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Whereas 90% of patients with Wilms tumor (WT) reach cure, approximately half of patients developing a recurrent tumor die of the disease. Therefore, to disclose events leading to recurrence represents a clinical need. To study paired primary/recurrent tumor samples, being aware of the intra-tumoral heterogeneity, might help finding these answers. We previously suggested that mutations in SIX1 and DROSHA underlie WT recurrence. With the aim to better investigate this scenario, we collected 19 paired primary/recurrent tumors and 10 primary tumors from relapsing patients and searched for mutations in the SIX1/2 genes and microRNA processing genes (miRNAPGs). We found SIX1 mutation in one case, miRNAPGs mutations in seven cases, and the co-occurrence of SIX1 and miRNAPG mutations in one case. We could observe that, whereas in primary tumors the mutations could be heterogeneously present, in all cases they were positively selected and homogeneously present in the recurrent disease, as also indicated by a “moderate” and “almost perfect” agreement (according to the Landis and Koch classification criteria) between paired samples. Analysis of SIX1/2 genes and miRNAPGs in 50 non-relapsing WTs disclosed SIX2 mutation in one case and miRNAPGs mutations in seven. A borderline statistically significant association was observed between miRNAPGs mutations and the occurrence of relapse (p value: 0.05). These data suggest that SIX1 and miRNAPGs mutations may provide an advantage during tumor progression to recurrence and can represent oncogenic drivers in WT development.

TW2003, the third Italian prospective study on Wilms tumor, aimed to improve survival in patients with stage III-IV tumors, de-escalate therapy for stage I-II nonanaplastic tumors, refine the risk stratification of therapy, and develop a national infrastructure for biobanking and central pathology review.TW2003 recruited children 18 years old or younger with primary intrarenal tumors. Local physicians chose nephrectomy with or without preoperative chemotherapy as the initial treatment based on the risk of unsafe and/or incomplete immediate surgery. The main drivers for adjuvant therapy were tumor stage and diffuse anaplasia. A new risk stratification schema was investigated, incorporating patient age, reason for stage III designation and completeness of lung nodule response in stage IV disease.We report on 453 patients with unilateral Wilms tumor. Preoperative chemotherapy was administered to 42% of patients. The 5-year event-free survival and overall survival rates were 89.1% (95% CI 83.6-94.9) and 97.0% (93.7-100) for stage I; 85.1% (79.6-91.1) and 94.0% (90.1-98.1) for stage II (160); 82.7% (75.3-90.8) and 90.9% (85.0-97.1) for stage III (101); and 72.1% (61.9-84.0) and 82.5% (73.1-93.1) for stage IV (69), respectively. On multivariable analysis only anaplasia was significant for event-free survival (HR 2.68, 95% CI 1.48-4.86, p=0.001; bias corrected c-index 0.580) and overall survival (HR 5.29, 95% CI 2.52-11.12, p0.001; bias corrected c-index 0.697).The survival rates achieved and the proposed risk stratification schema provide a basis for future comparisons of Wilms tumor treatment burden and patient outcome.

// Annalisa Astolfi 1, 2 , Fraia Melchionda 2 , Daniela Perotti 3 , Maura Fois 2 , Valentina Indio 1 , Milena Urbini 1, 2 , Chiara Giusy Genovese 1 , Paola Collini 4 , Nunzio Salfi 5 , Marilina Nantron 6 , Paolo D’Angelo 7 , Filippo Spreafico 8 , Andrea Pession 2 1 “Giorgio Prodi” Cancer Research Center, University of Bologna, Bologna, Italy 2 Pediatric Hematology and Oncology Unit, S.Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy 3 Unit of Molecular Bases of Genetic Risk and Genetic Testing, Department of Preventive and Predictive Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 4 Soft Tissue and Bone Pathology, Histopathology, and Pediatric Pathology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 5 Pathology Unit, S.Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy 6 Department of Pediatric Hematology and Oncology, Istituto G. Gaslini, Genova, Italy 7 Pediatric Hematology and Oncology Unit, A.R.N.A.S. Civico, Di Cristina and Benfratelli Hospital, Palermo, Italy 8 Pediatric Oncology Unit, Department of Hematology and Pediatric Onco-Hematology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy Correspondence to: Fraia Melchionda, e-mail: fraia.melchionda@aosp.bo.it Keywords: CCSK, whole transcriptome sequencing, BCOR Received: August 10, 2015 Accepted: September 28, 2015 Published: October 22, 2015 ABSTRACT Purpose: Clear cell sarcoma of the kidney (CCSK) is a rare pediatric renal tumor that is frequently difficult to distinguish among other childhood renal tumors due to its histological heterogeneity. This work evaluates genetic abnormalities carried by a series of CCSK samples by whole transcriptome sequencing (WTS), to identify molecular biomarkers that could improve the diagnostic process. Methods: WTS was performed on tumor RNA from 8 patients with CCSK. Bioinformatic analysis, with implementation of a pipeline for detection of intragenic rearrangements, was executed. Sanger sequencing and gene expression were evaluated to validate BCOR internal tandem duplication (ITD). Results: WTS did not identify any shared SNVs, Ins/Del or fusion event. Conversely, analysis of intragenic rearrangements enabled the detection of a breakpoint within BCOR transcript recurrent in all samples. Three different in-frame ITD in exon15 of BCOR, were detected. The presence of the ITD was confirmed on tumor DNA and cDNA, and resulted in overexpression of BCOR. Conclusion: WTS coupled with specific bioinformatic analysis is able to detect rare genetic events, as intragenic rearrangements. ITD in the last exon of BCOR is recurrent in all CCSK samples analyzed, representing a valuable molecular marker to improve diagnosis of this rare childhood renal tumor.

Background Children with Wilms’ tumor (WT) aged under 24 months (infants) have a better prognosis than older patients. Our aim was to study the epidemiology of this age group, with focus on the modality of diagnosis, tumor size, and association with malformations/syndromes, seeking to understand if any of these factors might be related to prognosis. Patients and methods Infants diagnosed with WT between 2003 and February 2010 were evaluated. A query form was used to collect data on the modality of WT diagnosis (symptomatic or incidental), tumor volume, maximum diameter, site, and stage. Results Data were collected for 117 of 124 WT infants registered. Twenty-four cases had an incidental diagnosis (ID) of renal mass, usually arising from an abdominal ultrasound performed for other reasons, and 93 had been diagnosed based on clinical signs/symptoms. The incidental cohort displayed unifocal disease, mean tumor diameter 5.52 cm, mean tumor volume 84.30 ml, and 14 patients showed associated malformations. Symptomatic patients had mean maximum tumor diameter of 10.18 cm, mean tumor volume of 451.18 ml, and six had associated malformations. Conclusions Our study showed that 20% of the infants had an ID of WT; they had a relatively smaller nonmetastatic tumor and a higher rate of malformations than infants of the symptomatically diagnosed group, but we did not detect any difference in age at diagnosis between the two groups. Conversely, we found a significant difference in the 5-year event-free survival rate (P = 0.018) between infants under 1 year (96%), more frequently associated with congenital malformations, and infants 1–2 years (80%).

Despite the excellent survival rate of Wilms tumor (WT) patients, only approximately one-half of children who suffer tumor recurrence reach second durable remission. This underlines the need for novel markers to optimize initial treatment. We investigated 77 tumors using Illumina 370CNV-QUAD genotyping BeadChip arrays and compared their genomic profiles to detect copy number (CN) abnormalities and allelic ratio anomalies associated with the following clinicopathological variables: relapse (yes vs. no), age at diagnosis (≤24 months vs. >24 months), and disease stage (low stage, I and II, vs. high stage, III and IV). We found that CN gains at chromosome region 1q21.1-q31.3 were significantly associated with relapse. Additional genetic events, including allelic imbalances at chromosome arms 1p, 1q, 3p, 3q, and 14q were also found to occur at higher frequency in relapsing tumors. Interestingly, allelic imbalances at 1p and 14q also showed a borderline association with higher tumor stages. No genetic events were found to be associated with age at diagnosis. This is the first genome wide analysis with single nucleotide polymorphism (SNP) arrays specifically investigating the role of genetic anomalies in predicting WT relapse on cases prospectively enrolled in the same clinical trial. Our study, besides confirming the role of 1q gains, identified a number of additional candidate genetic markers, warranting further molecular investigations. © 2012 Wiley Periodicals, Inc.

For many years the precise genetic etiology of the majority of Wilms' tumors has remained unexplained. Recently, the WTX gene, mapped to chromosome Xq11.1, has been reported to be lost or mutated in approximately one-third of Wilms' tumors. Moreover, in female cases, the somatically inactivated alleles were found to invariantly derive from the active chromosome X. Consequently, WTX has been proposed as a 'one-hit' tumor suppressor gene. To provide further insights on the contribution of WTX to the development of the disease, we have examined 102 Wilms' tumors, obtained from 43 male and 57 female patients. Quantitative PCR analyses detected WTX deletions in 5 of 45 (11%) tumors from males, whereas loss of heterozygosity at WTX-linked microsatellites was observed in 9 tumors from 50 informative females (19%). However, in the latter group, using a combination of HUMARA assay and bisulfite-modified DNA sequencing, we found that the deletion affected the active chromosome X only in two cases (4%). Sequence analyses detected an inactivating somatic mutation of WTX in a single tumor, in which a strongly reduced expression of the mutant allele respect to the wild-type allele was observed, a finding not consistent with its localization on the active chromosome X. Overall, a functional somatic nullizygosity of the WTX gene was ascertained only in seven of the Wilms' tumors included in the study (approximately 7%). Our findings indicate that previously reported estimates on the proportion of Wilms' tumors due to WTX alterations should be reconsidered.

Wilms Tumor (WT), originating through perturbation of developmental processes in embryonic kidney, is the most frequent pediatric genitourinary malignancy. Despite its overall survival rate has reached 90%, several patients develop relapses and, among them, approximately 50% will suffer of fatal disease. Hence, reduction of the relapse burden is key to improve WT outcome, and incorporation of novel prognostic markers is currently explored to achieve this goal. Chromosomal alterations and gene expression signatures in WT cohorts are thus examined to identify patients with risk disease. By evaluating gene expression profiles of 58 untreated primary tumor tissues from patients enrolled in the AIEOP multicenter protocol, we could identify four WT subtypes displaying enrichment for genes that were differentially expressed, and divergent clinical outcome. These patterns were validated against a previously published large WT cohort, confirming the accuracy of the prediction. A correlation analysis based on the expression of WT1, which deregulation is a crucial event in this disease, allowed us to verify known WT1 targets and to identify novel potential effectors implicated in this disease. However, although the majority or primary tumors from relapsing patients (7 out of 11) features lower WT1 level and increased 11p13 copy number (CN) losses, its expression did not significantly correlate with specific pathological parameters, confirming its poor prognostic impact as an independent disease predictor. Therefore, in order to reliably associate the risk of recurrence to a specific gene signature, we performed class comparison analysis of primary tumors between relapsing and non-relapsing patients. Unexpectedly, the pattern of genes up-regulated in relapsing tumors showed a negative enrichment of genes expressed in early embryonic kidney structures, such as the metanephric and cap mesenchyme, and a positive enrichment for genes expressed in nephrogenic structures developed upon mesenchymal-to-epithelial transition. These findings underscore the clinical heterogeneity of the disease and indicate that disruption of post-inductive renal processes occurs in high-risk WTs. Moreover, a few transcripts mapping at chromosome 1q21-44, with CN gain or allelic imbalance in 5/11 relapsing vs. 6/47 non-relapsing tumors, were found up-regulated in these patients. Remarkably, a few genes known to be implicated in metastatic disease in adult cancers, including SPP1, MUC1, MYC, CLDN1, and MAOA, showed up-regulation in relapsing WTs. Overall, our data suggests a gene signature associated to naive primary recidivant tumors, to be exploited as a potential predictor of disease at higher risk of relapse. Citation Format: Antonio Fiorino, Loris De Cecco, Beatrice Gamba, Edoardo Marchesi, Paola Collini, Andrea Pession, Marilina Nantron, Maurizio Bianchi, Filippo Spreafico, Silvana Canevari, Paolo Radice, Daniela Perotti. Gene expression associated to relapsing disease in Wilms tumor indicates a more differentiated phenotype unveiling a distinct transformation process for patients with a higher risk of relapse. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3268. doi:10.1158/1538-7445.AM2015-3268

BACKGROUND Intracranial immature teratomas (IT) are very rare germ cell tumors (GCT). The value of chemotherapy in their treatment has not been defined. METHODS A child was referred to our hospital for consultation regarding the need for adjuvant treatment after being operated upon twice (at the age of 7 months and 11 months) for a large supratentorial intracerebral mass. The patient was staged and the tumor specimens reviewed. Histology was that of an IT with no other type of malignant GCT. RESULTS Considering the age, lack of any sign of residual tumor by computed tomography and magnetic resonance imaging, absence of tumor cells in the spinal fluid or elevation of tumor markers, and based on the reported poor response of such tumors to chemotherapy, a policy of “wait and see” was adopted. One month later, the child presented with a rapidly growing intracranial mass invading the cranial bones on the left side. Chemotherapy consisting of 4 cycles of carboplatin, etoposide, and bleomycin followed by another 4 cycles of ifosfamide, vincristine, and dactinomycin alternating with carboplatin and etoposide, achieved complete remission, and 24 months after discontinuation of treatment, the patient continued free of disease. CONCLUSIONS This report indicates that chemotherapy may be effective therapy for intracranial IT. In our patient, chemotherapy modified the natural aggressive behavior of this disease and achieved a persistent, complete remission. Given the minimal information available in the literature concerning the response of IT to chemotherapy, this case addresses the issue of whether chemotherapy alone is adequate to treat intracranial germ cell tumors. Cancer 1996;77:977-82.

9549 Background: More effective regimens with acceptable toxicity are needed to improve prognosis of children with high-risk neuroblastoma. Iodine-131–metaiodobenzylguanidine (131I-MIBG) has been u...

Neuroblastoma, a tumor of post-ganglionic sympathetic neurons, may be associated with a variety of genetic defects and congenital malformations (1). We report a case of neuroblastoma (NB) stage IV-S (2) in an infant with bilateral microphthalmia and other ocular malformations.

10031 Background: Children with metastases at diagnosis for Wilms tumor (WT) still display a worse prognosis compared to localized disease. Methods: We analyzed survival results in patients with stage IV WT enrolled in the AIEOP CNR92 and TW2003 clinical trials (3/1992–3/2008). Treatment strategy across these trials evolved in terms of sparing whole lung radiotherapy (RT) in case of complete disappearance of lung metastases after primary chemotherapy and a doxorubicin cumulative dose reduction from 360 mg/m2 to 240 mg/m2 in TW2003. Results: Of 553 in-study patients aged less than 18 years, 68 (12%) were classified as stage IV (38 patients in CNR92, 30 in TW2003; median age 58 months). Children displaying millimetric nodules visible only on computed tomography scan were excluded. Initial treatment consisted of 6-week 3-drug (vincristine, dactinomycin, doxorubicin) phase in 60 cases, while 8 had up-front nephrectomy. Adjuvant therapy included 8-month 3-drug chemotherapy for non anaplastic “local” tumor stage I to III (flank RT for stage III), or an intensified regimen for anaplastic histology, adding etoposide, carboplatinum and ifosfamide (6 patients). Overall 19 tumor failure occurred (3 in anaplastic tumors): metastases progression 9, abdominal relapse 5 (combined to liver and mediastinum in 1 case each), lung 4, liver 1. After a median follow-up of 53 months 5-yr RFS and OS were 71% ±6 and 77% ±5 for the whole cohort of children, respectively. Overall, RFS was 86% in patients who achieved a complete metastases remission (in 2 cases by surgery) compared to 55% in patients who did not (Logrank p No significant financial relationships to disclose.

Wilms tumour (WT), the most frequent malignant childhood renal tumour, shows a high degree of genetic and epigenetic heterogeneity. Loss of imprinting on chromosome 11p15 is found in a large fraction of cases and mutations in a few genes, including WT1, CTNNB1, WTX, TP53 and, more recently, SIX1, SIX2 and micro RNA processing genes (miRNAPGs), have been observed. However, these alterations are not sufficient to describe the entire spectrum of genetic defects underlying WT development. We inspected data obtained from a previously performed genome-wide single nucleotide polymorphism (SNP) array analysis on 96 WT samples. By selecting focal regions commonly involved in chromosomal anomalies, we identified genes with a possible role in WT development, based on the prior knowledge of their biological relevance, including MYCN, DIS3L2, MIR562, HACE1, GLI3, CDKN2A and CDKN2B, PALB2, and CHEK2. The MYCN hotspot mutation c.131C>T was detected in seven cases (7.3%). Full sequencing of the remaining genes disclosed 16 rare missense variants and a splicing mutation. Most of these were present at the germline level. Promoter analysis of HACE1, CDKN2A and CDKN2B disclosed partial methylation affecting HACE1 in a consistent fraction of cases (85%). Interestingly, of the four missense variants identified in CHEK2, three were predicted to be deleterious by in silico analyses, while an additional variant was observed to alter mRNA splicing, generating a functionally defective protein. Our study adds additional information on putative WT genes, and adds evidences involving CHEK2 in WT susceptibility.