Marilina Nantron

Functional inactivation of the WTX gene is not a frequent event in Wilms' tumors

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.

Whole transcriptome sequencing identifies BCOR internal tandem duplication as a common feature of clear cell sarcoma of the kidney

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.

Genomic profiling by whole-genome single nucleotide polymorphism arrays in Wilms tumor and association with relapse

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.

Loss of Heterozygosity Analysis at Different Chromosome Regions in Wilms Tumor Confirms 1p Allelic Loss as a Marker of Worse Prognosis: A Study from the Italian Association of Pediatric Hematology and Oncology

PURPOSE The specific aims of the AIEOP-TW-2003 protocol included prospectively investigating a possible association of tumor loss of heterozygosity with outcomes in children treated for Wilms tumor. MATERIALS AND METHODS We analyzed 125 unilateral favorable histology Wilms tumors registered between 2003 and 2008 in the Italian cooperative protocol for microsatellite markers mapped to chromosomes 1p, 7p, 11q, 16q and 22q. RESULTS The 3-year disease-free survival and overall survival probabilities were 0.87 (95% CI 0.81-0.93) and 0.98 (95% CI 0.96-1.0), respectively. Loss of heterozygosity at 1p was significantly associated with a worse disease-free survival (probability 0.67 for patients with and 0.92 for those without 1p loss of heterozygosity, p = 0.0009), as confirmed also by multivariate analysis adjusting for tumor stage and patient age at diagnosis. There was no difference in disease-free survival probability among children with loss of heterozygosity in the other chromosomal regions tested. The worse outlook for children older than 2 years at diagnosis did not seem to be influenced by the loss of heterozygosity patterns considered. CONCLUSIONS Chromosome 1p loss of heterozygosity seems to be a risk factor for nonanaplastic Wilms tumor, possibly regardless of other clinical factors. Our findings were uninformative regarding loss of heterozygosity in the other chromosomal regions tested.

Heterogeneity of Disease Classified as Stage III in Wilms Tumor: A Report From the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP)

PURPOSE We analyzed whether the prognosis can differ among Wilms tumors (WT) labeled as Stage III according to currently adopted classification systems. METHODS AND MATERIALS Patients with nonanaplastic Stage III WT consecutively registered in two Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) trials (CNR-92, TW-2003) were the subjects in the present analysis. The steady mainstay of therapy was primary nephrectomy, followed by three-drug chemotherapy with vincristine, dactinomycin, doxorubicin, and abdominal radiotherapy (RT). RESULTS Ninety-nine WT patients met the criteria for classification as Stage III according to a revised version of the National Wilms Tumor Study-3 staging system (51 patients in CNR-92, 48 patients in TW-2003). Regional lymph nodes (LN) were not biopsied in 16 patients. After a median follow-up of 66 months, the 4-year disease-free survival (DFS) and overall survival (OS) rates were 85% ± 4% and 92% ± 3%, respectively, for the whole group. For 38 children with positive LN, the 4-year DFS rate was 73% ± 7%, as opposed to 98% ± 2% for the 45 children with Stage III WT according to the other criteria but with negative biopsied LN (p = 0.001). The subgroup with the worst prognosis consisted of children more than 2 years old with positive LN (DFS 67% ± 8%). A delay between surgery and RT > 30 days had an adverse impact on the abdominal tumor relapse rate. CONCLUSIONS This study provides further evidence that Stage III tumors with LN metastases might be distinguished from WTs meeting the other criteria for classification as Stage III. The worse outcome of the former may warrant a prospective study on the effects of intensified therapy. A subclassification of Stage III tumors is discussed.

Results of the Third AIEOP Cooperative Protocol on Wilms Tumor (TW2003) and Related Considerations

Purpose: 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. Materials and Methods: 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. Results: 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, p <0.001; bias corrected c‐index 0.697). Conclusions: The survival rates achieved and the proposed risk stratification schema provide a basis for future comparisons of Wilms tumor treatment burden and patient outcome.

Genetic and epigenetic analyses guided by high resolution whole-genome SNP array reveals a possible role of CHEK2 in Wilms tumour susceptibility

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.

Factors possibly affecting prognosis in children with Wilms’ tumor diagnosed before 24 months of age: A report from the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) Wilms Tumor Working Group

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.

Abstract 3268: 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

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

Toxicity of 131I-MIBG combined with high-dose chemotherapy in children with refractory neuroblastoma.

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 used for targeted radiotherapy of neural crest tumors, including neuroblastoma. By this retrospective study, we have evaluated feasibility and toxicity of 131I-MIBG combined to myeloablative chemotherapy (HD-CHT) with peripheral blood stem cell (PBSC) rescue in children with refractory neuroblastoma. METHODS Between October 1983 and October 2005 a total of 53 patients with refractory neuroblastoma received therapeutic 131I-MIBG combined with HD-CHT. Patients were divided in 2 cohorts. First cohort (FC) patients (n=34) received 131I-MIBG before HD-CHT. Second cohort (SC) patients (n=19) received 131I-MIBG after HD-CHT. Median age at diagnosis was 39 months (range, 7-198). FC patients received 131I-MIBG at the median dose of 7.26 mCi/kg (range, 3.75-16.66), at 16-45 days (median, 20) before HD-CHT. SC patients received 131I-MIBG at the median dose of 6 mCi/kg (range, 2.28-10), at 27-103 days (median, 57) after HD-CHT. Ten types of regimens were used as HD-CHT. RESULTS Both 131I-MIBG infusion and HD-CHT were well tolerated. Hematologic toxicity was comparable for the 2 cohorts. Median times to neutrophil (>0.5 x 103/µL) and platelet (> 50 x 103/µL) engraftment were 13 days (range, 8-30) and 21 days (range, 12->60), respectively. The predominant non hematopoietic toxicity was oral mucositis, of grade 4 in 23 patients (16 in the FC and 7 in the SC patients) and grade 3 in 9 (3 in the FC and 6 in the SC). Pulmonary complications occurred in 2 children. The predominant long-term toxicity was hypothyroidism which occurred in 9 FC and 6 SC patients. CONCLUSIONS Treatment with 131I-MIBG in combination to HD-CHT was feasible with acceptable toxicity. Future studies are warranted to evaluate the efficacy of this combination.