Jean Michon

Somatic and germline activating mutations of the ALK kinase receptor in neuroblastoma

Neuroblastoma, a tumour derived from the peripheral sympathetic nervous system, is one of the most frequent solid tumours in childhood. It usually occurs sporadically but familial cases are observed, with a subset of cases occurring in association with congenital malformations of the neural crest being linked to germline mutations of the PHOX2B gene. Here we conducted genome-wide comparative genomic hybridization analysis on a large series of neuroblastomas. Copy number increase at the locus encoding the anaplastic lymphoma kinase (ALK) tyrosine kinase receptor was observed recurrently. One particularly informative case presented a high-level gene amplification that was strictly limited to ALK, indicating that this gene may contribute on its own to neuroblastoma development. Through subsequent direct sequencing of cell lines and primary tumour DNAs we identified somatic mutations of the ALK kinase domain that mainly clustered in two hotspots. Germline mutations were observed in two neuroblastoma families, indicating that ALK is a neuroblastoma predisposition gene. Mutated ALK proteins were overexpressed, hyperphosphorylated and showed constitutive kinase activity. The knockdown of ALK expression in ALK-mutated cells, but also in cell lines overexpressing a wild-type ALK, led to a marked decrease of cell proliferation. Altogether, these data identify ALK as a critical player in neuroblastoma development that may hence represent a very attractive therapeutic target in this disease that is still frequently fatal with current treatments.

A new member of the ETS family fused to EWS in Ewing tumors

As a result of chromosome translocations, the EWS gene is fused to a variety of transcription factors in human solid neoplasia. In Ewing tumors EWS can be fused to four different members of the ETS family, namely FLI-1, ERG, ETV1 and E1AF. We have identified a new member of the ETS family, called FEV, which is fused to EWS in a subset of Ewing tumors. FEV encodes a 238 amino acid protein which contains an ETS DNA binding domain closely related to that of FLI-1 and ERG. However, the N-terminal portion of FEV is only 42 amino acids long which suggests that FEV is lacking important transcription regulatory domains contained in FLI-1 and ERG N-terminal parts. The C-terminal end of FEV is rich in alanine residues which may indicate that FEV is a transcription repressor. The FEV gene is encoded by three exons and is located on chromosome 2. FEV expression was only detected in adult prostate and small intestine but not in other adult nor in fetal tissues, thus indicating that FEV has a restricted expression pattern. Following a scheme similar to previously described translocations in Ewing tumors, a t(2;22) chromosome translocation fuses the N-terminal domain of EWS to the ETS DNA binding domain of FEV.

Primary Disseminated Multifocal Ewing Sarcoma: Results of the Euro-EWING 99 Trial

PURPOSE To improve the poor prognosis of patients with primary disseminated multifocal Ewing sarcomas (PDMES) with a dose-intense treatment concept. PATIENTS AND METHODS From 1999 to 2005, 281 patients with PDMES were enrolled onto the Euro-EWING 99 R3 study. Median age was 16.2 years (range, 0.4 to 49 years). Recommended treatment consisted of six cycles of vincristine, ifosfamide, doxorubicin, and etoposide (VIDE), one cycle of vincristine, dactinomycin, and ifosfamide (VAI), local treatment (surgery and/or radiotherapy), and high-dose busulfan-melphalan followed by autologous stem-cell transplantation (HDT/SCT). RESULTS After a median follow-up of 3.8 years, event-free survival (EFS) and overall survival (OS) at 3 years for all 281 patients were 27% +/- 3% and 34% +/- 4% respectively. Six VIDE cycles were completed by 250 patients (89%); 169 patients (60%) received HDT/SCT. The estimated 3-year EFS from the start of HDT/SCT was 45% for 46 children younger than 14 years. Cox regression analyses demonstrated increased risk at diagnosis for patients older than 14 years (hazard ratio [HR] = 1.6), a primary tumor volume more than 200 mL (HR = 1.8), more than one bone metastatic site (HR = 2.0), bone marrow metastases (HR = 1.6), and additional lung metastases (HR = 1.5). An up-front risk score based on these HR factors identified three groups with EFS rates of 50% for score <or= 3 (82 patients), 25% for score more than 3 to less than 5 (102 patients), and 10% for score >or= 5 (70 patients; P < .0001). CONCLUSION PDMES patients may survive with intensive multimodal therapy. Age, tumor volume, and extent of metastatic spread are relevant risk factors. A score based on these factors may facilitate risk-adapted treatment approaches.

Overall Genomic Pattern Is a Predictor of Outcome in Neuroblastoma

PURPOSE For a comprehensive overview of the genetic alterations of neuroblastoma, their association and clinical significance, we conducted a whole-genome DNA copy number analysis. PATIENTS AND METHODS A series of 493 neuroblastoma (NB) samples was investigated by array-based comparative genomic hybridization in two consecutive steps (224, then 269 patients). RESULTS Genomic analysis identified several types of profiles. Tumors presenting exclusively whole-chromosome copy number variations were associated with excellent survival. No disease-related death was observed in this group. In contrast, tumors with any type of segmental chromosome alterations characterized patients with a high risk of relapse. Patients with both numerical and segmental abnormalities clearly shared the higher risk of relapse of segmental-only patients. In a multivariate analysis, taking into account the genomic profile, but also previously described individual genetic and clinical markers with prognostic significance, the presence of segmental alterations with (HR, 7.3; 95% CI, 3.7 to 14.5; P < .001) or without MYCN amplification (HR, 4.5; 95% CI, 2.4 to 8.4; P < .001) was the strongest predictor of relapse; the other significant variables were age older than 18 months (HR, 1.8; 95% CI, 1.2 to 2.8; P = .004) and stage 4 (HR, 1.8; 95% CI, 1.2 to 2.7; P = .005). Finally, within tumors showing segmental alterations, stage 4, age, MYCN amplification, 1p and 11q deletions, and 1q gain were independent predictors of decreased overall survival. CONCLUSION The analysis of the overall genomic pattern, which probably unravels particular genomic instability mechanisms rather than the analysis of individual markers, is essential to predict relapse in NB patients. It adds critical prognostic information to conventional markers and should be included in future treatment stratification.

Safety assessment of intensive induction with vincristine, ifosfamide, doxorubicin, and etoposide (VIDE) in the treatment of Ewing tumors in the EURO‐E.W.I.N.G. 99 clinical trial

The EUROpean Ewing tumour Working Initiative of National Groups 1999 (EURO‐E.W.I.N.G. 99) protocol prescribes six courses of vincristine, ifosfamide, doxorubicin, and etoposide (VIDE) as intensive induction chemotherapy for Ewing tumors (ET). Granulocyte‐colony stimulating factor (G‐CSF) is recommended. Adverse reactions (AR) were evaluated; quality assurance of data collection reviewed.

N-Myc gene amplification is a major prognostic factor in localized neuroblastoma: results of the French NBL 90 study. Neuroblastoma Study Group of the Société Francaise d'Oncologie Pédiatrique.

PURPOSE To assess the relevance of N-Myc gene amplification (NMA) as a prognostic factor in localized neuroblastoma (NB) and to evaluate whether less intensive adjuvant treatment is advisable in infants without NMA. PATIENTS AND METHODS Assessment of NBs included clinical and imaging data to allow tumor-node-metastasis (TNM) staging, biologic determinations (N-Myc gene analysis), and standard histology and work-up to eliminate metastatic spread (metaiodobenzylguanidine [MIBG] scintigraphy and extensive bone marrow staging). Resectability was defined according to imaging findings. Chemotherapy was indicated in children older than 1 year at diagnosis who had postoperative residual disease or lymph node (LN) involvement, in infants with NMA, or as primary treatment in children with an unresectable NB, including dumbbell tumors. Radiotherapy was recommended in children older than 1 who presented with persistent gross residual disease at the end of therapy. RESULTS Between 1990 and 1994, 316 consecutive children who presented with a localized NB were registered in the NBL 90 study. The median age was 12 months, and 42 patients had dumbbell tumors (13%). NMA was found in 22 of 225 assessable children (10%) and correlated with adverse prognostic indicators such as age older than 1 year, an abdominal primary tumor, a large tumor (T3), and unresectability. Among 186 children who had primary excision, five died of surgery-related complications. Primary chemotherapy was given to 130 patients, which allowed removal of the tumor in all but four. The 5-year overall survival (OS) and event-free survival (EFS) rates were, respectively, 91% and 84% with a median follow-up time of 36 months. The outcome of infants and older children was similar (P = .2). EFS of patients with resectable tumors was slightly better than with unresectable primary tumors (EFS, 89% v 78%; P = .02). In dumbbell NBs, neurologic recovery was achieved in 74% of cases that presented with symptoms, and initial laminectomy was avoided in 75% of children. In a univariate analysis, large tumors, high neuron-specific enolase (NSE) and lactate dehydrogenase (LDH) levels, positive LNs, macroscopic residue, and NMA adversely influenced outcome. In the multivariate analysis, NMA was the most powerful unfavorable predictive indicator: OS and EFS rates for these children were 36% and 32%, compared with 98% and 90% in nonamplified tumors (P < .001). CONCLUSION Our data confirm the overall good prognosis of localized NBs, even when unresectable. NMA is the most relevant adverse prognostic factor in localized NBs, and more intensive treatment should be investigated in these patients. Prospective studies of other biologic factors are warranted to tailor therapy more accurately. The EFS of children who underwent primary surgery was excellent, and further justifies elimination of adjuvant treatment provided they have no NMA. Despite the elimination of postoperative therapy, infants with non-NMA tumors have an excellent outcome, which suggests that initial chemotherapy can be further reduced in case of unresectable NBs.

Prognostic factors in localized Ewing's tumours and peripheral neuroectodermal tumours: the third study of the French Society of Paediatric Oncology (EW88 study)

Purpose: (1) To improve survival rates in patients with Ewings sarcoma (ES) or peripheral neuroectodermal tumours (PNET) using semi-continuous chemotherapy and aiming to peform surgery in all; (2) To identify early prognostic factors to tailor therapy for future studies. Patients and methods One hundred and forty-one patients were entered onto the trial between January 1988 and December 1991. Induction therapy consisted of five courses of Cytoxan, 150 mg/m2 × 7 days, followed by Doxorubicin, 35 mg/m2 i.v on day 8 given at short intervals. Surgery was recommended whenever possible. The delivery of radiation therapy was based on the quality of resection and the histological response to CT. Maintenance chemotherapy consisted of vincristine + actinomycin and cytoxan + doxorubicin. The total duration of therapy was 10 months. Results After a median follow-up of 8.5 years, the projected overall survival at 5 years was 66% and disease-free survival (DFS) was 58%. In patients treated by surgery, only the histological response to CT had an influence on survival: 75% DFS for patients with a good histological response (less than 5% of cells), 48% for intermediate responders and only 20% for poor responders (≥ 30% of cells), P < 0.0001. The initial tumor volume by itself had no influence on DFS in these patients. In contrast, the tumour volume had a strong impact on DFS in patients treated by radiation therapy alone. Age had no impact on outcome. Conclusion Therapeutic trials for localized Ewings sarcoma should be based on the histological response to chemotherapy or on the tumour volume according to the modality used for local therapy.

Multivariate analysis of risk factors in stage 4 neuroblastoma patients over the age of one year treated with megatherapy and stem-cell transplantation: a report from the European Bone Marrow Transplantation Solid Tumor Registry.

PURPOSE The European Bone Marrow Transplantation (EBMT) Solid Tumor Registry (STR) contains detailed information on children with advanced neuroblastoma who, after standard-dose induction chemotherapy and surgery, received myeloablative megatherapy (MGT) followed by stem-cell transplantation (SCT). This data base was analyzed to identify factors that predict event-free survival (EFS). PATIENTS AND METHODS Eligibility criteria were stage IV neuroblastoma, age over 1 year at diagnosis, and no relapse before MGT/SCT. Between February 1978 and July 1992, 549 patients were registered by 36 European transplant centers. The median age at diagnosis was 36 months (range, 13 to 216 months) and the male-female ratio was 1:45. Before MGT, 157 patients were in complete remission (CR), 156 in very good partial remission (VGPR), and 208 in partial remission (PR), whereas 24 had had only a minor response (MR). One hundred ten of 546 patients had undergone two successive MGT procedures. The median observation time was 60 months (range, 12 to 187 months). RESULTS Actuarial EFS is 26% at 5 years. Multivariate analysis by the Cox proportional hazards regression model included 529 patients with complete data sets. After adjustment for treatment duration before MGT and double MGT procedures, two adverse, independent risk factors that influenced EFS were identified: (1) persisting skeletal lesions before MGT as defined by technetium (99TC) scans and/or meta-iodobenzylguanidine (mIBG) scans (P = .004) and (2) persisting bone marrow involvement before MGT (P = .03). CONCLUSION After induction treatment, persisting skeletal disease as defined above and persisting bone marrow involvement may be predictive of a particularly poor outcome. Physicians may consider this an additional important tool to decide the patients management.

Ovarian function after bone marrow transplantation during childhood

Ovarian failure is often brought about by the conditioning protocol used for bone marrow transplantation (BMT). We monitored ovarian function in 31 girls conditioned for BMT at 10.3 ±  0.6 (s.e., 3.2–17.5) years by chemotherapy alone (group 1, n = 8) or chemotherapy plus body irradiation (12 Gy, fractionated in group 2, n = 9, or 10 Gy single total body in group 3, n = 7, and 5 or 6 Gy single thoraco-abdominal in group 4, n = 7, irradiation) at 13.4 ± 0.4 (11.7–18.6) years. Breast development was normal (n = 11), did not occur (n = 14), or did not progress spontaneously (n = 2) after BMT. The other four girls who menstruated before BMT had permanent amenorrhea. Basal plasma gonadotropin concentrations were measured in 29; follicle- stimulating hormone was increased in them all and luteinizing hormone in 23. At the last clinical evaluation at 16.3 ± 0.4 (12.1–21.6) years, 23 girls had complete ovarian failure, two had partial ovarian failure, and six had normal ovarian function. Three of these were the youngest group 1 patients and those who had not received busulfan. We conclude that conditioning for BMT given during childhood frequently prevents normal estrogen secretion at puberty. Adequate substitutive treatment may be necessary to induce growth acceleration and sexual development.

Genomic landscape of Ewing sarcoma defines an aggressive subtype with co-association of STAG2 and TP53 mutations

UNLABELLED Ewing sarcoma is a primary bone tumor initiated by EWSR1-ETS gene fusions. To identify secondary genetic lesions that contribute to tumor progression, we performed whole-genome sequencing of 112 Ewing sarcoma samples and matched germline DNA. Overall, Ewing sarcoma tumors had relatively few single-nucleotide variants, indels, structural variants, and copy-number alterations. Apart from whole chromosome arm copy-number changes, the most common somatic mutations were detected in STAG2 (17%), CDKN2A (12%), TP53 (7%), EZH2, BCOR, and ZMYM3 (2.7% each). Strikingly, STAG2 mutations and CDKN2A deletions were mutually exclusive, as confirmed in Ewing sarcoma cell lines. In an expanded cohort of 299 patients with clinical data, we discovered that STAG2 and TP53 mutations are often concurrent and are associated with poor outcome. Finally, we detected subclonal STAG2 mutations in diagnostic tumors and expansion of STAG2-immunonegative cells in relapsed tumors as compared with matched diagnostic samples. SIGNIFICANCE Whole-genome sequencing reveals that the somatic mutation rate in Ewing sarcoma is low. Tumors that harbor STAG2 and TP53 mutations have a particularly dismal prognosis with current treatments and require alternative therapies. Novel drugs that target epigenetic regulators may constitute viable therapeutic strategies in a subset of patients with mutations in chromatin modifiers.