Hiroyuki Shimada, MD

Abstract

Tumor-associated macrophages (TAM) and cancer-associated fibroblasts (CAF) and their precursor mesenchymal stromal cells (MSC) are often detected together in tumors, but how they cooperate is not well understood. Here, we show that TAM and CAF are the most abundant nonmalignant cells and are present together in untreated human neuroblastoma (NB) tumors that are also poorly infiltrated with T and natural killer (NK) cells. We then show that MSC and CAF-MSC harvested from NB tumors protected human monocytes (MN) from spontaneous apoptosis in an interleukin (IL)-6 dependent mechanism. The interactions of MN and MSC with NB cells resulted in a significant induction or increase in the expression of several pro-tumorigenic cytokines/chemokines (TGF-1, MCP-1, IL-6, IL-8, and IL-4) but not of anti-tumorigenic cytokines (TNF-, IL-12) by MN or MSC, while also inducing cytokine expression in quiescent NB cells. We then identified a TGF-1/IL-6 pathway where TGF-1 stimulated the expression of IL-6 in NB cells and MSC, promoting TAM survival. Evidence for the contribution of TAM and MSC to the activation of this pathway was then provided in xenotransplanted NB tumors and patients with primary tumors by demonstrating a direct correlation between the presence of CAF and p-SMAD2 and p-STAT3. The data highlight a new mechanism of interaction between TAM and CAF supporting their pro-tumorigenic function in cancer.

View details for DOI 10.1080/2162402X.2022.2146860

View details for PubMedID 36479153

View details for PubMedCentralID PMC9721439

Abstract

The Warburg effect is the major metabolic hallmark of cancer. According to Warburg himself, the consequence of the Warburg effect is cell dedifferentiation. Therefore, reversing the Warburg effect might be an approach to restore cell differentiation in cancer. In this study, we used a mitochondrial uncoupler, niclosamide ethanolamine (NEN), to activate mitochondrial respiration, which induced neural differentiation in neuroblastoma cells. NEN treatment increased the nicotinamide adenine dinucleotide (NAD)+/NADH and pyruvate/lactate ratios and also the alpha-ketoglutarate (alpha-KG)/2- hydroxyglutarate (2-HG) ratio. Consequently, NEN treatment induced promoter CpG island demethylation and epigenetic landscape remodeling, activating the neural differentiation program. In addition, NEN treatment upregulated p53 but downregulated N-Myc and beta-catenin signaling in neuroblastoma cells. Importantly, even under hypoxia, NEN treatment remained effective in inhibiting 2-HG generation, promoting DNA demethylation, and suppressing hypoxia-inducible factor signaling. Dietary NEN intervention reduced tumor growth rate, 2-HG levels, and expression of N-Myc and beta-catenin in tumors in an orthotopic neuroblastoma mouse model. Integrative analysis indicated that NEN treatment upregulated favorable prognosis genes and downregulated unfavorable prognosis genes, which were defined using multiple neuroblastoma patient datasets. Altogether, these results suggest that mitochondrial uncoupling is an effective metabolic and epigenetic therapy for reversing the Warburg effect and inducing differentiation in neuroblastoma.

View details for DOI 10.1158/0008-5472.CAN-22-1029

View details for PubMedID 36318118

View details for DOI 10.1097/01.XCS.0000894496.27882.d8

View details for Web of Science ID 000867889300377

Abstract

INTRODUCTION: 123 I-MIBG scan is used in neuroblastoma (NB) to monitor treatment response. Time to resolution of 123 I-MIBG avidity after radiation therapy (RT) is unknown. We sought to determine time to resolution of 123 I-MIBG avidity after RT and local failure (LF) rate.METHODS: We performed a retrospective review of children with high-risk NB who underwent 123 I-MIBG scans pre- and post-RT from 2003 to 2019. Time from RT to resolution of 123 I-MIBG activity was analysed. LF and cumulative incidence of local progression (CILP) after RT stratified by site, presence of residual disease and use of boost RT were determined.RESULTS: Forty-two patients with median age 3.9years (1.9-4.7years) were included, with median follow-up time 3.9years (1.4-6.9). Eighty-six lesions were treated with RT to median dose of 21.6Gy. Eighteen of 86 lesions were evaluable for time to resolution of MIBG avidity after RT, with median resolution time of 78days (36-208). No LF occurred among 26 patients who received RT to primary sites after GTR, versus 4/12 (25%) patients treated with residual primary disease. 2-year CILP was 19% (12% primary disease 25% metastatic disease (P=0.18)). 2-year CILP for non-residual primary, residual primary, non-residual metastatic and residual metastatic lesions was 0%, 42%, 11% and 30% respectively (P=0.01) and for boosted and non-boosted residual lesions was 29% and 35% (P=0.44).CONCLUSION: Median time to MIBG resolution after RT was 78days. Primary lesions without residual disease had excellent local control. LF rate was higher after RT for residual disease, with no benefit for boost RT.

View details for DOI 10.1111/1754-9485.13487

View details for PubMedID 36300562

Abstract

PURPOSE: Postconsolidation immunotherapy including dinutuximab, granulocyte-macrophage colony-stimulating factor, and interleukin-2 improved outcomes for patients with high-risk neuroblastoma enrolled on the randomized portion of Children's Oncology Group study ANBL0032. After random assignment ended, all patients were assigned to immunotherapy. Survival and toxicities were assessed.PATIENTS AND METHODS: Patients with a pre-autologous stem cell transplant (ASCT) response (excluding bone marrow) of partial response or better were eligible. Demographics, stage, tumor biology, pre-ASCT response, and adverse events were summarized using descriptive statistics. Event-free survival (EFS) and overall survival (OS) from time of enrollment (up to day +200 from last ASCT) were evaluated.RESULTS: From 2009 to 2015, 1,183 patients were treated. Five-year EFS and OS for the entire cohort were 61.1 1.9% and 71.9 1.7%, respectively. For patients 18 months old at diagnosis with International Neuroblastoma Staging System stage 4 disease (n = 662) 5-year EFS and OS were 57.0 2.4% and 70.9 2.2%, respectively. EFS was superior for patients with complete response/very good partial response pre-ASCT compared with those with PR (5-year EFS: 64.2 2.2% v 55.4 3.2%, P = .0133); however, OS was not significantly different. Allergic reactions, capillary leak, fever, and hypotension were more frequent during interleukin-2-containing cycles than granulocyte-macrophage colony-stimulating factor-containing cycles (P < .0001). EFS was superior in patients with higher peak dinutuximab levels during cycle 1 (P = .034) and those with a high affinity FCGR3A genotype (P = .0418). Human antichimeric antibody status did not correlate with survival.CONCLUSION: Analysis of a cohort assigned to immunotherapy after cessation of random assignment on ANBL0032 confirmed previously described survival and toxicity outcomes. EFS was highest among patients with end-induction complete response/very good partial response. Among patients with available data, higher dinutuximab levels and FCGR3A genotype were associated with superior EFS. These may be predictive biomarkers for dinutuximab therapy.

View details for DOI 10.1200/JCO.21.02478

View details for PubMedID 35839426

View details for Web of Science ID 000863680300006

Abstract

Neuroblastoma is the most common extracranial childhood solid tumor. The majority of high-risk neuroblastoma is resistant/refractory to the current high intensity therapy. Neuroblastoma lacks classical HLA Class I expression and exhibits low mutation burden, allowing neuroblastoma cells to evade CD8+ T cell-mediated immunity. Neuroblastoma cells do not express PD-L1, and tumor-associated macrophages are the predominant PD-L1+ cells in the tumor. In this study, we performed gene expression profiling and survival analyses on large neuroblastoma datasets to address the prognostic effect of PD-L1 gene expression and the possible involvement of the SLAMF7 pathway in the anti-neuroblastoma immunity. High-level expression of PD-L1 was found significantly associated with better outcome of high-risk neuroblastoma patients; two populations of PD-1+ PD-L1+ macrophages could be present in high-risk tumors with PD-1/PD-L1 ratios, 1 and >1. Patients with the PD-1/PD-L1 ratio >1 tumor showed inferior survival. High-level co-expression of SLAMF7 and SH2D1B was significantly associated with better survival of the high-risk neuroblastoma patients. Together, this study supports the hypothesis that macrophages are important effector cells in the anti-high-risk neuroblastoma immunity, that PD-1 blockade therapy can be beneficial to the high-risk neuroblastoma subset with the PD-1/PD-L1 expression ratio >1, and that SLAMF7 is a new therapeutic target of high-risk neuroblastoma.

View details for DOI 10.1038/s41435-022-00172-w

View details for PubMedID 35525858

View details for Web of Science ID 000691614600017

Abstract

OBJECTIVE: Composite neuroblastoma is a tumor composed of multiple tumoral clones within the neuroblastoma family. To date, establishing this unique histopathologic diagnosis has required the evaluation of the primary tumor mass. We report a case of composite neuroblastoma diagnosed by evaluation of a metastatic lymph node.METHODS: One abdominal lymph node involved by tumor was evaluated in a 6-year-old boy. The primary abdominal mass was not examined. Following histopathologic examination, clonality studies using comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) were also performed.RESULTS: Two distinct tumor components were identified by histopathologic evaluation and classified as differentiating neuroblastoma (component A) and poorly differentiated neuroblastoma (component B). Based on the patient's age, each clone was further classified as Unfavorable Histology. The presence of these two different tumoral clones was confirmed by CGH and FISH.CONCLUSION: This case affirms the histopathologic approach to evaluating composite tumors, as established by the International Neuroblastoma Pathology Classification (INPC) model for ganglioneuroblastoma, nodular tumors. Also, when both components are metastatic, this case demonstrates that composite tumors can be diagnosed by the evaluation of metastatic lesions alone. Finally, it supports the addition of composite neuroblastoma to a future version of the INPC.

View details for PubMedID 34452899

Abstract

INTRODUCTION: High-risk neuroblastoma is a deadly disease; poor prognosticators are MYCN-amplification and TERT-overexpression. We hypothesized that Gene Set Enrichment Analysis (GSEA) could identify pathways associated with MYCN-amplification and that inhibition of these pathways could decrease tumor growth.METHODS: We analyzed the Neuroblastoma-Kocak dataset (GSE45547, n=649) and identified pathways associated with MYCN-amplification. Inhibitors were selected from upregulated gene sets for in vitro cytotoxicity testing using ST16-patient-derived primary neuroblastoma cells and in vivo testing using orthotopic ST16-patient-derived xenografts (PDX) in mice. Tumor volume was measured with ultrasound and tumor sections examined after H&E staining.RESULTS: GSEA identified significantly overexpressed gene sets in MYCN-amplified tumors including MYC targets, cell cycle mitotic genes, TERT associated genes, loss of RB1 gene sets, and E2Fs targets. Several genes were potential Bromodomain-containing protein 4 (Brd4) targets, making Brd4 inhibitors - JQ1, AZD5153 - and cyclin-dependent kinase (Brd4's binding partner) inhibitors - dinaciclib - potential therapeutic agents. JQ1 and dinaciclib were synergistic in inducing cytotoxicity in vitro. Dinaciclib-AZD5153 in vivo decreased tumor size compared to control, and increased tumor lymphocyte infiltration and necrosis on histology.CONCLUSIONS: GSEA is a powerful approach to identify upregulated genes and potential therapeutic targets. Dinaciclib-AZD5153 combination therapy can be effective against MYCN-amplified and TERT-overexpressing neuroblastoma tumors.

View details for DOI 10.1016/j.jpedsurg.2021.03.037

View details for PubMedID 33838899

View details for DOI 10.1016/j.pathol.2020.10.012

View details for PubMedID 33454141

Abstract

Neuroblastoma is the most common extracranial childhood solid tumor. The majority of high-risk neuroblastoma is resistant/refractory to the current high intensity therapy, and the survival of these patients remains poor for the last three decades. To effectively treat these extremely unfavorable neuroblastomas, innovative immunotherapy approaches would be the most promising. In this article, we discuss the identity of tumor-infiltrating effector cells and immunosuppressive cells in high-risk neuroblastoma. Neuroblastoma is unique in that it expresses little or no classical HLA Class I and II. In contrast, high-risk neuroblastomas express the stress-responsive non-classical Class I, HLA-E molecule. HLA-E is the ligand of activating receptors NKG2C/E that are expressed on memory NK cells, CD8+T cells and CD4 CTLs. By examining a comprehensive RNA-seq gene expression dataset, we detected relatively high levels of CD4 expression in high-risk neuroblastoma tissues. The majority of CD4+ cells were CD3+, and thus they were likely tumor-associated CD4+T cells. In addition, high-level of both CD4 and NKG2C/E expression was associated with prolonged survival of the high-risk neuroblastoma patients, but CD8 levels were not, further suggesting that the CD4+ NKG2C/E+ T cells or CD4 CTL conferred cytotoxicity against the neuroblastoma cells. However, this T cell mediated- "protective effect" declined over time, in part due to the progressive formation of immunosuppressive tumor microenvironment. These observations suggest that to improve survival of high-risk neuroblastoma patients, it is essential to gain insights into how to enhance CD4 CTL cytotoxicity and control the immunosuppressive tumor microenvironment during the course of the disease.

View details for DOI 10.3389/fimmu.2021.650427

View details for PubMedID 33968044

Abstract

131I-metaiodobenzylguanidine (MIBG) is an active radiotherapeutic for neuroblastoma. The primary aim of this trial was to identify which of three MIBG regimens was likely associated with the highest true response rate.Patients 1-30 years were eligible if they had relapsed or refractory neuroblastoma, at least one MIBG-avid site, and adequate autologous stem cells. Patients received MIBG 18 mCi/kg on day 1 and autologous stem cell on day 15. Patients randomly assigned to arm A received only MIBG; patients randomly assigned to arm B received intravenous vincristine on day 0 and irinotecan daily on days 0-4; patients randomly assigned to arm C received vorinostat (180 mg/m2/dose) orally once daily on days 1 to 12. The primary end point was response after one course by New Approaches to Neuroblastoma Therapy criteria. The trial was designed with 105 patients to ensure an 80% chance that the arm with highest response rate was selected.One hundred fourteen patients were enrolled, with three ineligible and six unevaluable, leaving 105 eligible and evaluable patients (36 in arm A, 35 in arm B, and 34 in arm C; 55 boys; and median age 6.5 years). After one course, the response rates (partial response or better) on arms A, B, and C were 14% (95% CI, 5 to 30), 14% (5 to 31), and 32% (18 to 51). An additional five, five, and four patients met New Approaches to Neuroblastoma Therapy Minor Response criteria on arms A, B, and C, respectively. On arms A, B, and C, rates of any grade 3+ nonhematologic toxicity after first course were 19%, 49%, and 35%.Vorinostat and MIBG is likely the arm with the highest true response rate, with manageable toxicity. Vincristine and irinotecan do not appear to improve the response rate to MIBG and are associated with increased toxicity.

View details for DOI 10.1200/JCO.21.00703

View details for PubMedID 34270348

View details for Web of Science ID 000582792300362

View details for Web of Science ID 000560368300241

View details for Web of Science ID 000560368300219

Abstract

MYCN amplification (MNA) is associated with poor outcomes in neuroblastoma. Less is known about heterogeneous MNA within a tumour. We compared clinical characteristics, biologic features and clinical outcomes of patients with heterogeneous MNA to patients with either homogeneous MNA or MYCN wild-type tumours.In this retrospective cohort study, we categorized patients as having tumours with MYCN wild-type, homogeneous MNA (>20% amplified tumour cells) or heterogeneous MNA (20% amplified tumour cells). We used chi-squared or Fisher's exact tests to compare features between groups. We used log-rank tests and Cox models to compare event-free survival (EFS) and overall survival (OS) between groups.MYCN status and heterogeneity status (if amplified) could be ascertained in diagnostic tumour samples from 5975 patients, including 57 (1%) with heterogeneous MNA, 981 (16.4%) with homogeneous MNA, and 4937 (82.6%) with MYCN wild-type tumours. Multiple clinical and biological features differed between patients with heterogeneous vs. homogeneous MNA, including enrichment for thoracic primary sites and paucity of 1p loss of heterozygosity with heterogeneous MNA (p<0.0001). Importantly, EFS and OS were not significantly different between patients with heterogeneous vs. homogeneous MNA. Further, EFS and OS for patients with heterogeneous MNA were significantly inferior to patients with wild-type MYCN.Although neuroblastomas with heterogeneous MNA demonstrate significantly different biological and clinical patterns compared with homogeneous MNA, prognosis is similar between the two groups. These results support current practice that treats patients with heterogeneous MNA similarly to patients with homogeneous MNA.

View details for DOI 10.1016/j.ejca.2020.04.007

View details for PubMedID 32492633

Abstract

(131)I-metaiodobenzylguanidine (MIBG) is specifically taken up in neuroblastoma, with a response rate of 20%-37% in relapsed disease. Nonradioactive carrier MIBG molecules inhibit uptake of (131)I-MIBG, theoretically resulting in less tumor radiation and increased risk of cardiovascular toxicity. Our aim was to establish the maximum tolerated dose of no-carrier-added (NCA) (131)I-MIBG, with secondary aims of assessing tumor and organ dosimetry and overall response.Eligible patients were 1-30 y old with resistant neuroblastoma, (131)I-MIBG uptake, and cryopreserved hematopoietic stem cells. A diagnostic dose of NCA (131)I-MIBG was followed by 3 dosimetry scans to assess radiation dose to critical organs and soft-tissue tumors. The treatment dose of NCA (131)I-MIBG (specific activity, 165 MBq/g) was adjusted as necessary on the basis of critical organ tolerance limits. Autologous hematopoietic stem cells were infused 14 d after therapy to abrogate prolonged myelosuppression. Response and toxicity were evaluated on day 60. The NCA (131)I-MIBG was escalated from 444 to 777 MBq/kg (12-21 mCi/kg) using a 3 + 3 design. Dose-limiting toxicity (DLT) was failure to reconstitute neutrophils to greater than 500/L within 28 d or platelets to greater than 20,000/L within 56 d, or grade 3 or 4 nonhematologic toxicity by Common Terminology Criteria for Adverse Events (version 3.0) except for predefined exclusions.Three patients each were evaluable at 444, 555, and 666 MBq/kg without DLT. The dose of 777 MBq/kg dose was not feasible because of organ dosimetry limits; however, 3 assigned patients were evaluable for a received dose of 666 MBq/kg, providing a total of 6 patients evaluable for toxicity at 666 MBq/kg without DLT. Mean whole-body radiation was 0.23 mGy/MBq, and mean organ doses were 0.92, 0.82, and 1.2 mGy/MBq of MIBG for the liver, lung, and kidney, respectively. Eight patients had 13 soft-tissue lesions with tumor-absorbed doses of 26-378 Gy. Four of 15 patients had a complete (n = 1) or partial (n = 3) response, 1 had a mixed response, 4 had stable disease, and 6 had progressive disease.NCA (131)I-MIBG with autologous peripheral blood stem cell transplantation is feasible at 666 MBq/kg without significant nonhematologic toxicity and with promising activity.

View details for DOI 10.2967/jnumed.111.098624

View details for Web of Science ID 000306164600033

View details for PubMedID 22700000

View details for Web of Science ID 000318009800569

Abstract

PURPOSE Ewing sarcoma family tumors (ESFTs) exhibit chromosomal translocations that lead to the creation of chimeric fusion oncogenes. Combinatorial diversity among chromosomal breakpoints produces varying fusions. The type 1 EWS-FLI1 transcript is created as a result of fusion between exons 7 of EWS and 6 of FLI1, and retrospective studies have reported that type 1 tumors are associated with an improved outcome. We have re-examined this association in a prospective cohort of patients with ESFT treated according to current Children's Oncology Group (COG) treatment protocols. METHODS Frozen tumor tissue was prospectively obtained from patients diagnosed with ESFT, and reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine translocation status. Analysis was confined to patients with localized tumors who were diagnosed after 1994 and treated according to COG protocols. Translocation status was correlated with disease characteristics, event-free survival (EFS), and overall survival (OS). Results RT-PCR identified chimeric fusion oncogenes in 119 of 132 ESFTs. Eighty-nine percent of identified transcripts were EWS-FLI1, and of these, 58.8% were type 1. Five-year EFS and OS rates for patients with type 1 and non-type 1 fusions diagnosed between 2001 and 2005 were equivalent (type 1: EFS, 63% +/- 7%; OS, 83% +/- 6%; non-type 1: EFS, 71% +/- 9%; OS, 79% +/- 8%). CONCLUSION Current intensive treatment protocols for localized ESFT have erased the clinical disadvantage that was formerly observed in patients with non-type 1 EWS-FLI1 fusions.

View details for DOI 10.1200/JCO.2009.24.5845

View details for Web of Science ID 000276764000006

View details for PubMedID 20308669

View details for PubMedCentralID PMC2860404

Abstract

Alveolar rhabdomyosarcomas (ARMS) are highly malignant soft-tissue sarcomas that arise in children, adolescents, and young adults. Although formation and expression of the PAX-FKHR fusion genes is thought to be the initiating event in this cancer, the role of PAX-FKHR in the neoplastic process remains largely unknown in a progenitor cell that is undefined. We hypothesize that PAX-FKHR determine the ARMS progenitor to the skeletal muscle lineage, which when coupled to the inactivation and/or activation of critical cell signaling pathways leads to the formation of ARMS. Because a number of studies have proposed that mesenchymal stem cells (MSC) are the progenitor for several of the sarcomas, we tested this hypothesis in MSCs. We show that PAX-FKHR induce skeletal myogenesis in MSCs by transactivating MyoD and myogenin. Despite exhibiting enhanced growth in vitro, the PAX-FKHR-expressing populations do not form colonies in soft agar or tumors in mice. Expression of dominant-negative p53, or the SV40 early region, elicits tumor formation in some of the PAX-FKHR-expressing populations. Additional activation of the Ras signaling pathway leads to highly malignant tumor formation for all of the populations. The PAX-FKHR-expressing tumors were shown to have histologic, immunohistochemical, and gene expression profiles similar to human ARMS. Our results show the critical role played by PAX-FKHR in determining the molecular, myogenic, and histologic phenotype of ARMS. More importantly, we identify MSCs as a progenitor that can give rise to ARMS.

View details for DOI 10.1158/0008-5472.CAN-08-0859

View details for Web of Science ID 000258548200015

View details for PubMedID 18701482

Abstract

The occurrence of rhabdomyosarcoma (RMS) primary in or metastatic to breast has been regarded as an uncommon event, associated with an unfavorable outcome. Records of 26 patients with diagnoses of breast RMS, either primary or secondary, entered in the Intergroup Rhabdomyosarcoma Study (IRS) (1972-1992) were reviewed and compared with data regarding 47 similar patients in published reports. Of the 26 IRS cases, the histologic subtype was alveolar in 24, embryonal in 1, and not determined in 1. All were female with ages ranging from 11.5 to 20.2 years (median, 15.2 years; mode, 14-16 years). This compact age distribution of both primary (n = 7) and metastatic (n = 19) breast RMS was seen in previously reported series. Among the 19 cases of RMS with initial dissemination to breast, primary tumor sites, were extremity (n = 8), nasopharynx/paranasal sinuses (n = 7), and trunk (n = 4). IRS treatment was risk-based according to site and extent of disease. Four of 7 patients with primary RMS remain disease free 2.9 to 7 years post diagnosis. Among 19 patients with RMS initially metastatic to breast, including 7 in IRS clinical group IV at original diagnosis, three are disease free at 7.6, 15.7 and 17.0 years. Conclusions: primary or metastatic RMS in breast is almost confined to adolescent females having tumors with alveolar histology. Approximately one-half of the patients with primary breast disease and 15% of those with metastatic breast disease as an initial recurrence are long-term survivors.

View details for Web of Science ID A1997XH25700004

View details for PubMedID 9212842