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

An extensive quality control and quality assurance (QC/QA) program significantly improves inter-laboratory concordance rates of flow-cytometric minimal residual disease assessment in acute lymphoblastic leukemia: An I-BFM-FLOW-network report

Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts

Risk factors in pediatric T-cell acute lymphoblastic leukemia: a collaborative study of the biological committee of the leukemia group of the Spanish Hematology and Oncology Pediatric Society

Background: Useful biomarkers for risk stratification in pediatric T-ALL are still needed. Several biological variables have been proposed as new prognostic biomarkers, including NOTCH1, FBXW7, PTEN & RAS mutations, expression of myeloid antigens and presence of copy number alterations (CNAs) of certain genes, but published results are controversial. Aims: To analyze the prognostic impact of mutations, CNAs and myeloid antigens expression in a series of 56 pediatric T-ALL patients.N

CD34+CD19-CD22+ B-cell progenitors might underlie phenotypic escape in patients treated with CD19-directed therapies

CD19-directed immunotherapies have revolutionized the treatment of advanced B-ALL. Despite initial impressive rates of complete remission (CR) many patients ultimately relapse. B-ALL patients successfully treated with CD19-directed T-cells eventually relapse, which coupled with the early onset of CD22 expression during B-cell development suggests that pre-existing CD34+CD22+CD19- (pre)-leukemic cells could represent an "early progenitor origin-related" mechanism underlying phenotypic escape to CD19-directed immunotherapies. We demonstrate that CD22 expression precedes CD19 expression during B-cell development. CD34+CD19-CD22+ cells are found in diagnostic and relapsed BM samples of ~70% B-ALL patients, and their frequency increases 2-fold in B-ALL patients in CR after CD19-CAR T-cell therapy. The median of CD34+CD19-CD22+ cells before treatment was 3-fold higher in B-ALL patients who relapse after CD19-directed immunotherapy (median follow-up of 24 months). FISH analysis in flow-sorted populations and xenograft modeling revealed that CD34+CD19-CD22+ cells harbor the genetic abnormalities present at diagnosis and initiate leukemogenesis in vivo. Our data suggest that pre-leukemic CD34+CD19-CD22+ progenitors may underlie phenotypic escape after CD19-directed immunotherapies and reinforce ongoing clinical studies aimed at CD19/CD22 dual-targeting as a strategy to reduce CD19- relapses. The implementation of such CD34/CD19/CD22 immunophenotyping in clinical laboratories for initial diagnosis and subsequent monitoring of B-ALL patients during CD19-targeted therapy is encouraged

Increased delivery of chemotherapy to the vitreous by inhibition of the blood-retinal barrier

Treatment of retinoblastoma -a pediatric cancer of the developing retina- might benefit from strategies to inhibit the blood-retinal barrier (BRB). The potent anticancer agent topotecan is a substrate of efflux transporters BCRP and P-gp, which are expressed at the BRB to restrict vitreous and retinal distribution of xenobiotics. In this work we have studied vitreous and retinal distribution, tumor accumulation and antitumor activity of topotecan, using pantoprazole as inhibitor of BCRP and P-gp. We used rabbit and mouse eyes as BRB models and patient-derived xenografts as retinoblastoma models. To validate the rabbit BRB model we stained BCRP and P-gp in the retinal vessels. Using intravitreous microdialysis we showed that the penetration of the rabbit vitreous by lactone topotecan increased significantly upon concomitant administration of pantoprazole (P = 0.0285). Pantoprazole also increased topotecan penetration of the mouse vitreous, measured as the vitreous-to-plasma topotecan concentration ratio at the steady state (P = 0.0246). Pantoprazole increased topotecan antitumor efficacy and intracellular penetration in retinoblastoma in vitro, but did not enhance intratumor drug distribution and survival in mice bearing the intraocular human tumor HSJD-RBT-2. Anatomical differences with the clinical setting likely limited our in vivo study, since xenografts were poorly vascularized masses that loaded most of the vitreous compartment. We conclude that pharmacological modulation of the BRB is feasible, enhances anticancer drug distribution into the vitreous and might have clinical implications in retinoblastoma.Fil: Pascual-Pasto, Guillem. Hospital Sant Joan de Deu Barcelona; España. Institut de Recerca Sant Joan de Deu; EspañaFil: Olaciregui, Nagore G.. Hospital Sant Joan de Deu Barcelona; España. Institut de Recerca Sant Joan de Deu; EspañaFil: Opezzo, Javier A. W.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología; ArgentinaFil: Castillo Ecija, Helena. Hospital Sant Joan de Deu Barcelona; España. Institut de Recerca Sant Joan de Deu; EspañaFil: Cuadrado Vilanova, Maria. Hospital Sant Joan de Deu Barcelona; España. Institut de Recerca Sant Joan de Deu; EspañaFil: Paco, Sonia. Hospital Sant Joan de Deu Barcelona; España. Institut de Recerca Sant Joan de Deu; EspañaFil: Rivero, Ezequiel Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Vila Ubach, Monica. Hospital Sant Joan de Deu Barcelona; España. Institut de Recerca Sant Joan de Deu; EspañaFil: Restrepo Perdomo, Camilo A.. Hospital Sant Joan de Deu Barcelona; EspañaFil: Torrebadell, Montserrat. Hospital Sant Joan de Deu Barcelona; España. Institut de Recerca Sant Joan de Deu; EspañaFil: Suñol, Mariona. Hospital Sant Joan de Deu Barcelona; EspañaFil: Schaiquevich, Paula Susana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Mora, Jaume. Hospital Sant Joan de Deu Barcelona; España. Institut de Recerca Sant Joan de Deu; EspañaFil: Bramuglia, Guillermo Federico. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología; ArgentinaFil: Chantada, Guillermo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina. Hospital Sant Joan de Deu Barcelona; España. Institut de Recerca Sant Joan de Deu; EspañaFil: Carcaboso, Angel M.. Hospital Sant Joan de Deu Barcelona; España. Institut de Recerca Sant Joan de Deu; Españ

Therapeutic targeting of the RB1 pathway in retinoblastoma with the oncolytic adenovirus VCN-01.

Retinoblastoma is a pediatric solid tumor of the retina activated upon homozygous inactivation of the tumor suppressor RB1 VCN-01 is an oncolytic adenovirus designed to replicate selectively in tumor cells with high abundance of free E2F-1, a consequence of a dysfunctional RB1 pathway. Thus, we reasoned that VCN-01 could provide targeted therapeutic activity against even chemoresistant retinoblastoma. In vitro, VCN-01 effectively killed patient-derived retinoblastoma models. In mice, intravitreous administration of VCN-01 in retinoblastoma xenografts induced tumor necrosis, improved ocular survival compared with standard-of-care chemotherapy, and prevented micrometastatic dissemination into the brain. In juvenile immunocompetent rabbits, VCN-01 did not replicate in retinas, induced minor local side effects, and only leaked slightly and for a short time into the blood. Initial phase 1 data in patients showed the feasibility of the administration of intravitreous VCN-01 and resulted in antitumor activity in retinoblastoma vitreous seeds and evidence of viral replication markers in tumor cells. The treatment caused local vitreous inflammation but no systemic complications. Thus, oncolytic adenoviruses targeting RB1 might provide a tumor-selective and chemotherapy-independent treatment option for retinoblastoma

Measurable Residual Disease Assessed by Flow-Cytometry Is a Stable Prognostic Factor for Pediatric T-Cell Acute Lymphoblastic Leukemia in Consecutive SEHOP Protocols Whereas the Impact of Oncogenetics Depends on Treatment

Altres ajuts: Fondo Europeo de Desarrollo Regional (FEDER); Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER).Robust and applicable risk-stratifying genetic factors at diagnosis in pediatric T-cell acute lymphoblastic leukemia (T-ALL) are still lacking, and most protocols rely on measurable residual disease (MRD) assessment. In our study, we aimed to analyze the impact of NOTCH1, FBXW7, PTEN, and RAS mutations, the measurable residual disease (MRD) levels assessed by flow cytometry (FCM-MRD) and other reported risk factors in a Spanish cohort of pediatric T-ALL patients. We included 199 patients treated with SEHOP and PETHEMA consecutive protocols from 1998 to 2019. We observed a better outcome of patients included in the newest SEHOP-PETHEMA-2013 protocol compared to the previous SHOP-2005 cohort. FCM-MRD significantly predicted outcome in both protocols, but the impact at early and late time points differed between protocols. The impact of FCM-MRD at late time points was more evident in SEHOP-PETHEMA 2013, whereas in SHOP-2005 FCM-MRD was predictive of outcome at early time points. Genetics impact was different in SHOP-2005 and SEHOP-PETHEMA-2013 cohorts: NOTCH1 mutations impacted on overall survival only in the SEHOP-PETHEMA-2013 cohort, whereas homozygous deletions of CDKN2A/B had a significantly higher CIR in SHOP-2005 patients. We applied the clinical classification combining oncogenetics, WBC count and MRD levels at the end of induction as previously reported by the FRALLE group. Using this score, we identified different subgroups of patients with statistically different outcome in both Spanish cohorts. In SHOP-2005, the FRALLE classifier identified a subgroup of high-risk patients with poorer survival. In the newest protocol SEHOP-PETHEMA-2013, a very low-risk group of patients with excellent outcome and no relapses was detected, with borderline significance. Overall, FCM-MRD, WBC count and oncogenetics may refine the risk-stratification, helping to design tailored approaches for pediatric T-ALL patients

Helpful Criteria When Implementing NGS Panels in Childhood Lymphoblastic Leukemia.

The development of Next-Generation Sequencing (NGS) has provided useful diagnostic, prognostic, and therapeutic strategies for individualized management of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. Consequently, NGS is rapidly being established in clinical practice. However, the technology's complexity, bioinformatics analysis, and the different available options difficult a broad consensus between different laboratories in its daily routine introduction. This collaborative study among Spanish centers was aimed to assess the feasibility, pros, and cons of our customized panel and other commercial alternatives of NGS-targeted approaches. The custom panel was tested in three different sequencing centers. We used the same samples to assess other commercial panels (OncomineTM Childhood Cancer Research Assay; Archer®FusionPlex® ALL, and Human Comprehensive Cancer Panel GeneRead Panel v2®). Overall, the panels showed a good performance in different centers and platforms, but each NGS approach presented some issues, as well as pros and cons. Moreover, a previous consensus on the analysis and reporting following international guidelines would be preferable to improve the concordance in results among centers. Our study shows the challenges posed by NGS methodology and the need to consider several aspects of the chosen NGS-targeted approach and reach a consensus before implementing it in daily practice.This study has been supported by a grant from “Fundación Unoentrecienmil”; grants from Instituto de Salud Carlos III (PI12/02417; PI16/00246; PI12/00816; PI16CIII/00026; PI16/0665); Asociación Pablo Ugarte (TPY-M 1149/13; TRPV 205/18), ASION (TVP 141/17); Fundación Sonrisa de Alex & Todos somos Iván (TVP 1324/15); Consejería de Educación, Junta de Castilla y León (SA271P18); Fondos FEDER (CIBERONC [CB16/12/00284]), Proyectos de Investigación del SACYL, GRS 1847/A/18, Generalitat Valenciana GV2019/077 and Fundación AMPILE. J.M.H.S. is supported with a research grant by FEHH (“Fundación Española de Hematología y Hemoterapia”), A.M. a grant from Junta provincial de Salamanca de la Asociación Española Contra el Cáncer (AECC), and M.M. holds the grant “Ayuda predoctoral de la Junta de Castilla y León” by the Fondo Social Europeo (JCYL-EDU/556/2019 Ph.D. scholarship).S