Karen Ernestus

Customized Oligonucleotide Microarray Gene Expression–Based Classification of Neuroblastoma Patients Outperforms Current Clinical Risk Stratification

PURPOSE To develop a gene expression-based classifier for neuroblastoma patients that reliably predicts courses of the disease. PATIENTS AND METHODS Two hundred fifty-one neuroblastoma specimens were analyzed using a customized oligonucleotide microarray comprising 10,163 probes for transcripts with differential expression in clinical subgroups of the disease. Subsequently, the prediction analysis for microarrays (PAM) was applied to a first set of patients with maximally divergent clinical courses (n = 77). The classification accuracy was estimated by a complete 10-times-repeated 10-fold cross validation, and a 144-gene predictor was constructed from this set. This classifiers predictive power was evaluated in an independent second set (n = 174) by comparing results of the gene expression-based classification with those of risk stratification systems of current trials from Germany, Japan, and the United States. RESULTS The first set of patients was accurately predicted by PAM (cross-validated accuracy, 99%). Within the second set, the PAM classifier significantly separated cohorts with distinct courses (3-year event-free survival [EFS] 0.86 +/- 0.03 [favorable; n = 115] v 0.52 +/- 0.07 [unfavorable; n = 59] and 3-year overall survival 0.99 +/- 0.01 v 0.84 +/- 0.05; both P < .0001) and separated risk groups of current neuroblastoma trials into subgroups with divergent outcome (NB2004: low-risk 3-year EFS 0.86 +/- 0.04 v 0.25 +/- 0.15, P < .0001; intermediate-risk 1.00 v 0.57 +/- 0.19, P = .018; high-risk 0.81 +/- 0.10 v 0.56 +/- 0.08, P = .06). In a multivariate Cox regression model, the PAM predictor classified patients of the second set more accurately than risk stratification of current trials from Germany, Japan, and the United States (P < .001; hazard ratio, 4.756 [95% CI, 2.544 to 8.893]). CONCLUSION Integration of gene expression-based class prediction of neuroblastoma patients may improve risk estimation of current neuroblastoma trials.

Localized infant neuroblastomas often show spontaneous regression: results of the prospective trials NB95-S and NB97.

PURPOSE The excellent prognosis of localized neuroblastoma in infants, the overdiagnosis observed in neuroblastoma screening studies, and several case reports of regression of localized neuroblastoma prompted us to initiate a prospective cooperative trial on observation of localized neuroblastoma without cytotoxic treatment. PATIENTS AND METHODS For infants with localized neuroblastoma without MYCN amplification, chemotherapy was scheduled only in cases with threatening symptoms; otherwise, the tumor was either resected or observed by ultrasound and magnetic resonance imaging (MRI). RESULTS Of 340 eligible participants, 190 underwent resection, 57 were treated with chemotherapy, and 93 were observed with gross residual tumor. Of those 93 patients with unresected tumors, spontaneous regression was seen in 44, local progression in 28, progression to stage 4S in seven, and progression to stage 4 in four. Time to regression was quite variable, with first signs of regression noted 1 to 18 months after diagnosis and in 15 of 44 patients even after the first year of life. So far, complete regression was observed in 17 of 44 patients 4 to 20 months after diagnosis. Known clinical risk factors were not able to differentiate between patients with regression and regional or metastatic progression. Overall survival (OS; 3-year OS, 0.99 +/- 0.01) and metastases-free survival (rate at 3 years, 0.94 +/- 0.03) for patients with unresected tumors was excellent and was not different from patients treated with surgery or chemotherapy. CONCLUSION Spontaneous regression is regularly seen in infants with localized neuroblastoma and is not limited to the first year of life. A wait-and-see strategy is justified in those patients.

Oligonucleotide array-based comparative genomic hybridization (aCGH) of 90 neuroblastomas reveals aberration patterns closely associated with relapse pattern and outcome.

The study of genomic alterations in neuroblastoma is of particular importance since several cytogenetic markers proved to be closely associated with the clinical phenotype. To disclose patterns of gains and losses, we performed high‐resolution oligonucleotide array‐based comparative genomic hybridization (aCGH). A total cohort of 90 patients was classified into 6 subsets according to tumor stage and outcome: Stages 1‐3+ (with event), Stage 1‐3− (no event), Stage 4+/−, and Stage 4S+/−. The aberration patterns in Stages 1‐3− and 4S− tumors differed from all other groups as they were predominantly characterized by losses (3, 4, 14, X) and gains (7, 17) of whole chromosomes. However, 59/65 (91%) tumors of Stages 1‐3+ or Stage 4 revealed numerous structural copy number alterations (sCNA). While deletions in chromosomes 1, 3, and 11 discriminated outcome in Stage 4, there were no specific sCNA that distinguished tumor stage within the subgroup of unfavorable tumors. sCNA in 1p, 3p, 11q, 17q, or MYCN amplification (MNA) was seen among 22/24 patients who died, 10/12 with metastatic relapses, and 5/9 with local recurrences. Detailed breakpoint analyses on chromosomes 1, 3, 11, and 17 disclosed preferred breaking areas, although breakpoints were not identical. Amplifications were found in 18 patients and involved 2p24 (MYCN) and other segments of chromosome 2, as well as regions on chromosome arms 6q, 12q, and 17q. One single feature in 21q21.1 (BU678720, without known function yet) attracted particular attention since five patients showed a homozygous loss of this sequence.

Expression of MHC class I, MHC class II, and cancer germline antigens in neuroblastoma.

Background: Neuroblastoma is the most common solid extracranial tumor in childhood, still with poor survival rates for metastatic disease. Neuroblastoma cells are of neuroectodermal origin and express a number of cancer germline (CG) antigens. These CG antigens may represent a potential target for immunotherapy such as peptide-based vaccination strategies. Objective: The purpose of this study was to analyze the presence of MAGE-A1, MAGE-A3/A6, and NY-ESO-1 on an mRNA and protein level and to determine the expression of MHC class I and MHC class II antigens within the same tumor specimens. Methods: A total of 68 tumors were available for RT-PCR, and 19/68 tumors were available for immunohistochemical (IHC) analysis of MAGE-A1, MAGE-A3/A6, and NY-ESO-1. In parallel, the same tumors were stained with a panel of antibodies for MHC class I and MHC class II molecules. Results: Screening of 68 tumor specimens by RT-PCR revealed expression of MAGE-A1 in 44%, MAGE-A3/A6 in 21%, and NY-ESO-1 in 28% of cases. Immunohistochemistry for CG antigens of selected tumors showed good agreement between protein and gene expression. However, staining revealed a heterogeneous expression of CG antigens. None of the selected tumors showed MHC class I or MHC class II expression. Conclusions: mRNA expression of MAGE-A1, MAGE-A3/A6, and NY-ESO-1 is congruent with the protein expression as determined by immunohistochemistry. The heterogeneous CG-antigen expression and the lack of MHC class I and II molecules may have implications for T-cell–mediated immunotherapy in neuroblastoma.

Differential Expression of Neuronal Genes Defines Subtypes of Disseminated Neuroblastoma with Favorable and Unfavorable Outcome

Purpose: Identification of molecular characteristics of spontaneously regressing stage IVS and progressing stage IV neuroblastoma to improve discrimination of patients with metastatic disease following favorable and unfavorable clinical courses. Experimental Design: Serial analysis of gene expression profiles were generated from five stage IVS and three stage IV neuroblastoma. Differential expression of candidate genes was evaluated by real-time quantitative reverse transcription-PCR in 76 pretreatment tumor samples (stage IVS n = 27 and stage IV n = 49). Gene expression-based outcome prediction was determined by Prediction Analysis for Microarrays using 38 tumors as a training set and 38 tumors as a test set. Results: Comparison of serial analysis of gene expression profiles from stage IV and IVS neuroblastoma revealed ∼500 differentially expressed transcripts. Genes related to neuronal differentiation were observed more frequently in stage IVS tumors as determined by associating transcripts to Gene Ontology annotations. Forty-one candidate genes were evaluated by quantitative reverse transcription-PCR and 18 were confirmed to be differentially expressed (P ≤ 0.001). Classification of patients according to expression patterns of these 18 genes using Prediction Analysis for Microarrays discriminated two subgroups with significantly differing event-free survival (96 ± 6% versus 40 ± 8% at 3 years; P < 0.0001) and overall survival (100% versus 72 ± 7% at 3 years; P = 0.0003). This classifier was the only independent covariate marker in a multivariate analysis considering the variables stage, age, MYCN amplification, and gene signature. Conclusions: Spontaneously regressing and progressing metastatic neuroblastoma differ by specific gene expression patterns, indicating distinct levels of neuronal differentiation and allowing for an improved risk estimation of children with disseminated disease.

High Skp2 Expression Characterizes High-Risk Neuroblastomas Independent of MYCN Status

Purpose: Amplified MYCN oncogene defines a subgroup of neuroblastomas with poor outcome. However, a substantial number of MYCN single-copy neuroblastomas exhibits an aggressive phenotype similar to that of MYCN-amplified neuroblastomas even in the absence of high MYCN mRNA and/or protein levels. Experimental Design: To identify shared molecular mechanisms that mediate the aggressive phenotype in MYCN-amplified and single-copy high-risk neuroblastomas, we defined genetic programs evoked by ectopically expressed MYCN in vitro and analyzed them in high-risk versus low-risk neuroblastoma tumors (n = 49) using cDNA microarrays. Candidate gene expression was validated in a separate cohort of 117 patients using quantitative PCR, and protein expression was analyzed in neuroblastoma tumors by immunoblotting and immunohistochemistry. Results: We identified a genetic signature characterized by a subset of MYCN/MYC and E2F targets, including Skp2, encoding the F-box protein of the SCFSkp2 E3-ligase, to be highly expressed in high-risk neuroblastomas independent of amplified MYCN. We validated the findings for Skp2 and analyzed its expression in relation to MYCN and E2F-1 expression in a separate cohort (n = 117) using quantitative PCR. High Skp2 expression proved to be a highly significant marker of dire prognosis independent of both MYCN status and disease stage, on the basis of multivariate analysis of event-free survival (hazard ratio, 3.54; 95% confidence interval, 1.56-8.00; P = 0.002). Skp2 protein expression was inversely correlated with expression of p27, the primary target of the SCFSkp2 E3-ligase, in neuroblastoma tumors. Conclusion: Skp2 may have a key role in the progression of neuroblastomas and should make an attractive target for therapeutic approaches.

Fluorescence in situ hybridization analyses of chromosome band 1p36 in neuroblastoma detect two classes of alterations.

Chromosomal alterations in 1p36 were investigated in 196 neuroblastoma tumors using fluorescence in situ hybridization. Additionally, by using the same technique, it was determined whether MYCN was amplified in 149 of these. The most frequent finding was a deletion in 1p36, leading to monosomy of this region (29 cases, 15%). Furthermore, we found tumors with at least two intact copies of chromosome 1 and additional 1p36‐deleted copies. Altogether, 21 tumors (11%) displayed this imbalance of 1p36. Similar to the cases with deletion, imbalances were predominantly found in stage 4 tumors (81%), and they were significantly associated with an increased patient age (P = 0.01). Nearly all 1p‐deleted tumors showed amplification of MYCN (24/27 analyzed samples, 89%), whereas only 8 of 21 (38%) with imbalance did. Eight cases with imbalance were investigated for loss of heterozygosity (LOH) using microsatellite markers in 1p35–36. Only 4 displayed 1p36 LOH, whereas the remaining 4 were heterozygous. Both patients with deletion of 1p and with imbalance had a poor outcome [3‐year rate of event‐free‐survival (EFS): 33±15% and 41±15%], which was significantly worse compared to the outcome of patients without 1p alterations (3‐year EFS: 70±5%; P = 0.01 and P = 0.0059). We conclude that besides monosomic short arm deletions, imbalance of 1p36 is a strong marker of a poor prognosis in neuroblastoma and not necessarily associated with MYCN amplification and LOH.

Frequency and diagnostic patterns of lymphomas in liver biopsies with respect to the WHO classification

The recent World Health Organization (WHO) classification of hematopoietic and lymphoid tissue tumors represents the first worldwide consensus classification of these malignancies. However, the applicability of this classification to a representative number of hepatic lymphomas in liver biopsy specimens has not yet been investigated. The frequency and infiltration pattern of a series of 205 liver biopsies with lymphoma manifestations was analyzed with the aid of immunohistochemical and molecular pathological analyses. Diffuse large B-cell lymphoma (DLBCL) was by far the most frequent entity, comprising 45% of the cases analyzed. Using a previously published immunohistochemical algorithm, 35% of 80 DLBCL were assigned to a germinal center B-cell-like (GCB) and 65% to a non-GCB group. Most B-cell lymphoma entities involving the liver revealed a characteristic infiltration pattern. Diagnostically challenging entities were T-cell-rich B-cell lymphomas, anaplastic large cell lymphomas and peripheral T-cell lymphomas, which frequently required additional molecular clonality assessment. Overall, the percentage of T-cell lymphomas in the liver (12%) was higher as compared to other extranodal sites except for the skin and the small intestine. This study provides relevant data on the distribution of hepatic lymphomas and demonstrates the applicability of the WHO classification proposing a diagnostic algorithm for liver biopsies.

Lacking immunocytological GD2 expression in neuroblastoma: report of 3 cases.

Immunocytological bone marrow assessment for contamination with neuroblastoma cells is based on their characteristic GD2 surface staining. Neuroblastoma without GD2 expression have been rarely and only after antibody therapy reported. Conventional cytology was performed using Pappenheim staining. For immunocytology, the APAAP method was utilized with the 14G2a anti‐GD2 mouse monoclonal antibody. 7 × 105 cells on cytospin preparations were investigated. In 2003, 288 bone marrow samples from 191 neuroblastoma patients were investigated by cytology and immunocytology. Three cases demonstrated GD2 negativity on cytologically unambiguous neuroblastoma cells. Two female cases (94 and 37 months of age) with stage 4 neuroblastoma had GD2 expressing neuroblastoma cells in bone marrow at diagnosis. At 2nd relapse 25 and 23 months after diagnosis and 8 months and 12 months after anti‐GD2 antibody treatment (ch14.18), the bone marrow infiltrating neuroblastoma cells lacked GD2 staining. The third patient, a 63‐month‐old girl with bone marrow replacement by neuroblastoma cells showed at diagnosis a mixture of GD2‐unstained tumor clumps and very weakly stained neuroblastoma cells. Neuroblastoma cells may lack GD2 expression at diagnosis and at recurrence. This observation has diagnostic and therapeutic implications.

TP53 alterations in Wilms tumour represent progression events with strong intratumour heterogeneity that are closely linked but not limited to anaplasia

TP53 mutations have been associated with anaplasia in Wilms tumour, which conveys a high risk for relapse and fatal outcome. Nevertheless, TP53 alterations have been reported in no more than 60% of anaplastic tumours, and recent data have suggested their presence in tumours that do not fulfil the criteria for anaplasia, questioning the clinical utility of TP53 analysis. Therefore, we characterized the TP53 status in 84 fatal cases of Wilms tumour, irrespective of histological subtype. We identified TP53 alterations in at least 90% of fatal cases of anaplastic Wilms tumour, and even more when diffuse anaplasia was present, indicating a very strong if not absolute coupling between anaplasia and deregulation of p53 function. Unfortunately, TP53 mutations do not provide additional predictive value in anaplastic tumours since the same mutation rate was found in a cohort of non‐fatal anaplastic tumours. When classified according to tumour stage, patients with stage I diffuse anaplastic tumours still had a high chance of survival (87%), but this rate dropped to 26% for stages II–IV. Thus, volume of anaplasia or possible spread may turn out to be critical parameters. Importantly, among non‐anaplastic fatal tumours, 26% had TP53 alterations, indicating that TP53 screening may identify additional cases at risk. Several of these non‐anaplastic tumours fulfilled some criteria for anaplasia, for example nuclear unrest, suggesting that such partial phenotypes should be under special scrutiny to enhance detection of high‐risk tumours via TP53 screening. A major drawback is that these alterations are secondary changes that occur only later in tumour development, leading to striking intratumour heterogeneity that requires multiple biopsies and analysis guided by histological criteria. In conclusion, we found a very close correlation between histological signs of anaplasia and TP53 alterations. The latter may precede development of anaplasia and thereby provide diagnostic value pointing towards aggressive disease.