Genotypic Characteristics of Hepatoblastoma as Detected by Next Generation Sequencing and Their Correlation With Clinical Efficacy
Authors: Yanyan Mei
Yuan Wen
Jing Li
Dongsheng Huang
Huimin Hu
Weiling Zhang
Tian Zhi
Fan Li
Journal: Frontiers in Oncology (2021)
Discipline: CLINICAL MEDICINE
Keywords: CTNNB1; PDL1; hepatoblastoma; next generation sequencing; tumor mutation burden.
Affiliations: Department of Pediatrics, Beijing Tongren Hospital, Capital Medical University, Beijing, China.

Abstract:  Background: Hepatoblastoma (HB) is the most common malignant embryonic liver tumor type in children under 3 years of age. In the present study, the next generation sequencing (NGS) method was used to detect the genotype characteristics of HB and summarize the correlation between the common mutation genotypes noted in this disease and the clinical treatment and prognosis. The results may aid clinical prognosis and the successful application of targeted drugs. Methods: Initially, DNA was extracted from tumor tissue specimens and peripheral blood derived from 19 pediatric patients with HB. Subsequently, DNA panel and NGS methods were used to detect tumor diagnosis and the expression levels of treatment-associated genes, followed by the summary of genotype characteristics. In addition, in order to further assess the application of immunotherapy in HB, immunohistochemical detection of programmed cell death 1 ligand 1 (PDL1) was performed in combination with tumor mutation burden (TMB) and DNA mismatch repair status analysis. Furthermore, the clinical treatment effect and prognosis of the pediatric patients were statistically analyzed according to the characteristics of the genotype. Overall prognosis and prognostic analyses in different groups were performed by Kaplan-Meier and log-rank tests, respectively. Finally, expression validation and diagnostic analysis of commonly reported genes were performed in the GSE75271 dataset, which was obtained from the Gene Expression Omnibus (GEO) database. Results: In the present study, certain mutated genes, including nuclear factor erythroid 2-related factor 2 (NFE2L2), catenin Œ≤1 (CTNNB1), MYCN, tumor protein p53, axis inhibition protein 1 (AXIN1) and adenomatous polyposis coli (APC) were associated with the pathogenesis of HB. During TMB and DNA mismatch repair status analyses, pediatric patients had a low TMB. All of them did not present with microsatellite instability. The immunohistochemical results indicated lower expression levels of PDL1 in HB. The complete remission (CR) rate of pediatric patients in the gene abnormality group was lower than that of the non-reported disease-associated gene abnormality group. The 2-year overall survival rate and disease-free survival rate of 19 pediatric patients with HB were 72.1% and 42.4%, respectively. Receiver operating characteristic (ROC) analysis demonstrated that CTNNB1, NFE2L2, AXIN1, APC, MYCN and insulin growth factor 2 (IGF2) may be potential biomarkers that could be used for the diagnosis of HB. Conclusion: The genotype changes in HB were more common and the CR rate of the pediatric patients with an altered genotype was lower than that of pediatric patients without an altered genotype. In addition, pediatric patients with HB exhibited lower TMB compared with adult patients. Moreover, the data indicated that CTNNB1, NFE2L2, AXIN1, APC, MYCN and IGF2 may be potential biomarkers that can be used for the diagnosis of HB. 

Retracted: Yes

PubPeer comments:
 Unexpectedly, this papers appears to have been published in Frontiers in Immunology and retracted in APMIS (as DOI: 10.1111 apm 13180).  Can the authors please clarify? Perhaps this was a double submission, i.e. the article might have been submitted to two different journals and accepted in both, with APMIS deciding to retract their copy? , 
 Maybe the authors can also clarify how the patients were recruited between November 1, 2018 and March 31, 2020, the paper was submitted in November 2020, but the patients were followed for 50 months? Can they look into the future?  , 
 Dear Pubpeer officer Thank you very much for receiving your inquiry email. Due to the busy clinical work, I am very sorry that I did not check your email and unable to reply timely. After seeing the email, we combed the questions raised in detail and checked the original data. In fact, we are also surprised by the concerns regarding two submissions for our paper on Pubpeer. We are also surprised that the manuscript was still online on the journal of AMPIS although we have repeatedly sent the request for the withdrawal application of the manuscript and the journal editor have confirmed and agreed to the withdrawal it, under the condition of without peer-reviewing and proof-reading of our manuscript. With regard to the ambiguity on the follow-up period of patients, we are sorry that we may have not previously clearly stated in the manuscript. I will specify the two questions in detail as follows: Firstly, Replies to the concern about two submissions for our paper 1. The paper was submitted to Frontier in Oncology for the first time on November 12, 2020, and no any information about submission progress was received before May 6, 2021. Under the context of Chinese culture, no response with regard to a submission for a long time ( 3 months) is considered a rejection. Therefore, we have switched to the journal of AMPIS. We are very sorry and regret that we did not notice and thoroughly realized the submission habits of English journals here. We must recognize that it is a flaw. 2. 2 weeks after switching to AMPIS journal, I received a rejection decision from the journal of Frontier in Oncology, but allows a rebuttal for consideration, in the editoru2019s tab, within 7 days of this decision. The decision letter stated that u201cThis does not guarantee that your manuscript will be further considered for peer review, and that no extensions can be granted at this stageu201d. Thus, we made a rebuttal as required, and provided the original data. Then, the journal added more reviewers and provided relatively positive review comments (the last review opinion was June 29, 2021). 3. After receiving the comments of the fourth reviewer and completing the corresponding modification, we then sent a request for the withdrawal of our manuscript from the journal of AMPIS, and then received an email confirming the approval of the withdrawal request. 4. Thereafter, after completing proof-reading and publication cost payments etc. according to the requirements of Frontier in Oncology, the article will be published online. 5. 10 days after the paper went online in Frontiers in Oncology, I received the decision letter regarding the acceptance for publication of our manuscript on the journal of AMPIS without receiving the peer-reviewing comments. Since the request for withdrawal had been sent before, I sent the withdrawn request again after receiving the decision letter of the manuscript. The journal also confirmed the request for withdrawal, agreed and confirmed the status of withdrawal on the journal submission system. 6. About 1 month after the withdrawal of the manuscript is confirmed, the journal of AMPIS put the article online without proof-reading. 7. In January 2022, we found that the publication was duplicated and applied for withdrawal, and then the journal of AMPIS withdraw the article. Above is the detailed process of our article, However, I must to say, anyhow, we have to change the habit of submitting articles at home and abroad to avoid the possible misunderstanding and concerns, although we have no subjective intention to the two submissions for one article. We will be greatly appreciated if you can understand and anticipate our more rigorous and international publication in future. Meanwhile, we are appreciated for your and all of the researcher efforts for the improvement of the whole scientific research. Secondly, with regard the concerns of follow-up time for clinical data of patients With regard to the concerns of follow-up time of clinical data in this series, we are very sorry for the ambiguity or misunderstanding that the lack of detailed description of the time of onset of illness of the case in the figure legend. In fact, we collected the clinical data of 19 children who were hospitalized in our hospital and underwent genetic testing from November 1, 2018 to March 31, 2020. This is the starting time point of the genetic testing, NOT the follow-up time. The onset time of 19 children was from March 28, 2016 to September 9, 2019. The start time point of follow-up was defined as the onset time to the end point of follow-up (May 31, 2020). Therefore, the follow-up time of 19 children was 9-50 months, and the median follow-up time was 21 months. Since this article focus mainly on genotypes, the initial information of clinical data is not described clearly in sufficient and detailed manner in the article. We guarantee the originality and authenticity of our data. Please refer to the attachment for specific original clinical data. Thanks again to the experts for our supervision, we will correct and improve our scientific research in the future. Regards, Dongsheng Huang  ,