mRNA Expression Level of ALK in Neuroblastoma Is Associated with Histological Subtype, ALK Mutations and ALK Immunohistochemical Protein Expression
, Marta F. Fiocco 1,2,3, Wendy W. J. de Leng 4, Lennart A. Kester 1, Karin P. S. Langenberg 1, Godelieve A. M. Tytgat 1, Max M. van Noesel 1,5, Marc H. W. A. Wijnen 1, Alida F. W. van der Steeg 1,†and Ronald R. de Krijger 1,4,†Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis study evaluates the association between mRNA expression levels of ALK in neuroblastoma and compares to histologic subtype, ALK mutations, and ALK IHC protein expression. The background nicely describes the role of ALK in neuroblastoma and the correlation with more aggressive disease.
While mRNA ALK expression was not associated with EFS or OS, it was associated with more undifferentiated tumors. Association between ALK mRNA expression and protein expression by IHC was also shown.
This paper would be stronger if it included clinical connection in terms of how the information would be best utilized for patient care. Would the mRNA be an addition to current risk classification?
Author Response
Comment 1: This study evaluates the association between mRNA expression levels of ALK in neuroblastoma and compares to histologic subtype, ALK mutations, and ALK IHC protein expression. The background nicely describes the role of ALK in neuroblastoma and the correlation with more aggressive disease. While mRNA ALK expression was not associated with EFS or OS, it was associated with more undifferentiated tumors. Association between ALK mRNA expression and protein expression by IHC was also shown. This paper would be stronger if it included clinical connection in terms of how the information would be best utilized for patient care. Would the mRNA be an addition to current risk classification?
Response 1: We would like to thank you for the comments on our manuscript. We appreciate the time and effort you have dedicated to provide us with feedback. We agree with your comment to emphasize the clinical connecting. Details regarding the clinical connection can be found in the conclusion (conclusion, paragraph 1, page 12, lines 414-422).
Again, we would like to thank you for giving us the opportunity to strengthen our manuscript.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors studied the relationship between mRNA expression levels of ALK and survival in children with neuroblastoma. They also investigated whether ALK mRNA expression levels correlate with its protein expression using IHC, and whether ALK expression is associated with differentiation, ALK amplification, or mutation.
Their findings show an association between ALK mRNA expression and both ALK immunohistochemical protein expression and histological subtypes of neuroblastomas. Furthermore, their results show that mRNA expression levels are higher in ALK mutated neuroblastomas and seem to be much higher in ALK amplified neuroblastomas.
Overall, this is a well-executed study with some interesting findings. However, the authors are urged to provide some Figures containing findings from the FISH, Single Nucleotide Polymorphism array, Whole exome sequencing, and Whole transcriptome sequencing experiments.
Author Response
Comment 1: The authors studied the relationship between mRNA expression levels of ALK and survival in children with neuroblastoma. They also investigated whether ALK mRNA expression levels correlate with its protein expression using IHC, and whether ALK expression is associated with differentiation, ALK amplification, or mutation. Their findings show an association between ALK mRNA expression and both ALK immunohistochemical protein expression and histological subtypes of neuroblastomas.
Furthermore, their results show that mRNA expression levels are higher in ALK mutated neuroblastomas and seem to be much higher in ALK amplified neuroblastomas. Overall, this is a well-executed study with some interesting findings.
However, the authors are urged to provide some Figures containing findings from the FISH, Single Nucleotide Polymorphism array, Whole exome sequencing, and Whole transcriptome sequencing experiments.
Response 1: We would like to thank you for the comments on our manuscript. We appreciate the time and effort you have dedicated to provide us with feedback. We agree that figures visualizing the findings of molecular diagnostics would improve our manuscript. We have added an image of a MYCN amplification detected by FISH (figure 2, materials & methods, section 2.3, page 5, lines 176-177) and an ALK amplification (and MYCN amplification) detected by SNP-array (figure 3, materials & methods, section 2.4, page 5, lines 193-194) and WES (figure 4, materials & methods, section 2.6, page 6, lines 228-229). The total amount of cases in which theses aberrations were detected can be found in table 2. For Whole Transcriptome Sequencing we cannot provide an image that would add to the manuscript.
Again, we would like to thank you for giving us the opportunity to strengthen our manuscript.
Reviewer 3 Report
Comments and Suggestions for AuthorsIn this manuscript, the authors characterize ALK expression in neuroblastoma and explore its clinical and pathologic significance. Overall, the study is very well done and very well-written. The main limitation of the study, which the authors acknowledge in line 391, is the relatively small study population, which limits the significance of the findings. Nonetheless, the study describes an important relationship between ALK mRNA expression, IHC findings, histological subtype, and other molecular findings. A few minor comments:
Line 195-198, the ACMG system is used to classify variants. This system is more designed for germline findings rather than classification of somatic alterations, as the criteria used in this classification system relies upon data, studies, and findings in the germline setting. The AMP/ASCO/CAP tiering system (Li et al, JMD 2017) may be more appropriate for classification in the somatic setting. However, other than this statement in the methods, the other results and conclusions of the study do not really depend on variant classification, and so perhaps a solution may be to just mention something like “mutations were classified using terminology analogous to the ACMG system for interpretation of sequence variants: Pathogenic, LP, etc.”
Table 2 (line 296) – why was only EFS examined for ALK mRNA expression and MYCN amplification?
Author Response
We would like to thank you for the comments on our manuscript. We appreciate the time and effort you have dedicated to provide us with feedback.
Comment 1: Line 195-198, the ACMG system is used to classify variants. This system is more designed for germline findings rather than classification of somatic alterations, as the criteria used in this classification system relies upon data, studies, and findings in the germline setting. The AMP/ASCO/CAP tiering system (Li et al, JMD 2017) may be more appropriate for classification in the somatic setting. However, other than this statement in the methods, the other results and conclusions of the study do not really depend on variant classification, and so perhaps a solution may be to just mention something like “mutations were classified using terminology analogous to the ACMG system for interpretation of sequence variants: Pathogenic, LP, etc.”
Response 2: We agree with your comment regarding the classification system of ALK mutations and we have adjusted our manuscript accordingly (Materials and Methods, section 2.5., paragraph 1, page 6, lines 198-202).
Comment 2: Table 2 (line 296) – why was only EFS examined for ALK mRNA expression and MYCN amplification?
Response 2: The statistician involved in our researched has indicated that we should only add one variable per ten events in the Cox Proportional Hazard model. Since the number of deaths was thirteen, we could only include one explanatory variable ( ALK mRNA expression) to the model when estimating the effect of mRNA expression on OS. However, twenty patients experienced an event and therefore we could add an additional explanatory variable to the model when estimating the effect of mRNA expression on EFS. We chose MYCN amplification, since this is the most important prognostic factor in neuroblastoma.
Again, we would like to thank you for giving us the opportunity to strengthen our manuscript.
