Overall comment: this article has significant methodological and interpretation issues which need to be dealt with.

Some specific comments

1 . Firstly, in terms of wording: The term hereditary tumor predisposition syndrome is not exactly right for 2 reasons:

1) It implies that it is ""hereditary"" as opposed to ""heritable""

2) All of their patients in this series are classified as ""HTPS"" patient 1, 2, 3, etc.. when many of these patients do not have a cancer predisposition syndrome (or have an ""unknown"" status for a CPS)

2 . One of the major points is that the study population could be better described in terms of eligibility for the study and in terms of clinical features. It is not clear to me how the patients got to genetic counselling in the first place. Are these children that were randomly sent by their treating oncologist and then were included in the study if they fit one of the following criteria? (fam Hx, rare tumor, multiple tumors, congenital anomalies, excessive treatment toxicities. How many children went to genetic counselling and didn't get tested? It is also confusing to me how patients were classified into group A/B or C.

For example, Why is HTPS-33 in group B and not group A? This may very well be compatible with LFS

Why is HTPS-31 in group A and HTPS-37 in group C? These are both Wilms tumors in young children with GDD and overgrowth.

I also believe there may be an error in Table 2, HTPS-5 is in group A and B.

3 . It also seems odd to have 10/45 patients with a suspicion of DICER1 syndrome. This seems very disproportionate to what is seen in clinics. What was the basis of consideration of DICER1 syndrome?

4 . In terms of the algorithm, how do you choose which ones from group A are considered as sporadic (case closed) and which ones go on to have WES/CGH? It would be helpful to add in the information about the genetic tests that were performed in the group A patients, thereby helping readers know if they would also have considered these patients as having a sporadic tumor. For example, a choroid plexus carcinoma that is negative for TP53 might be well accepted by the oncology and genetics community. A medulloblastoma that has negative testing for TP53 and PTCH1/SUFU may not be enough to call this case as ""sporadic"".

5 . What are the criteria of overgrowth?

6 . The authors make many assumptions in this manuscript without being able to confirm causality. The most difficult cases are the ones in which CNVs encompassing various genes are considered ""positive HTPS cases"". The ""likely pathogenic"" CNV is based on what criteria?

7 . I don't think the authors should assume that because one of their patients had a anaplastic sarcoma of the kidney and that the mother also had DICER1 and was asymptomatic, that this is a confirmation of a ""reduced penetrance of DICER1"" (page 7, line 8-9). It is supportive perhaps, but one pedigree with two persons tested can’t be confirmatory

8 . GLOW syndrome is not a ""formal"" syndrome, but more of an association of features seen in children with a specific mosaic DICER1 germline mutation located in RNAse IIIb domain. Moreover, since the only two previous cases of GLOW syndrome (Klein et al JMG) both had very unusual RNase IIIb missense mutations, occurring as mosaics, I seriously question whether this is the same disorder. Much more phenotypic information should be provided. If extended, this single case is worthy of publication alone, since as I say, only two such cases have been reported, and there are no non-IIIB, non-mosaic cases described.

9 . The case with the germline pathogenic variant in PTPRD in the patient with ALL and GBM is intriguing. However, the article the authors refer to describes 3 cases of metastatic EWS who were identified with PTPRD germline alterations. In 2 of the cases, these were SNPs (found in 2.5 and 7% of the population). This is the only article I could find that describes this association. What is the basis of the susceptibility in these 2 cases? Again, more work on this case could justify a separate publication.

10 . PARK2 has been associated with melanoma and Parkinson disease. Polymorphisms in PARK2 have been identified in colorectal cancer, but it isn't clear that there is causality here with NBL, especially in a child. To my knowledge (and with lit review), I do not see an association with palmoplantar keratoderma (unless one of the genes related to this disorder falls in the deleted part of the chromosome). Please explain.

11 . A few comments on wording: A desmoplastic astrocytoma is not the correct terminology. It is either a desmoplastic infantile or non-infantile astrocytoma. (as per the WHO CNS tumor classification). ""Oncologic patient"" is not correct English.

Thank you for the revisions. Some points remain.

1) ""Nine patients were suspected of DICER1 syndrome. In detail, 8/9 patients developed an embryonal rhabdomyosarcoma (ERMS), and one (Pt-15) was affected by an anaplastic sarcoma of the kidney.""

This needs to be changed, as ERMS by itself is not a situation where DICER1 mutations are likely. They have very rarely been found in ERMS outside the genitourinary tract, so since only 3 of the ERMS tumors arose there, I do not think it reasonable to say these eight cases were chosen because DICER1 syndrome was suspected. Three would be fair. The ASK is more obvious, since nearly all cases of ASK are DICER1-related.

2) ""We did not observe skewed coverage distribution for the reference allele compared with the alternate allele in the WES data from peripheral blood. However, a somatic mosaicism cannot be ruled out because no additional tissues were available to be analyzed for the variant"". Can the allele frequencies and the read depth be provided somewhere? This is an extremely unusual case, so the more information provided, the more it will help readers.

3) ""In our cohort, 9/45 patients were suspected of DICER1 syndrome"". Two things need to be changed here. First, this is not a cohort.(this also needs to be changed at other places in the text). It is a case series. Secondly, see my point about about DICER1. As 5 of the cases of ERMS are not G-U ERMS, I would reduce the n from 45 to 40.

4) Diagnostic groupings.

Your groupings are -- - group A, patients with the suspicion of a known CPS based on (i) clinical presentation (i.e. personal and/or familial cancer history) and (ii) tumor tissue features (i.e. immunohistochemistry data), and therefore eligible for a candidate gene/genes testing; - group B, cases with a strong suspicion of a genetic disease, but not readily fitting into a particular CPS, and eligible for a WES analysis; - group C, cases with a syndromic disease based on the presence of cancer together with either congenital anomalies, dysmorphic features, developmental delay and/or overgrowth. This classification has been included in the legend of table 1.

But from this system, some pts in group C could be in group A or B, since neither A nor B exclude children with congenital abnormalities. The groups should be mutually exclusive. So I suggest the following (please alter table 1 to reflect this), if you agree with my reasoning and subsequent groupings.

group A, patients with the suspicion of a known CPS based on (i) clinical presentation (i.e. personal and/or familial cancer history, and no congenital abnormalities) and (ii) tumor tissue features (i.e. immunohistochemistry data), and therefore eligible for a candidate gene/genes testing;

• group B, cases with a strong suspicion of a genetic disease without congenital anomalies, dysmorphic features, developmental delay or overgrowth features, but not readily fitting into a particular CPS and eligible for a WES analysis;

• group C, cases with a syndromic disease based on the presence of cancer together with either congenital anomalies, dysmorphic features, developmental delay and/or overgrowth.

see attached file.