Esophageal squamous cell carcinoma is a significant disease, with poor survival rates. In early stage disease, surgical removal of the cancer offers the best chance for cure. However, the lack of effective screening and surveillance programs can result in diagnosis at a late disease stage, which is not amenable to surgery.

Cheng et al present an interesting study describing whole-genome and exome sequencing complimented with target capture validation (TCS) on a reasonably large cohort of Stage I (n=51) and Stage III (n=53) esophageal squamous cell carcinoma (ESCC) cases. This in itself is a valuable and significant contribution.

However there are several major issues that need addressing.

Major Compulsory Revisions
1. The authors appear to be trying to identify targets involved in development of ESCC. However I do not believe that this can be achieved using the reported study design ie a comparison of Stage I and Stage III cancers. Such a study would require the analysis of premalignant disease.


2. According to the latest Edition of the American Joint Committee on Cancer, Cancer Staging Standards (7th Edition, 2010), Stage I disease is divided into Stage IA and IB, and Stage III into IIIA, IIIB and IIIC. Stage IA and IB disease is defined as:

Stage IA: This is the same as T1 cancer, in which the cancer is located in only the two inside layers of the esophagus (T1, N0, M0, G1).
Stage IB: Either of these two conditions:
The cancer is located in only the two inside layers of the esophagus, but the tumor cells are less differentiated (T1, N0, M0, G2 or G3).
The tumor is located in the lower part of the esophagus, and the cancer has spread to either of the two outer layers of the esophagus, but not to the lymph nodes or other parts of the body (T2 or T3, N0, M0, G1).

Given that Stage IB is considered to be more advanced, and is associated with decreased survival compared to Stage IA, and that the majority of cancers analysed in this study were Stage IB (n=48), it is inappropriate to refer to the Stage I cohort as “early Stage I” cancers.


3. In the abstract the authors state that “Knockdown of FAM84B significantly promoted cell proliferation and migration/invasion, indicating its new oncogenic roles in ESCC.” It is clear from the results that knockdown INHIBITED in vitro cell growth, invasion and migration of ESCC cell lines.


4. The authors state “This finding suggests that FAM84B amplification and the resultant increased protein contribute the genesis or development or both of ESCC and may be a potential diagnosis marker for susceptibility and early stage of ESCC.

There is no evidence presented to suggest that FAM84B contributes to the development of ESCC, as this hypothesis has not been adequately tested.

Rather, the data demonstrate that a) expression of FAM84B is increased in the progression from normal squamous epithelium to ESCC. This is an association, and in itself does not suggest that FAM84B contributes to development; b) that FAM84B promotes tumour cell growth (MTT assay), migration (migration assay), and invasion (invasion assay) in vitro.


5. The authors state “Likewise, NOTCH signaling was altered in 55% of stage I tumors versus 32% of stage III tumors (P＜0.02, Fisher’s test, , Figure 5a), indicating that NOTCH pathway may be early and necessary events in the development ESCC.”

This statement is not supported by the data. If the NOTCH pathway is necessary for the development of ESCC, then why were alterations only observed in 55% of stage I tumours, and not 100%?


Minor Essential Revisions
The obvious mistakes that I noted have been highlighted in the accompanying pdf file.

Level of interest An article of importance in its field
Quality of written English Needs some language corrections before being published
Statistical review No, the manuscript does not need to be seen by a statistician.
Declaration of competing interests No competing interests.

Manuscript with reviewer comments: (http://www.gigasciencejournal.com/manuscript/review/attachment/5051132331486671.pdf)

Authors' response to reviews: (http://www.gigasciencejournal.com/imedia/1682852505161404_comment.pdf) 

The reviewed version of the manuscript can be seen here:
http://www.gigasciencejournal.com/imedia/5235196031442538_manuscript.pdf
All revised versions are also available:
Draft - http://www.gigasciencejournal.com/imedia/5235196031442538_manuscript.pdf

Major Compulsory Revisions
None

Minor Essential Revisions
None

Discretionary Revisions
None

I am satisfied with the authors revised manuscript.

Level of interest An article of importance in its field
Quality of written English Acceptable
Statistical review No, the manuscript does not need to be seen by a statistician.
Declaration of competing interests I declare that I have no competing interests.

The reviewed version of the manuscript can be seen here:
http://www.gigasciencejournal.com/imedia/1408580998161405_manuscript.pdf
All revised versions are also available:
First revision - http://www.gigasciencejournal.com/imedia/1408580998161405_manuscript.pdf