CD44-high neural crest stem-like cells are associated with tumour aggressiveness and poor survival in neuroblastoma tumours

BACKGROUND: Neuroblastoma is a paediatric tumour originated from sympathoadrenal precursors and characterized by its heterogeneity and poor outcome in advanced stages. Intra-tumoral cellular heterogeneity has emerged as an important feature in neuroblastoma, with a potential major impact on tumour aggressiveness and response to therapy. CD44 is an adhesion protein involved in tumour progression, metastasis and stemness in different cancers; however, there has been controversies about the significance of CD44 expression in neuroblastoma and its relationship with tumour progression. METHODS: We have performed transcriptomic analysis on patient tumour samples studying the outcome of patients with high CD44 expression. Adhesion, invasion and proliferation assays were performed in sorted CD44high neuroblastoma cells. Tumoursphere cultures have been used to enrich in undifferentiated stem-like cells and to asses self-renewal and differentiation potential. We have finally performed in vivo tumorigenic assays on cell line-derived or Patient-derived xenografts. FINDINGS: We show that high CD44 expression is associated with low survival in high-grade human neuroblastoma, independently of MYCN amplification. CD44 is expressed in a cell population with neural crest stem-like features, and with the capacity to generate multipotent, undifferentiated tumourspheres in culture. These cells are more invasive and proliferative in vitro. CD44 positive cells obtained from tumours are more tumorigenic and metastatic, giving rise to aggressive neuroblastic tumours at high frequency upon transplantation. INTERPRETATION: We describe an unexpected intra-tumoural heterogeneity within cellular entities expressing CD44 in neuroblastoma, and propose that CD44 has a role in neural crest stem-like undifferentiated cells, which can contribute to tumorigenesis and malignancy in this type of cancer. FUNDING: Research supported by grants from the "Asociación Española contra el Cáncer" (AECC), the Spanish Ministry of Science and Innovation SAF program (SAF2016-80412-P), and the European Research Council (ERC Starting Grant to RP).Spanish Ministry of Science and Innovation SAF program (SAF2016-80412-P

Multidrug resistance transporter profile reveals MDR3 as a marker for stratification of blastemal Wilms tumour patients

Wilms tumour (WT) is the most common renal tumour in children. Most WT patients respond to chemotherapy, but subsets of tumours develop resistance to chemotherapeutic agents, which is a major obstacle in their successful treatment. Multidrug resistance transporters play a crucial role in the development of resistance in cancer due to the efflux of anticancer agents out of cells. The aim of this study was to explore several human multidrug resistance transporters in 46 WT and 40 nonneoplastic control tissues (normal kidney) from patients selected after chemotherapy treatment SIOP 93–01, SIOP 2001. Our data showed that the majority of the studied multidrug resistance transporters were downregulated or unchanged between tumours and control tissues. However, BCRP1, MDR3 and MRP1 were upregulated in tumours versus control tissues. MDR3 and MRP1 overexpression correlated with highrisk tumours (SIOP classification) (p = 0.0022 and p < 0.0001, respectively) and the time of disease-free survival was significantly shorter in patients with high transcript levels of MDR3 (p = 0.0359). MDR3 and MRP1 play a role in drug resistance in WT treatment, probably by alteration of an unspecific drug excretion system. Besides, within the blastemal subtype, we observed patients with low MDR3 expression were significantly associated with a better outcome than patients with high MDR3 expression. We could define two types of blastemal WT associated with different disease outcomes, enabling the stratification of blastemal WT patients based on the expression levels of the multidrug resistance transporter MDR3.Ministerio de Economía y Competitividad PI1401466, RD06/0020/0059, PI1100018, CD06/00001Red Tematica de Investigacion Cooperativa en Cancer RD12/0036/0017Unión Europea FP7-HEALTH- 2011-two-stage, Project ID 278742 EUROSARCInstituto de Salud Carlos III FIS PI13/0228

Outcome of Stage IV Completely Necrotic Wilms Tumour and Local Stage III Treated According to the SIOP 2001 Protocol

Objective: Wilms tumour (WT) patients with a localised completely necrotic nephroblastoma after preoperative chemotherapy are a favourable outcome group. Since the introduction of the SIOP 2001 protocol, the SIOP– Renal Tumour Study Group (SIOP–RTSG) has omitted radiotherapy for such patients with low-risk, local stage III in an attempt to reduce treatment burden. However, for metastatic patients with local stage III, completely necrotic WT, the recommendations led to ambiguous use. The purpose of this descriptive study is to demonstrate the outcomes of patients with metastatic, completely necrotic and local stage III WT in relation to the application of radiotherapy or not. Methods and materials: all metastatic patients with local stage III, completely necrotic WT after 6 weeks of preoperative chemotherapy who were registered in the SIOP 2001 study were included in this analysis. The pattern of recurrence according to the usage of radiation treatment and 5 year event-free survival (EFS) and overall survival (OS) was analysed. Results: seven hundred and three metastatic WT patients were registered in the SIOP 2001 database. Of them, 47 patients had a completely necrotic, local stage III WT: 45 lung metastases (11 combined localisations), 1 liver/peritoneal, and 1 tumour thrombus in the renal vein and the inferior vena cava with bilateral pulmonary arterial embolism. Abdominal radiotherapy was administered in 29 patients (62%; 29 flank/abdominal irradiation and 9 combined with lung irradiation). Eighteen patients did not receive radiotherapy. Median follow-up was 6.6 years (range 1–151 months). Two of the 47 patients (4%) developed disease recurrence in the lung (one combined with abdominal relapse) and eventually died of the disease. Both patients had received abdominal radiotherapy, one of them combined with lung irradiation. Five-year EFS and OS were 95% and 95%, respectively. Conclusions: the outcome of patients with stage IV, local stage III, completely necrotic Wilms tumours is excellent. Our results suggest that abdominal irradiation in this patient category may not be of added value in first-line treatment, consistent with the current recommendation in the SIOP–RTSG 2016 UMBRELLA protocol

Diagnostic MRI characteristics of pediatric clear cell sarcoma of the kidney and rhabdoid tumor of the kidney: A retrospective multi-center SIOP-RTSG Radiology panel study

Introduction: Clear cell sarcoma of the kidney (CCSK) and rhabdoid tumor of the kidney (RTK) are rare malignant pediatric renal tumors, both accounting for 1–5% of diagnoses. Within the International Society of Pediatric Oncology-Renal Tumor Study Group (SIOP-RTSG) protocols diagnostic invasive procedures to determine histology are discouraged. MRI has become the preferred imaging modality, however, non-invasive discrimination of CCSK and RTK remains challenging. Therefore, this study aims to identify diagnostic MRI-characteristics of CCSK and RTK, in the largest series of patients to date. Material and methods: Five SIOP-RTSG national review radiologists identified national diagnostic MRIs of histologically proven CCSKs and RTKs. Scan-protocols were based on MRI-guidelines following SIOP-RTSG protocols. Radiologists assessed their national cases using a validated case report form (CRF). Results: Retrospectively, 59 patients were identified (n = 38 CCSK, n = 21 RTK). CCSKs showed a high median volume (576 cm3, range 54–4414), which was lower for RTKs (290 cm3, range 20–761) (p = 0.006 *10-3). Fifty-two percent (11/21) of RTK-patients showed disease at other locations, predominantly the lungs and brain, compared to 8% of CCSK-patients (3/38). RTKs showed ill-defined margins (12/21, 57%) and infiltrative growth pattern (13/21, 62%). CCSKs appeared hyper-intense on T2-weighted imaging (27/38, 71%) with a characteristics band-like enhancement (13/38, 34%), whereas RTK cases often showed T2-weighted hypo-intensity (11/21, 52%). The overall mean ADC-value for CCSK was 1.11 * 10-3 mm2/s (range 0.57–2.07 *10-3 mm2/s), and 0.72 * 10-3 mm2/s (range 0.53–0.90 *10-3 mm2/s) for RTK. Conclusions: This retrospective study suggests a small size, T2-weighted hypo-intensity and an aggressive growth pattern may be characteristic for RTK. CCSK often showed a typical band-like enhancement pattern with relatively high ADC-values. Identified MRI-characteristics may be used in future studies focusing on validation and discrimination from especially WT, aiming for an early non-invasive diagnosis

Diagnostic magnetic resonance imaging characteristics of congenital mesoblastic nephroma: a retrospective multi-center International Society of Pediatric Oncology-Renal Tumor Study Group (SIOP-RTSG) radiology panel study

Background: Congenital mesoblastic nephroma is the most common solid renal tumor in neonates. Therefore, patients <3 months of age are advised to undergo upfront nephrectomy, whereas invasive procedures at diagnosis in patients ≥3 months of age are discouraged by the International Society of Pediatric Oncology-Renal Tumor Study Group (SIOP-RTSG). Nevertheless, discriminating congenital mesoblastic nephroma, especially from the more common Wilms tumor, solely based on imaging remains difficult. Recently, magnetic resonance imaging (MRI) has become the preferred modality. Studies focusing on MRI characteristics of congenital mesoblastic nephroma are limited. Objective: This study aims to identify diagnostic MRI characteristics of congenital mesoblastic nephroma in the largest series of patients to date. Materials and methods: In this retrospective multicenter study, five SIOP-RTSG national review radiologists identified 52 diagnostic MRIs of histologically proven congenital mesoblastic nephromas. MRI was performed following SIOP-RTSG protocols, while radiologists assessed their national cases using a validated case report form. Results: Patients (24/52 classic, 11/52 cellular, and 15/52 mixed type congenital mesoblastic nephroma, 2/52 unknown) had a median age of 1 month (range 1 day–3 months). Classic type congenital mesoblastic nephroma appeared homogeneous with a lack of hemorrhage, necrosis and/or cysts, showing a concentric ring sign in 14 (58.3%) patients. Cellular and mixed type congenital mesoblastic nephroma appeared more heterogeneous and were larger (311.6 and 174.2 cm3, respectively, versus 41.0 cm3 for the classic type (P<0.001)). All cases were predominantly T2-weighted isointense and T1-weighted hypointense, and mean overall apparent diffusion coefficient values ranged from 1.05–1.10×10−3 mm2/s. Conclusion: This retrospective international collaborative study showed classic type congenital mesoblastic nephroma predominantly presented as a homogeneous T2-weighted isointense mass with a typical concentric ring sign, whereas the cellular type appeared more heterogeneous. Future studies may use identified MRI characteristic of congenital mesoblastic nephroma for validation and for exploring the discriminative non-invasive value of MRI, especially from Wilms tumor. Graphical Abstract: (Figure presented.

Rationale for the treatment of children with CCSK in the UMBRELLA SIOP-RTSG 2016 protocol

The International Society of Paediatric Oncology-Renal Tumour Study Group (SIOP-RTSG) has developed a new protocol for the diagnosis, treatment, and follow-up monitoring of childhood renal tumours-the UMBRELLA SIOP-RTSG 2016 protocol (the UMBRELLA protocol). This protocol has been designed to continue international collaboration in the treatment of childhood renal tumours and will be implemented in over 50 different countries. Clear cell sarcoma of the kidney, which is a rare paediatric renal tumour that most commonly occurs in childre

HMGA2 overexpression predicts relapse susceptibility of blastemal Wilms tumor patients

Wilms tumor (WT) is an embryonal malignant neoplasm of the kidney that accounts for 6–7% of all childhood cancers. WT seems to derive from multipotent embryonic renal stem cells that have failed to differentiate properly. Since mechanisms underlying WT tumorigenesis remain largely unknown, the aim of this study was to explore the expression of embryonic stem cell (ESC) markers in samples of WT patients after chemotherapy treatment SIOP protocol, as the gene expression patterns of ESC are like those of most cancer cells. We found that expression of ESC markers is heterogeneous, and depends on histological WT components. Interestingly, among ESC markers, HMGA2 was expressed significantly stronger in the blastemal component than in the stromal and the normal kidney. Moreover, two subsets of patients of WT blastemal type were identified, depending on the expression levels of HMGA2. High HMGA2 expression levels were significantly associated with a higher proliferation rate (p=0.0345) and worse patient prognosis (p=0.0289). The expression of HMGA2 was a stage-independent factor of clinical outcome in blastemal WT patients. Our multivariate analyses demonstrated the association between LIN28B–LET7A–HMGA2 expression, and the positive correlation between HMGA2 and SLUG expression (p=0.0358) in blastemal WT components. In addition, patients with a poor prognosis and high HMGA2 expression presented high levels of MDR3 (multidrug resistance transporter). Our findings suggest that HMGA2 plays a prominent role in the pathogenesis of a subset of blastemal WT, strongly associated with relapse and resistance to chemotherapy.LHP and DJGD are supported by Red Temática de Investigación Cooperativa en Cáncer (RD12/0036/0017). CS is supported by a contract from Nicolás Monardes Program, Consejería de Salud, Junta de Andalucía. Research in Enrique de Alava’s lab is supported by the Ministry of Economy and Competitiveness of Spain-FEDER (CIBERONC, PI1700464, RD06/0020/0059, FMGE) and the European Commission (FP7-HEALTH-2011-two-stage, Project ID 278742 EUROSARC). DJGD is a PhD researcher funded by the Consejería de Salud, Junta de Andalucía (PI-0197-2016). Research in Carmen Sáez’s lab is also supported by research grants from the Instituto de Salud Carlos III, FIS PI13/02282.Peer reviewe

Multidrug resistance transporter profile reveals MDR3 as a marker for stratification of blastemal Wilms tumour patients

Wilms tumour (WT) is the most common renal tumour in children. Most WT patients respond to chemotherapy, but subsets of tumours develop resistance to chemotherapeutic agents, which is a major obstacle in their successful treatment. Multidrug resistance transporters play a crucial role in the development of resistance in cancer due to the efflux of anticancer agents out of cells. The aim of this study was to explore several human multidrug resistance transporters in 46 WT and 40 non-neoplastic control tissues (normal kidney) from patients selected after chemotherapy treatment SIOP 93–01, SIOP 2001. Our data showed that the majority of the studied multidrug resistance transporters were downregulated or unchanged between tumours and control tissues. However, BCRP1, MDR3 and MRP1 were upregulated in tumours versus control tissues. MDR3 and MRP1 overexpression correlated with high-risk tumours (SIOP classification) (p = 0.0022 and p < 0.0001, respectively) and the time of disease-free survival was significantly shorter in patients with high transcript levels of MDR3 (p = 0.0359). MDR3 and MRP1 play a role in drug resistance in WT treatment, probably by alteration of an unspecific drug excretion system. Besides, within the blastemal subtype, we observed patients with low MDR3 expression were significantly associated with a better outcome than patients with high MDR3 expression. We could define two types of blastemal WT associated with different disease outcomes, enabling the stratification of blastemal WT patients based on the expression levels of the multidrug resistance transporter MDR3.LHP and DJGD are supported by Red Temática de Investigación Cooperativa en Cáncer (RD12/0036/0017). CS was supported by a contract from Nicolás Monardes Program, Consejería de Salud, Junta de Andalucía. Research in Enrique de Alava’s lab is also supported by the Ministry of Economy and Competitiveness of Spain-FEDER (PI1401466, RD06/0020/0059, PI1100018, ISCIII postdoc grant CD06/00001), María García-Estrada Foundation and the European Commission (FP7-HEALTH-2011-two-stage, Project ID 278742 EUROSARC). Research in Carmen Sáez lab is also supported by research grants from the Instituto de Salud Carlos III, FIS PI13/02282.Peer reviewe