429. Moreover, we demonstrate that OX40 expression is up-regulated on both CD4+ and CD8+ T cells shortly after administration of a GM-CSF expressing vaccine. These studies highlight the increased efficacy of OX40 costimulation when combined with a GM-CSF-secreting vaccine, and define a new role for OX40 costimulation of CD8+ T cells in overcoming tolerance and boosting antitumor immunity.

Recruitment of latent pools of high-avidity CD8(+) T cells to the antitumor immune response.

A major barrier to successful antitumor vaccination is tolerance of high-avidity T cells specific to tumor antigens. In keeping with this notion, HER-2/neu (neu)-targeted vaccines, which raise strong CD8(+) T cell responses to a dominant peptide (RNEU(420-429)) in WT FVB/N mice and protect them from a neu-expressing tumor challenge, fail to do so in MMTV-neu (neu-N) transgenic mice. However, treatment of neu-N mice with vaccine and cyclophosphamide-containing chemotherapy resulted in tumor protection in a proportion of mice. This effect was specifically abrogated by the transfer of neu-N-derived CD4(+)CD25(+) T cells. RNEU(420-429)-specific CD8(+) T cells were identified only in neu-N mice given vaccine and cyclophosphamide chemotherapy which rejected tumor challenge. Tetramer-binding studies demonstrated that cyclophosphamide pretreatment allowed the activation of high-avidity RNEU(420-429)-specific CD8(+) T cells comparable to those generated from vaccinated FVB/N mice. Cyclophosphamide seemed to inhibit regulatory T (T reg) cells by selectively depleting the cycling population of CD4(+)CD25(+) T cells in neu-N mice. These findings demonstrate that neu-N mice possess latent pools of high-avidity neu-specific CD8(+) T cells that can be recruited to produce an effective antitumor response if T reg cells are blocked or removed by using approaches such as administration of cyclophosphamide before vaccination.