Management of High-Risk Neuroblastoma with Soft-Tissue-Only Disease in the Era of Anti-GD2 Immunotherapy.

Neuroblastoma presents with two patterns of disease: locoregional or systemic. The poor prognostic risk factors of locoregional neuroblastoma (LR-NB) include age, MYCN or MDM2-CDK4 amplification, 11q, histology, diploidy with ALK or TERT mutations, and ATRX aberrations. Anti-GD2 immunotherapy has significantly improved the outcome of high-risk (HR) NB and is mostly effective against osteomedullary minimal residual disease (MRD), but less so against soft tissue disease. The question is whether adding anti-GD2 monoclonal antibodies (mAbs) benefits patients with HR-NB compounded by only soft tissue. We reviewed 31 patients treated at SJD for HR-NB with no osteomedullary involvement at diagnosis. All tumors had molecular genetic features of HR-NB. The outcome after first-line treatment showed 25 (80.6%) patients achieving CR. Thirteen patients remain in continued CR, median follow-up 3.9 years. We analyzed whether adding anti-GD2 immunotherapy to first-line treatment had any prognostic significance. The EFS analysis using Cox models showed a HR of 0.20, p = 0.0054, and an 80% decrease in the risk of relapse in patients treated with anti-GD2 immunotherapy in the first line. Neither EFS nor OS were significantly different by CR status after first-line treatment. In conclusion, adding treatment with anti-GD2 mAbs at the stage of MRD helps prevent relapse that unequivocally portends poor survival.

DRI Triton SS-OCT applied to detect choroidal nodules in paediatric patients affected by NF1.

Purpose: To examine whether image processing of non-mydriatic DRI Triton SS-OCT (Topcon Corporation, Tokyo, Japan) using the red free filter could assess the presence of choroidal nodules and thus include their detection as a diagnostic criterion in neurofibromatosis type 1 (NF1). Material and methods: We included 417 eyes from 210 patients, 377 - from 190 patients diagnosed with NF1 according to the criteria established by the National Institutes of Health Consensus Development Conference (NIH) and 40 from 20 healthy patients as a control group. The mean age was 9.4 years (range 2 years-18 years). All patients had their visual acuity measured by a test according to age, were examined for the presence of lisch nodules and an Optical Coherence Tomography (OCT) of the macular area was performed. All the OCT images were analysed to check if visible nodules could be identified. Results: Ages 14 (95% CI=(9.7,18.3)) and 12 years (95% CI=(9.1,14)) are the cut-off points that best separate those with choroidal nodules with Triton OCT and lisch with slit lamp, respectively, from those without. lisch nodules were detected in 50% of cases of NF1 patients. The presence of choroidal nodules did not present a statistically significant correlation with the occurrence of optic pathway glioma (p = 0.96) nor with the patient's visual worsening (p = 0.072). A statistically significant correlation was observed between the presence of choroidal nodules and the presence of lisch nodules (p < 0.05). Conclusion: The Topcon Triton OCT red free tool would not be a good tool to detect choroidal nodules in patients with NF1 because of its low sensitivity. If the presence of choroidal nodules were to be included in the diagnostic criteria for NF1, it would be convenient to use a device with red and infrared radiations.

Vitamin D Status in Children With High-risk Neuroblastoma.

BACKGROUND: Vitamin D deficiency has become a matter of concern in pediatric cancer patients. A relationship between neuroblastoma and Vitamin D signaling pathways has been revealed with interest in the antiproliferative and antiinvasive properties of vitamin D. Our aim is to describe the prevalence of Vitamin D deficiency among children with high-risk neuroblastoma (HR-NB) and to explore its association with disease status. MATERIALS AND METHODS: In all, 182 patients with HR-NB were managed at our center from 2017 to 2021. Serum 25(OH)D levels were tested at the first blood analysis performed and correlated with clinical data and disease status. RESULTS: One hundred forty-eight (81.4%) had low 25(OH)D levels (48.4% categorized as deficiency (25(OH)D below 20 ng/mL) and 33.0% as insufficiency (25(OH)D 20 to 30 ng/mL). Median Vitamin D level was 20.2 ng/mL. Vitamin D levels were not associated with race or sex. Although malnourished patients had lower median 25(OH)D levels(11.1 ng/mL), no statistical association was observed with Vitamin D deficiency. There was no association between Vitamin D levels and disease status. An inverse correlation was found between age and vitamin D levels ( P =0.0040). CONCLUSION: A concerning high prevalence of low Vitamin D levels affects more than two-thirds of patients with HR-NB in our cohort, regardless of the disease status at the time of evaluation. Older children are at a higher risk for deficient levels of vitamin D.

Early Salvage Chemo-Immunotherapy with Irinotecan, Temozolomide and Naxitamab Plus GM-CSF (HITS) for Patients with Primary Refractory High-Risk Neuroblastoma Provide the Best Chance for Long-Term Outcomes.

Patients with high-risk neuroblastoma (HR-NB) who are unable to achieve a complete response (CR) to induction therapy have worse outcomes. We investigated the combination of humanized anti-GD2 mAb naxitamab (Hu3F8), irinotecan (I), temozolomide (T), and sargramostim (GM-CSF)-HITS-against primary resistant HR-NB. Eligibility criteria included having a measurable chemo-resistant disease at the end of induction (EOI) treatment. Patients were excluded if they had progressive disease (PD) during induction. Prior anti-GD2 mAb and/or I/T therapy was permitted. Each cycle, administered four weeks apart, comprised Irinotecan 50 mg/m2/day intravenously (IV) plus Temozolomide 150 mg/m2/day orally (days 1-5); naxitamab 2.25 mg/kg/day IV on days 2, 4, 8 and 10, (total 9 mg/kg or 270 mg/m2 per cycle), and GM-CSF 250 mg/m2/day subcutaneously was used (days 6-10). Toxicity was measured using CTCAE v4.0 and responses through the modified International Neuroblastoma Response Criteria (INRC). Thirty-four patients (median age at treatment initiation, 4.9 years) received 164 (median 4; 1-12) HITS cycles. Toxicities included myelosuppression and diarrhea, which was expected with I/T, and pain and hypertension, expected with naxitamab. Grade ≥3-related toxicities occurred in 29 (85%) of the 34 patients; treatment was outpatient. The best responses were CR = 29% (n = 10); PR = 3% (n = 1); SD = 53% (n = 18); PD = 5% (n = 5). For cohort 1 (early treatment), the best responses were CR = 47% (n = 8) and SD = 53% (n = 9). In cohort 2 (late treatment), the best responses were CR = 12% (n = 2); PR = 6% (n = 1); SD = 53% (n = 9); and PD = 29% (n = 5). Cohort 1 had a 3-year OS of 84.8% and EFS 54.4%, which are statistically significant improvements (EFS p = 0.0041 and OS p = 0.0037) compared to cohort 2. In conclusion, naxitamab-based chemo-immunotherapy is effective against primary chemo-resistant HR-NB, increasing long-term outcomes when administered early during the course of treatment.

EpiGe: A machine-learning strategy for rapid classification of medulloblastoma using PCR-based methyl-genotyping.

Molecular classification of medulloblastoma is critical for the treatment of this brain tumor. Array-based DNA methylation profiling has emerged as a powerful approach for brain tumor classification. However, this technology is currently not widely available. We present a machine-learning decision support system (DSS) that enables the classification of the principal molecular groups-WNT, SHH, and non-WNT/non-SHH-directly from quantitative PCR (qPCR) data. We propose a framework where the developed DSS appears as a user-friendly web-application-EpiGe-App-that enables automated interpretation of qPCR methylation data and subsequent molecular group prediction. The basis of our classification strategy is a previously validated six-cytosine signature with subgroup-specific methylation profiles. This reduced set of markers enabled us to develop a methyl-genotyping assay capable of determining the methylation status of cytosines using qPCR instruments. This study provides a comprehensive approach for rapid classification of clinically relevant medulloblastoma groups, using readily accessible equipment and an easy-to-use web-application.t.

Naxitamab Combined with Granulocyte-Macrophage Colony-Stimulating Factor as Consolidation for High-Risk Neuroblastoma Patients in First Complete Remission under Compassionate Use-Updated Outcome Report.

Naxitamab is an anti-GD2 antibody approved for the treatment of relapsed/refractory HR-NB. We report the survival, safety, and relapse pattern of a unique set of HR-NB patients consolidated with naxitamab after having achieved first CR. Eighty-two patients were treated with 5 cycles of GM-CSF for 5 days at 250 µg/m2/day (-4 to 0), followed by GM-CSF for 5 days at 500 µg/m2/day (1-5) and naxitamab at 3 mg/kg/day (1, 3, 5), on an outpatient basis. All patients but one were older than 18 months at diagnosis and had stage M; 21 (25.6%) pts had MYCN-amplified (A) NB; and 12 (14.6%) detectable MRD in the BM. Eleven (13.4%) pts had received high-dose chemotherapy and ASCT and 26 (31.7%) radiotherapy before immunotherapy. With a median follow-up of 37.4 months, 31 (37.8%) pts have relapsed. The pattern of relapse was predominantly (77.4%) an isolated organ. Five-year EFS and OS were 57.9% (71.4% for MYCN A) 95% CI = (47.2, 70.9%); and 78.6% (81% for MYCN A) 95% CI = (68.7%, 89.8%), respectively. EFS showed significant differences for patients having received ASCT (p = 0.037) and pre-immunotherapy MRD (p = 0.0011). Cox models showed only MRD as a predictor of EFS. In conclusion, consolidation with naxitamab resulted in reassuring survival rates for HR-NB patients after end-induction CR.

Novel infusion strategy reduces severe adverse events caused by the anti-GD2 monoclonal antibody naxitamab.

Introduction: Anti-disialoganglioside 2 (anti-GD2) monoclonal antibodies (mAbs) are associated with Grade ≥3 (≥G3) adverse events (AEs) such as severe pain, hypotension, and bronchospasm. We developed a novel method of administering the GD2-binding mAb naxitamab, termed "Step-Up" infusion (STU), to reduce the risk of AEs of severe pain, hypotension, and bronchospasm. Methods: Forty-two patients with GD2-positive tumors received naxitamab under "compassionate use" protocols and administered via either the standard infusion regimen (SIR) or the STU regimen. The SIR comprises a 60-min infusion of 3 mg/kg/day on Day 1 of cycle 1 and a 30- to 60-min infusion on Day 3 and Day 5, as tolerated. The STU regimen uses a 2-h infusion on Day 1, initiated at a rate of 0.06 mg/kg/h during 15 min (0.015 mg/kg) and which increases gradually to a cumulative dose of 3 mg/kg; on Days 3 and 5, the 3-mg/kg dose is initiated at 0.24 mg/kg/h (0.06 mg/kg) and delivered in 90 min according to the same gradual-increase strategy. AEs were graded according to Common Terminology Criteria for Adverse Events version 4.0. Results: The frequency of infusions with an associated G3 AE was reduced from 8.1% (23/284 infusions) with SIR to 2.5% (5/202 infusions) with STU. The odds of an infusion being associated with a G3 AE reduced by 70.3% with STU vs. SIR (odds ratio: 0.297; p = 0.037). Mean serum naxitamab levels pre- and post-STU (11.46 µg/ml pre-infusion; 100.95 µg/ml post-infusion) were within the range reported for SIR. Discussion: The comparable pharmacokinetics of naxitamab during SIR and STU may indicate that switching to STU reduces G3 AEs without impact on efficacy.

Prognostic value of xenograft engraftment in patients with metastatic high-risk neuroblastoma.

BACKGROUND: Successful engraftment of human cancer biopsies in immunodeficient mice correlates with the poor prognosis of patients. This was reported 30 years ago for children with neuroblastoma, but the standard of care treatment evolved significantly during the last 15 years, leading to improved survival of these patients. Here, we evaluated the association of patient-derived xenograft (PDX) engraftment and prognosis in patients receiving up-to-date treatments for cancers classified as metastatic (stage M) high-risk neuroblastoma (HR-NB) by the International Neuroblastoma Risk Group Staging System (INRGSS). METHODS: We obtained biopsies from patients with stage M HR-NB. We inoculated biopsy fragments subcutaneously in mice. We studied the association of PDX engraftment with event-free survival (EFS) and overall survival (OS) of patients. RESULTS: Since 2009, we established 17 PDX from 97 samples of 66 patients with stage M HR-NB, with a follow-up of at least two years. Factors associated with higher probability of engraftment were the death as outcome (p = .0006) and the amplification of the gene MYCN in tumors (p = .0271). Patients whose biopsies established a PDX had significantly shorter EFS and OS (p = .0039 and .0002, respectively) than patients whose samples did not engraft. The association of PDX engraftment and OS was significant in patients without MYCN amplification (p = .0041), but not in patients with MYCN amplification (p = .2707). CONCLUSION: Positive PDX engraftment is a factor related to poor prognosis and fatal outcome in patients with stage M HR-NB treated with up-to-date therapies.

A miRNA signature related to stemness identifies high-risk patients in paediatric acute myeloid leukaemia.

Clinical and biological variables like genetic aberrations at diagnosis and the levels of measurable residual disease (MRD) are the most powerful biomarkers to predict the outcome of paediatric leukaemia. Recently, a model integrating the genetic abnormalities, transcriptional identity, and leukaemia stemness measured as leukaemic stem cell score (pLSC6) has been proposed to identify high-risk paediatric acute myeloid leukaemia (AML) patients. However, the role of epigenetics in defining prognosis still needs to be established. We evaluated the role of 89 miRNAs regulating stemness and their contribution to predicting outcomes in 110 paediatric patients with acute leukaemia. We identified a 24-miRNA signature capable of distinguishing paediatric AML patients with excellent or poor outcomes. We validated these results in an independent cohort using public repository-based data. The 24-miRNA signature was significantly associated with the leukaemic stemness scores and the underlying genetics of patients. Notably, the combination of classical prognostic factors (MRD and genetics), the pLSC6 score and the 24-miRNA signature had a higher capacity to predict the overall and event-free survival than each variable individually. Our 24-miRNA signature provides epigenetic data to integrate into genetics, MRD and stemness-related leukaemic scores to refine risk stratification in paediatric AML patients.

Low Bcl-2 is a robust biomarker of sensitivity to nab-paclitaxel in Ewing sarcoma.

Nanoparticle albumin-bound paclitaxel (nab-paclitaxel) shows potent preclinical anticancer activity in pediatric solid tumors such as Ewing sarcoma, rhabdomyosarcoma and neuroblastoma, but responses in clinical trials have been modest. In this work, we aimed to discover a rational biomarker-based approach to select the right candidate patients for this treatment. We assessed the efficacy of nab-paclitaxel in 27 patient-derived xenografts (PDX), including 14 Ewing sarcomas, five rhabdomyosarcomas and several other pediatric solid tumors. Response rate (partial or complete response) was remarkable in rhabdomyosarcomas (four of five) and Ewing sarcomas (four of 14). We addressed several predictive factors of response to nab-paclitaxel such as the expression of the secreted protein acidic and rich in cysteine (SPARC), chromosomal stability of cancer cells and expression of antiapoptotic members of the B-cell lymphoma-2 (Bcl-2) family of proteins such as Bcl-2, Bcl-xL, Bcl-W and Mcl-1. Protein (immunoblotting) and gene expression of SPARC correlated positively, while immunoblotting and immunohistochemistry expression of Bcl-2 correlated negatively with the efficacy of nab-paclitaxel in Ewing sarcoma PDX. The negative correlation of Bcl-2 immunoblotting signal and activity was especially robust (r = 0.8352; P = 0.0007; Pearson correlation). Consequently, we evaluated pharmacological strategies to inhibit Bcl-2 during nab-paclitaxel treatment. We observed that the Bcl-2 inhibitor venetoclax improved the activity of nab-paclitaxel in highly resistant Bcl-2-expressing Ewing sarcoma PDX. Overall, our results suggest that low Bcl-2 expression could be used to select patients with Ewing sarcoma sensitive to nab-paclitaxel, and Bcl-2 inhibitors could improve the activity of this drug in Bcl-2-expressing Ewing sarcoma.

Follow-up of intraocular retinoblastoma through the quantitative analysis of conserved nuclear DNA sequences in aqueous humor from patients.

Fundoscopy is the standard method for diagnosis and follow-up of intraocular retinoblastoma, but it is sometimes insufficient to discern whether tumors are inactivated following treatments. In this work, we hypothesized that the amount of conserved nuclear DNA sequences in the cell-free DNA (cfDNA) fraction of the aqueous humor (AH) might complement fundoscopy for retinoblastoma follow-up. To address our hypothesis, we developed highly sensitive droplet digital polymerase chain reaction (ddPCR) methods to quantify highly conserved DNA sequences of nucleus-encoded genes (GAPDH and B4GALNT1) and of a mitochondrial gene, MT-ATP6. We obtained AH samples during intravitreal treatments. We analyzed 42 AH samples from 25 patients with intraocular retinoblastoma and 11 AH from controls (non-cancer patients). According to clinical criteria, we grouped patients as having progression-free or progressive retinoblastoma. cfDNA concentration in the AH was similar in both retinoblastoma groups. Copy counts for nucleus-derived sequences of GAPDH and B4GALNT1 were significantly higher in the AH from patients with progressive disease, compared to the AH from progression-free patients and control non-cancer patients. The presence of mitochondrial DNA in the AH explained that both retinoblastoma groups had similar cfDNA concentration in AH. The optimal cut-off point for discriminating between progressive and progression-free retinoblastomas was 108 GAPDH copies per reaction. Among patients having serial AH samples analyzed during their intravitreal chemotherapy, GAPDH copies were high and decreased below the cut-off point in those patients responding to chemotherapy. In contrast, one non-responder patient remained with values above the cut-off during follow-up, until enucleation. We conclude that the measurement of conserved nuclear gene sequences in AH allows follow-up of intraocular retinoblastoma during intravitreal treatment. The method is applicable to all patients and could be relevant for those in which fundoscopy evaluation is inconclusive.

MIF/CXCR4 signaling axis contributes to survival, invasion, and drug resistance of metastatic neuroblastoma cells in the bone marrow microenvironment.

BACKGROUND: The bone marrow (BM) is the most common site of dissemination in patients with aggressive, metastatic neuroblastoma (NB). However, the molecular mechanisms underlying the aggressive behavior of NB cells in the BM niche are still greatly unknown. In the present study, we explored biological mechanisms that play a critical role in NB cell survival and progression in the BM and investigated potential therapeutic targets. METHODS: Patient-derived bone marrow (BM) primary cultures were generated using fresh BM aspirates obtained from NB patients. NB cell lines were cultured in the presence of BM conditioned media containing cell-secreted factors, and under low oxygen levels (1% O2) to mimic specific features of the BM microenvironment of high-risk NB patients. The BM niche was explored using cytokine profiling assays, cell migration-invasion and viability assays, flow cytometry and analysis of RNA-sequencing data. Selective pharmacological inhibition of factors identified as potential mediators of NB progression within the BM niche was performed in vitro and in vivo. RESULTS: We identified macrophage migration inhibitory factor (MIF) as a key inflammatory cytokine involved in BM infiltration. Cytokine profiling and RNA-sequencing data analysis revealed NB cells as the main source of MIF in the BM, suggesting a potential role of MIF in tumor invasion. Exposure of NB cells to BM-conditions increased NB cell-surface expression of the MIF receptor CXCR4, which was associated with increased cell viability, enhanced migration-invasion, and activation of PI3K/AKT and MAPK/ERK signaling pathways. Moreover, subcutaneous co-injection of NB and BM cells enhanced tumor engraftment in mice. MIF inhibition with 4-IPP impaired in vitro NB aggressiveness, and improved drug response while delayed NB growth, improving survival of the NB xenograft model. CONCLUSIONS: Our findings suggest that BM infiltration by NB cells may be mediated, in part, by MIF-CXCR4 signaling. We demonstrate the antitumor efficacy of MIF targeting in vitro and in vivo that could represent a novel therapeutic target for patients with disseminated high-risk NB.

Identification of immunosuppressive factors in retinoblastoma cell secretomes and aqueous humor from patients.

The microenvironment of retinoblastoma, the solid malignancy of the developing retina, is immunosuppressive. To study the interactions between tumor-associated microglia/macrophages (TAMs) and tumor cells in retinoblastomas, we analyzed immunohistochemistry markers in 23 patient samples and characterized 105 secreted cytokines of 11 retinoblastoma cell models in culture. We detected profuse infiltration of CD163+ protumoral M2-like polarized TAMs in eyes enucleated due to cancer progression. Previous treatment of patients increased the number of TAMs but did not affect M2-like polarization. M2-like microglia/macrophages were almost absent in five eyes obtained from children enucleated due to nontumoral causes. CD8+ tumor-infiltrating lymphocytes (TILs) were moderately abundant in tumor eyes and very scarce in nontumoral ones. The expression of the immune checkpoint molecule PD-L1 was absent in 95% of the tumor samples, which is concordant with the finding of FOXP3+ Tregs infiltrating tumors. We confirmed the pathology results using single-cell transcriptome analysis of one tumor. We identified the cytokines extracellular matrix metalloproteinase inducer (EMMPRIN) and macrophage migration inhibitory factor (MIF), both with reported immunosuppressive activity, secreted at high levels in retinoblastoma primary cell cultures. Gene expression analysis of a large retinoblastoma cohort and single-cell transcriptome analysis confirmed that MIF and EMMPRIN were significantly upregulated in retinoblastomas, which led us to quantify both proteins by immunoassays in liquid biopsies (aqueous humor obtained from more than 20 retinoblastoma patients). We found a significant increase in the concentration of MIF and EMMPRIN in cancer patients, compared to 12 noncancer ones. Finally, we showed that macrophages derived from peripheral blood mononuclear cells increased the expression of markers of M2-like polarization upon exposure to retinoblastoma-conditioned medium or recombinant MIF. Overall, our findings suggest that retinoblastoma cell secretions induce the protumoral phenotype of this tumor. Our results might have clinical impact in the fields of biomarkers and treatment. © 2022 The Pathological Society of Great Britain and Ireland.

Lower incidence of clinical allergy with PEG-asparaginase upfront versus the sequential use of native E. coli asparaginase followed by PEG-ASP in pediatric patients with acute lymphoblastic leukemia.

Asparaginase (ASP) is an essential component for the acute lymphoblastic leukemia (ALL) treatment, but toxicities, such as allergy, frequently limit its use. Although the potentially lower PEG-ASP formulation immunogenicity, few studies with conflicting results have compared the allergy incidence between Escherichia coli-ASP and PEG-ASP in the same protocol. We aimed at comparing the allergy incidence in children receiving native E. coli-ASP versus PEG-ASP within the same clinical protocol (Spanish Society of Pediatric Hematology and Oncology ALL-SEHOP-PETHEMA 2013). One hundred and twenty-six children (1-19 years) diagnosed with ALL from 2013 to 2020 were included. Patients in group 1 received a sequential scheme of native E. coli-ASP 10,000 IU/m2 intramuscularly (IM) followed by PEG-ASP 1000 IU/m2 IM. Patients in group 2 received PEG-ASP 1000 IU/m2 IM upfront. Clinical allergy incidence was compared between both groups. Serum ASP activity (SAA) was measured in a subgroup of patients, and silent inactivation was recorded. The cumulative incidence of clinical allergy was significantly higher in group 1 (native followed by PEG-ASP) than in group 2 (PEG-ASP upfront), 24.7% versus 4.1% (p = 0.0085). Adequate ASP activity was achieved with PEG-ASP 1000 IU/m2 dose in most patients (median SAA 412.5 and 453.0 IU/L at days 7 and 14). The incidence of silent inactivation in PEG-ASP upfront patients was very low. PEG-ASP-used upfront was associated with a lower incidence of clinical allergy than that observed in the sequential use of native E. coli-ASP followed by PEG-ASP. PEG-ASP at 1000 IU/m2 was effective in achieving enough ASP activity in most patients.

Naxitamab combined with granulocyte-macrophage colony-stimulating factor as consolidation for high-risk neuroblastoma patients in complete remission.

BACKGROUND: Naxitamab is a humanized anti-disialoganglioside (GD2) monoclonal antibody approved for treatment of bone/bone marrow refractory high-risk neuroblastoma (HR-NB). Compassionate use (CU) expanded access program at Hospital Sant Joan de Deu permitted treatment of patients in complete remission (CR). We here report the survival, toxicity, and relapse pattern of patients in first or second CR treated with naxitamab and sargramostim (GM-CSF). PROCEDURE: Seventy-three consecutive patients with HR-NB (stage M at age >18 months or MYCN-amplified stages L1/L2 at any age) were treated in first or second CR. Treatment comprised five cycles of subcutaneous (SC) GM-CSF for 5 days at 250 µg/m2 /day (days -4 to 0), followed by naxitamab + SC GM-CSF for 5 days at 500 µg/m2 /day (days 1-5). Naxitamab was infused over 30 minutes at 3 mg/kg/day, days 1, 3, and 5, outpatient. RESULTS: Fifty-five patients were in first CR and 18 in second CR. Seventeen patients had MYCN-amplified NB and 11 detectable minimal residual disease in the bone marrow. Fifty-eight (79.5%) patients completed therapy. Four (5%) experienced grade 4 toxicities and 10 (14%) early relapse. Three-year event-free survival (EFS) 58.4%, 95% CI = (43.5%, 78.4%) and overall survival (OS) 82.4%, 95% CI = (66.8%, 100%). First CR patients 3-year EFS 74.3%, 95% CI = (62.7%, 88.1%), and OS 91.6%, 95% CI = (82.4%, 100%). EFS is significantly different between first and second CR (p = .0029). The pattern of relapse is predominantly (75%) of an isolated organ, mainly bone (54%). Univariate Cox models show prior history of relapse as the only statistically significant predictor of EFS but not OS. CONCLUSIONS: Consolidation with naxitamab and GM-CSF resulted in excellent survival rates for HR-NB patients in CR.

Prognostic value of patient-derived xenograft engraftment in pediatric sarcomas.

The goals of this work were to identify factors favoring patient-derived xenograft (PDX) engraftment and study the association between PDX engraftment and prognosis in pediatric patients with Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma. We used immunodeficient mice to establish 30 subcutaneous PDX from patient tumor biopsies, with a successful engraftment rate of 44%. Age greater than 12 years and relapsed disease were patient factors associated with higher engraftment rate. Tumor type and biopsy location did not associate with engraftment. PDX models retained histology markers and most chromosomal aberrations of patient samples during successive passages in mice. Model treatment with irinotecan resulted in significant activity in 20 of the PDXs and replicated the response of rhabdomyosarcoma patients. Successive generations of PDXs responded similarly to irinotecan, demonstrating functional stability of these models. Importantly, out of 68 tumor samples from 51 patients with a median follow-up of 21.2 months, PDX engraftment from newly diagnosed patients was a prognostic factor significantly associated with poor outcome (p = 0.040). This association was not significant for relapsed patients. In the subgroup of patients with newly diagnosed Ewing sarcoma classified as standard risk, we found higher risk of relapse or refractory disease associated with those samples that produced stable PDX models (p = 0.0357). Overall, our study shows that PDX engraftment predicts worse outcome in newly diagnosed pediatric sarcoma patients.

Clinical and Pathological Evidence of Anti-GD2 Immunotherapy Induced Differentiation in Relapsed/Refractory High-Risk Neuroblastoma.

BACKGROUND: Neuroblastic tumors (NBTs) originate from a block in the process of differentiation. Histologically, NBTs are classified in neuroblastoma (NB), ganglioneuroblastoma (GNB), and ganglioneuroma (GN). Current therapy for high-risk (HR) NB includes chemotherapy, surgery, radiotherapy, and anti-GD2 monoclonal antibodies (mAbs). Anti-GD2 mAbs induce immunological cytoxicity but also direct cell death. METHODS: We report on patients treated with naxitamab for chemorefractory NB showing lesions with long periods of stable disease. Target lesions with persisting 123I-Metaiodobenzylguanidine (MIBG) uptake after 4 cycles of immunotherapy were further evaluated by functional Magnetic Resonance Imaging (MRI) and/or Fluorodeoxyglucose (FDG)-positron emission tomography (PET). MIBG avid lesions that became non-restrictive on MRI (apparent diffusion coefficient (ADC) > 1) and/or FDG-PET negative (SUV < 2) were biopsied. RESULTS: Twenty-seven relapse/refractory (R/R) HR-NB patients were enrolled on protocol Ymabs 201. Two (7.5%) of the 27 showed persistent bone lesions on MIBG, ADC high, and/or FDG-PET negative. Forty-four R/R HR-NB patients received chemo-immunotherapy. Twelve (27%) of the 44 developed persistent MIBG+ but FDG-PET- and/or high ADC lesions. Twelve (86%) of the 14 cases identified were successfully biopsied producing 16 evaluable samples. Histology showed ganglioneuroma maturing subtype in 6 (37.5%); ganglioneuroma mature subtype with no neuroblastic component in 4 (25%); differentiating NB with no Schwannian stroma in 5 (31%); and undifferentiated NB without Schwannian stroma in one (6%). Overall, 10 (62.5%) of the 16 specimens were histopathologically fully mature NBTs. CONCLUSIONS: Our results disclose an undescribed mechanism of action for naxitamab and highlight the limitations of conventional imaging in the evaluation of anti-GD2 immunotherapy clinical efficacy for HR-NB.

The Role of Autologous Stem-Cell Transplantation in High-Risk Neuroblastoma Consolidated by anti-GD2 Immunotherapy. Results of Two Consecutive Studies.

Background: Treatment of HR-NB comprise induction, consolidation with autologous stem cell transplant (ASCT) followed by anti-GD2 immunotherapy and isotretinoin. Childrens Oncology Group and SIOPEN studies used dinutuximab and cytokines to treat patients in complete remission or refractory Bone/Bone marrow (B/BM) disease after ASCT. Methods: HR-NB patients referred to Hospital Sant Joan de Déu for anti-GD2 immunotherapy were eligible for two consecutive studies (dinutuximab for EudraCT 2013-004864-69 and naxitamab for 017-001829-40) and naxitamab/Sargramostim CU with or without prior ASCT. Patients enrolled in first complete remission or with primary refractory B/BM disease. We accrued a study population of two groups whose therapy, aside from ASCT, was similar. This is a retrospective analysis of their outcome calculated from study entry. Results: From December 2014-2019, 67 patients were treated with dinutuximab and cytokines (n = 21) in the Hospital Sant Joan de Déu-HRNB-Ch14.18 study or with naxitamab and Sargramostim either in the Ymabs study 201 (n = 12) or CU (n = 34). 23 patients were treated with primary refractory disease in the B/BM (11 with dinutuximab and 12 with naxitamab), and 44 in first CR (10 with dinutuximab and 34 with naxitamab). Study patients included 13 (19.4%) treated following single ASCT and 54 following conventional chemotherapy. Median follow-up for all patients is 16.2 months. Two-year rates for ASCT and non-ASCT patients were, respectively, EFS 64.1% vs. 54.2% (p = 0.28), and OS 66.7% vs. 84.1% (p = 0.81). For the 44 pts in first CR, 2-years rates for ASCT and non-ASCT patients were, respectively, EFS 65.5% vs. 58.7% (p = 0.48), and OS 71.4% vs. 85.4% (p = 0.63). Conclusions: In this retrospective, single center study, ASCT did not provide survival benefit when anti-GD2 immunotherapy was used after induction chemotherapy.

Treatment-driven selection of chemoresistant Ewing sarcoma tumors with limited drug distribution.

Ewing sarcoma is a bone and soft tissue tumor predominantly affecting adolescents and young adults. To characterize changes in anticancer drug activity and intratumor drug distribution during the evolution of Ewing sarcomas, we used immunodeficient mice to establish pairs of patient-derived xenografts (PDX) at early (initial diagnosis) and late (relapse or refractory progression) stages of the disease from three patients. Analysis of copy number alterations (CNA) in early passage PDX tissues showed that two tumor pairs established from patients which responded initially to therapy and relapsed more than one year later displayed similar CNAs at early and late stages. For these two patients, PDX established from late tumors were more resistant to chemotherapy (irinotecan) than early counterparts. In contrast, the tumor pair established at refractory progression showed highly dissimilar CNA profiles, and the pattern of response to chemotherapy was discordant with those of relapsed cases. In mice receiving irinotecan infusions, the level of SN-38 (active metabolite of irinotecan) in the intracellular tumor compartment was reduced in tumors at later stages compared to earlier tumors for those pairs bearing similar CNAs, suggesting that distribution of anticancer drug shifted toward the extracellular compartment during clonal tumor evolution. Overexpression of the drug transporter P-glycoprotein in late tumor was likely responsible for this shift in drug distribution in one of the cases.