Background: In neuroblastoma (NB), the ALK receptor tyrosine kinase can be
constitutively activated either through genomic amplification or activating point
mutations. We studied ALK genetic alterations in high-risk NB patients to determine
their frequency and prognostic impact.N/

Ambros, Inge

Ambros, Peter  F.

Avigad, Smadar

Baulande, Sylvain

Beiske, Klaus

Bellini, Angela

Bernard, Virginie

Betts, David

Bown, Nick

Chicard, Mathieu

Clement, Nathalie

Combaret, Valérie

de Preter, Katleen

Defferrari, Raffaella

Delattre, Olivier

Font de Mora, Jaime

Irene, Jimenez

Jaydutt, Bhalshankar

Jeison, Marta

Ladenstein, Ruth

Lapouble, Eve

Leprovost, Nada

Marques, Barbara

Martinsson, Tommy

Mazzocco, Katia

Michon, Jean

Morini, Martina

Mühlethaler-Mottet, Annick

Noguera, Rosa

Pierron, Gaelle

Rossing, Caroline Maria

Schleiermacher, Gudrun

Tweddle, Deborah

Valteau-Couanet, Dominique

Vicha, Ales

English

Background: In neuroblastoma (NB), the ALK receptor tyrosine kinase can be constitutively activated through genomic amplification or activating point mutations. We studied ALK genetic alterations in high-risk NB patients to determine their frequency and prognostic impact.

Methods: Diagnostic NB samples from 1039 patients enrolled in the SIOPEN-HR-NBL1 trial were studied to determine the ALK amplification status (copy number analysis; n=337), the ALK mutational profile (Sanger and/or NGS including deep sequencing, n=203) or both (n=499).
The sensitivity of ALK mutated/ALK amplified or ALK wildtype NB cell lines ((CLB-GA (R1275Q), CLB-GE (F1174V; ALK-A), SKNBE-2C (ALK wt)) to simultaneous or consecutive combinations of ALK TKIs (crizotinib/lorlatinib) and/or chemotherapy (Etoposide and Doxorubicin) was then tested. 

Results: Genomic ALK amplifications were detected in 4.4% of cases (37/836); all but 2 showed MYCN amplification. ALK mutations were detected at a clonal level (>20% mutated allele fraction, MAF) in 9.8% of cases (69/702) (F1174 n=25, R1275 n=32, both F1174 and R1275 n=1, F1245 n=6, others n=5) and at a subclonal level (MAF 0.5-20%) in 3.7% of patients (22/586) (F1174 n=11, R1275 n=6, both F1174 and R1275 or F1174 and F1245 n=3, other n=2). 

A significantly poorer OS and EFS was observed in cases with clonal ALK mutations, versus all others (3-years OS 47% +/-6.4% versus 65% +/-2%, logrank, p< 0.0001) and in those with ALK amplifications, versus all others (3-years OS 31% +/-8.5% versus 66% +/- 1.9%; logrank, p<0.0001). 

A Cox proportional hazards procedure (450 patients with complete clinical/biological datasets) retained stage 4 disease (as opposed to non-stage 4) and ALK amplification as factors with a higher hazard of relapse/progression (hazard 2.3 and 2.2, respectively), whereas ALK mutation, MYCN amplification and age>18 months were not retained.

The consecutive treatment of Doxorubicin followed by Lorlatinib had a synergistic effect in ALK mutated/amplified NB cell lines.

Conclusion: Genetic alterations of ALK (clonal mutations, amplifications) in high-risk NB patients are associated with poorer survival. Further preclinical data are required to determine optimal treatment modalities for integration of TKI in upfront treatment strategies of HR NB patients with ALK alterations.N/

F. Ambros, Peter

Scientific Repository of the National Health Institute Doutor Ricardo Jorge

Genetic alterations of ALK in high-risk neuroblastoma patients. A SIOPEN study

Repositório Científico do Instituto Nacional de Saúde

Abstract ANR 2018: ALK SIOPEN   Genetic alterations of ALK in high-risk neuroblastoma patients. A SIOPEN study.  Angela Bellini1, Virginie Bernard1, Inge Ambros2, Peter F. Ambros2, Katleen de Preter3, Valérie Combaret4, Klaus Beiske5, Marta Jeison6, Barbara Marques7, Martina Morini8, Katia Mazzocco8, Raffaella Defferrari8, David Betts9, Tommy Martinsson10, Annick Mühlethaler-Mottet11, Rosa Noguera12, Jaime Font de Mora12, Ales Vicha13, Ruth Ladenstein14, Dominique Valteau-Couanet15, Caroline Maria Rossing16, Nick Bown17, Deborah Tweddle18, Smadar Avigad19, Eve Lapouble1, Mathieu Chicard1, Nada Leprovost1, Nathalie Clement1, Sylvain Baulande1, Gaelle Pierron1, Jimenez Irene1, Bhalshankar Jaydutt1, Olivier Delattre1, Jean Michon1, Gudrun Schleiermacher1  1Institut Curie, Paris, France 2Children’s Cancer Research Institute, Vienna, Austria 3Ghent University, Ghent, Belgium 4Centre Léon Berard, Lyon, France 5Oslo University Hospital, Oslo, Norway 6Schneider Children's Medical Center of Israel, Tel Aviv University, Israel 7 Instituto Nacional de Saúde Doutor Ricardo Jorge, Lisboa, Portugal 8Istituto G. Gaslini, Genoa, Italy 9 Department of Clinical Genetics, Our Lady's Children's Hospital, Dublin, Ireland 10Clinical Genetics, Sahlgrenska Academy at the University of Gothenburg 11University Hospital CHUV, Lausanne, Switzerland 12Hospital Universitario y Politécnico La Fe, Valencia, Spain 13Charles University and University Hospital Kralovske Vinohrady, Prague, Czech Republic 14St Anna Children’s Hospital, Vienna, Austria 15Gustave Roussy, Villejuif, France 16Center for Genomic Medicine, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark 17Northern Genetics Service, Newcastle upon Tyne, Newcastle, UK 18Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne NE1 7RU, UK 19Schneider Children's Medical Center of Israel, Pediatric Hematology Oncology, Petah Tikva, Israel.   Background: In neuroblastoma (NB), the ALK receptor tyrosine kinase can be constitutively activated either through genomic amplification or activating point mutations. We studied ALK genetic alterations in high-risk NB patients to determine their frequency and prognostic impact.  Methods: Diagnostic NB samples from 1039 patients enrolled in the SIOPEN-HR-NBL1 trial were studied to determine the ALK amplification status (copy number analysis; n=337), the ALK mutational profile (Sanger and/or NGS including deep sequencing covering hotspots in exons 23-25, n=203) or both (n=499).  Results: High level genomic ALK amplifications were detected in 4.4% of cases (37/836); all but 2 showed MYCN amplification. As for MYCN amplification, ALK amplification was more frequently observed in children aged<18 months at diagnosis (p=0.01). No correlation with the primary tumor site was observed.  ALK mutations were detected at a clonal level (>10% mutated allele fraction, MAF) in 9.8% of cases (69/702) (F1174 n= 25, R1275 n=32, both F1174 and R1275 n=1, F1245 n=6, others n=5). Additionally, 3.7% of patients (22/586 by NGS) harbored ALK mutations at a subclonal level (MAF 0.5-10%) (F1174 n=11, R1275 n=6, both F1174 and R1275 or F1174 and F1245 n=3, other n=2). Although not statistically significant, ALK mutations were observed slightly more frequently in non-adrenal compared to adrenal primary tumors (p=0.08).   Whereas no statistically significant difference in survival was observed between patients with and without ALK mutations, patients with ALK amplification had a significantly poorer event free (EFS) and overall survival compared to those without ALK amplification (logrank, p<0.0001).   A multivariate analysis was performed to determine which parameters independently predicted EFS in this high risk population. Among 450 patients with complete datasets, a Cox proportional hazards procedure retained stage 4 disease (as opposed to non-stage 4) and ALK amplification as factors with a higher hazard of relapse/progression (hazard 2.3 and 2.2, respectively), whereas ALK mutation, MYCN amplification and age>18 months were not retained.  Conclusion: Taking into account amplifications, clonal and subclonal mutations, genetic alterations of ALK were observed in tumor samples of 17% of high-risk NB patients, of importance when considering ALK targeted therapies. Among the different genetic alterations, only ALK amplification predicted poorer survival.   

Genetic alterations of ALK in high-risk neuroblastoma patients: a SIOPEN study

Abstract SIOP 2019: ALK SIOPEN   Genetic alterations of ALK in high-risk neuroblastoma patients. A SIOPEN study.  Angela Bellini1, Virginie Bernard1, Inge Ambros2, Peter F. Ambros2, Katleen de Preter3, Valérie Combaret4, Klaus Beiske5, Marta Jeison6, Barbara Marques7, Martina Morini8, Katia Mazzocco8, Raffaella Defferrari8, David Betts9, Tommy Martinsson10, Annick Mühlethaler-Mottet11, Rosa Noguera12, Jaime Font de Mora12, Ales Vicha13, Ruth Ladenstein14, Dominique Valteau-Couanet15, Caroline Maria Rossing16, Nick Bown17, Deborah Tweddle18, Smadar Avigad19, Eve Lapouble1, Mathieu Chicard1, Nada Leprovost1, Nathalie Clement1, Sylvain Baulande1, Gaelle Pierron1, Jimenez Irene1, Bhalshankar Jaydutt1, Olivier Delattre1, Jean Michon1, Gudrun Schleiermacher1  1Institut Curie, Paris, France 2Children’s Cancer Research Institute, Vienna, Austria 3Ghent University, Ghent, Belgium 4Centre Léon Berard, Lyon, France 5Oslo University Hospital, Oslo, Norway 6Schneider Children's Medical Center of Israel, Tel Aviv University, Israel 7 Instituto Nacional de Saúde Doutor Ricardo Jorge, Lisboa, Portugal 8Istituto G. Gaslini, Genoa, Italy 9 Department of Clinical Genetics, Our Lady's Children's Hospital, Dublin, Ireland 10Clinical Genetics, Sahlgrenska Academy at the University of Gothenburg 11University Hospital CHUV, Lausanne, Switzerland 12Hospital Universitario y Politécnico La Fe, Valencia, Spain 13Charles University and University Hospital Kralovske Vinohrady, Prague, Czech Republic 14St Anna Children’s Hospital, Vienna, Austria 15Gustave Roussy, Villejuif, France 16Center for Genomic Medicine, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark 17Northern Genetics Service, Newcastle upon Tyne, Newcastle, UK 18Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne NE1 7RU, UK 19Schneider Children's Medical Center of Israel, Pediatric Hematology Oncology, Petah Tikva, Israel.   Background: In neuroblastoma (NB), the ALK receptor tyrosine kinase can be constitutively activated through genomic amplification or activating point mutations. We studied ALK genetic alterations in high-risk NB patients to determine their frequency and prognostic impact.  Methods: Diagnostic NB samples from 1039 patients enrolled in the SIOPEN-HR-NBL1 trial were studied to determine the ALK amplification status (copy number analysis; n=337), the ALK mutational profile (Sanger and/or NGS including deep sequencing, n=203) or both (n=499). The sensitivity of ALK mutated/ALK amplified or ALK wildtype NB cell lines ((CLB-GA (R1275Q), CLB-GE (F1174V; ALK-A), SKNBE-2C (ALK wt)) to simultaneous or consecutive combinations of ALK TKIs (crizotinib/lorlatinib) and/or chemotherapy (Etoposide and Doxorubicin) was then tested.   Results: Genomic ALK amplifications were detected in 4.4% of cases (37/836); all but 2 showed MYCN amplification. ALK mutations were detected at a clonal level (>20% mutated allele fraction, MAF) in 9.8% of cases (69/702) (F1174 n=25, R1275 n=32, both F1174 and R1275 n=1, F1245 n=6, others n=5) and at a subclonal level (MAF 0.5-20%) in 3.7% of patients (22/586) (F1174 n=11, R1275 n=6, both F1174 and R1275 or F1174 and F1245 n=3, other n=2).   A significantly poorer OS and EFS was observed in cases with clonal ALK mutations, versus all others (3-years OS 47% +/-6.4% versus 65% +/-2%, logrank, p< 0.0001) and in those with ALK amplifications, versus all others (3-years OS 31% +/-8.5% versus 66% +/- 1.9%; logrank, p<0.0001).   A Cox proportional hazards procedure (450 patients with complete clinical/biological datasets) retained stage 4 disease (as opposed to non-stage 4) and ALK amplification as factors with a higher hazard of relapse/progression (hazard 2.3 and 2.2, respectively), whereas ALK mutation, MYCN amplification and age>18 months were not retained.  The consecutive treatment of Doxorubicin followed by Lorlatinib had a synergistic effect in ALK mutated/amplified NB cell lines.  Conclusion: Genetic alterations of ALK (clonal mutations, amplifications) in high-risk NB patients are associated with poorer survival. Further preclinical data are required to determine optimal treatment modalities for integration of TKI in upfront treatment strategies of HR NB patients with ALK alterations.   

http://repositorio.insa.pt/bitstream/10400.18/5900/1/Abstract%20ALK%20SIOPEN%20SIOPEM%20Oct2018_submittedVers.pdf

Genetic alterations of ALK in high-risk neuroblastoma patients: a SIOPEN study

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Scientific Repository of the National Health Institute Doutor Ricardo Jorge

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Repositório Científico do Instituto Nacional de Saúde