The Polemic Diagnostic Role of TP53 Mutations in Liquid Biopsies from Breast, Colon and Lung Cancers

Round 1

Reviewer 1 Report

This Garrido-Navas et al. summarize current knowledge of the use of liquid biopsies(circulating tumor cells and cell-free DNA) for treating p53 mutant tumours.

Overall, the review is written nicely and is well structured, and references are appropriate. The tables are clear to follow.

Minor comments:

Authors should elaborate more in the discussion about improvement and/or standardization of the protocols for detection of the p53 status in liquid biopsies in order to have more reproducible results.

Line 124 tp53 should be TP53

Line 138, 156, 167, 169, 212,287,298,314 347,352, 358 Authors should check how they reference the studies

Author Response

We thank reviewer 1 for identifying places where our manuscript could be improved and made more accessible to readers. 

We have included a comment (lines 383-389) about protocols used for TP53 mutation detection to explain better the issue about standardization and reproducibility.

We have also capitalized "TP53" on line 124 and referenced properly all studies as suggested by the reviewer.

Reviewer 2 Report

In this review, the authors summarized the main studies done on two types of liquid biopsies: circulating tumor cells(CTCs)
and cell-free DNA(cfDNA). They evaluated correlation for mutation status between liquid biopsies and tumor tissue, suggesting possible sources of discrepancies, as well as evaluating the clinical utility of using liquid biopsies for the analysis of TP53 mutation status and the future actions that need to be undertaken to make liquid biopsy analysis a reality for the evaluation of TP53 mutations. To my impression, this review is presented in a well-organized and logical manner. All the experimental results show reasonable consistency. In addition, these studies provide insightful knowledge of TP53 mutations and will contribute to further studies on its applications. I would therefore strongly recommend this manuscript for publication in Cancers.

.

Author Response

We very much appreciate the interest of reviewer 2 in our manuscript.

Reviewer 3 Report

In this review, the authors introduced the diagnostic role of TP53 mutations in liquid biopsies in lung, colon and breast cancers and discussed some controversies arising from the study of liquid biopsies compared to tissue samples. I think the authors' review covers a wide range of research in this area. However, the target is limited to lung, colon, and breast cancer, and I think it is better to include this in the title. I also felt it would be better if there was an explanation about the detection limit and cost effectiveness of liquid biopsy in these cancers.

Author Response

We thank reviewer 3 for raising the idea of cost-effectiveness and limit of detection that might have not been assessed properly in our manuscript. 

We have added an explanation of limit of detection (LoD) for CTCs (lines 128-129) and for cfDNA (lines 189-192). Furthermore, a paragraph regarding cost-effectiveness was added (lines 406-415).

Finally, we have added the three tumor types ("breast, colon and lung cancer") in which we focussed our review in the title as suggested by the reviewer.

Reviewer 4 Report

This is a potentially useful review on a subject of extreme interest for clinical oncology, that is in fact a bit confusing for its technical challenges and intrinsic complexity. Regrettably, the manuscript is hard to read, and contains much information that is reported with some detail but is not developed into a clear message related to the main object of the paper.

I think the authors too often lose the focus on p53 as a biomarker in liquid biopsies, deviating towards a more general discussion of liquid biopsy as a prognostic or diagnostic tool.

Overall the paper has a good potential but I recommend a substantial revision.

1) I am not convinced that “Mutations in TP53 occur very early during tumor development…” as the authors write in the abstract and elsewhere in the paper. With the exception of hereditary cancer syndromes such as Li-Fraumeni, in many models (e.g. colon) it is believed that p53 mutation occurs at later stages of tumor progression. Unless authors mean that p53 mutation occurs prior to metastatic behavior (and thus the increase in CTCs). I think the meaning of such statement should be clarified and eventually supported by appropriate references.

2) As authors know well, there are two possible types of TP53 mutation: those that cause loss of the protein (i.e. deletions, frameshift, splicing aberrations) and those that cause production of a stable mutant p53 protein with oncogenic properties (gain of function). Since this review is focused on p53 as a biomarker, it would be important to understand whether the various studies that authors describe could actually discriminate between the types of TP53 mutations detected. If not, the absence of such a distinction in those studies should be at least mentioned, emphasized, and commented for its implications.

3) Authors review numerous studies that obtained contradictory evidence for correlation between mutations in cfDNA, CTCs, and tumor tissue, and their respective prognostic potential. However, I feel authors do not help the reader understand the possible reasons for this phenomenon, and the first part of the manuscript fails to draw a “broad picture” of the state-of-the art, that I’m sure would be appreciated by the readers.

I think paragraphs 2.1 and 2.2 should be extensively edited to provide a clearer take-home message. Authors should try to be more descriptive, perhaps even reducing the details, and try to highlight the basis and possible explanations of confusing results and variable correlation between solid and liquid biopsies. And the future that lays ahead.

4) Table 1 includes a few studies in which only CTC or cfDNA were analyzed. Those data are interesting per se, but have limited relevance in this specific context because they provide no info on the correlation between liquid biopsy and solid biopsy, nor between the two different kinds of liquid biopsy. In my opinion, they can be omitted.

Minor points

Line 112. Check the meaning of “translational status”

Line 169. The study by Zhang et al. (2014) was earlier, not later

Line 172. Please define VAF here since this is the first time it’s mentioned.

Lines 174-179. Important closing paragraph, not so clear. Consider rewriting.

257-261. Unclear, please re-write the paragraph. The prognostic role was of p53 mutation in CTCs or in the number of CTCs? Was it correlated with worst or better outcome? What does it mean “baseline ctDNA levels”? Was it ctDNA or cfDNA?

262-266. As above. What was analyzed: the amount of cfDNA or specific mutations in cfDNA? Correlation was associated with better o worse overall survival?

Line 337-341. Repetition of a notion already discussed elsewhere. It seems out of context here.

Line 359. Please specify that mutations were found in liquid biopsy

Line 373. please define dPCR

Lines 390-391. An important concept. Should be described in a clearer way.

Author Response

We thank reviewer 4 for all interesting comments that will improve our manuscript. 

Regarding comment 1: we have not changed the sentence "Mutations in TP53 occur very early during tumor development..." in the abstract because TP53 is the most commonly mutated gene in virtually all solid tumors not only in hereditary cancer syndromes like Li-Fraumeni but also in sporadic cancers. As it is a tumor suppressor, not a metastasis suppressor gene, mutations in TP53 actually occur early during tumor development facilitating cell proliferation and progression in breast cancer^A. We agree with the reviewer that in colorectal cancer, if following the model proposed by Fearon and Voglestein, TP53 mutations might appear later during tumor development^B; however, some studies have identified TP53 mutations in pre-cancerous lesions such as ulcerative colitis^C and colitis-associated neoplasia^D being an early event promoting carcinogenesis. Likewise, TP53 mutations have long been detected in lung pre-malignant lesions^{E, F} and early cancer stages^G. Thus, although TP53 mutation frequency might increase during cancer progression, we indicated in the abstract: "Mutations in TP53 occur very early during tumor development" because highly specific and sensitive technologies such as the study of cfDNA or CTCs has demonstrated their occurrence at those very early stages.

REFERENCES:

^AW Zhou, AA Muggerud, P Vu, EU Due, T Sørlie, A Børresen-Dale, F Wärnberg and A Langerød. TP53 mutation is an early event in breast cancer progression. Cancer Res January 15 2009 (69) (2 Supplement) 1047

^BFearon ER and Vogelstein B: A genetic model for colorectal tumorigenesis. Cell. 61:759–767. 1990

^CHyung-Joon Kim, Sae-Kyung Chang. p53 mutation in patients with ulcerative colitis in rectal biopsy. The Korean Journal of Internal Medicine 1998;13(2):110-116.

^DWen-Chi L.Chang, Renata A.Coudry, Margie L.Clapper, Xiaoyan Zhang, Kara-Lynn Williams, Cynthia S. Spittle, Tianyu Li and Harry S.Cooper. Loss of p53 enhances the induction of colitis-associated neoplasia by dextran sulfate sodium. Carcinogenesis vol.28 no.11 pp.2375–2381, 2007

^EKitamura, H. et al. Atypical adenomatous hyperplasia and bronchoalveolar lung carcinoma. Analysis by morphometry and the expressions of p53 and carcinoembryonic antigen. Am. J. Surg. Pathol. 20, 553–562 (1996).

^FKerr, K. M., Carey, F. A., King, G. & Lamb, D. Atypical alveolar hyperplasia: relationship with pulmonary adenocarcinoma, p53, and c-erbB-2 expression. J. Pathol. 174, 249–256 (1994).

^GIzumchenko, E., Chang, X., Brait, M. et al. Targeted sequencing reveals clonal genetic changes in the progression of early lung neoplasms and paired circulating DNA. Nat Commun 6, 8258 (2015).

Regarding comment 2: A paragraph was added in lines 378-382 to clarify that the type of TP53 (either gain of function or loss of function) was not taken into account in the reviewed studies.

Regarding comment 3: Closing paragraph in part 2.1 was rewritten to better explain source of discrepancies between solid and liquid biopsies. Furthermore, "...as discrepancies might be intrinsic to tumor evolution itself" was added at the end of paragraph 2.2 to clarify the source of discrepancies regarding cfDNA.

Regarding comment 4: Raws in table 1 for which concordance is N/A as only one type of biopsy (either solid or liquid) was analyzed were removed as suggested by the reviewer.

Minor points

Line 112. Check the meaning of “translational status” This was a typological error. We have changed it to "transcriptional status"

Line 169. The study by Zhang et al. (2014) was earlier, not later. We agree with the reviewer and have changed the order of the references in the paragraph to follow a logic time order.

Line 172. Please define VAF here since this is the first time it’s mentioned. We have included definition of VAS in line 172

Lines 174-179. Important closing paragraph, not so clear. Consider rewriting. Rewritten accordingly

257-261. Unclear, please re-write the paragraph. The prognostic role was of p53 mutation in CTCs or in the number of CTCs? the word "numbers" was added to clarify Was it correlated with worst or better outcome? the words "bad prognostic marker" were added to clarify. What does it mean “baseline ctDNA levels”? The words "before treatment" were added for explanation. Was it ctDNA or cfDNA? in this paper they calculated ctDNA based on the proportion of TP53 mutated DNA with respect to the total cfDNA using LINE1 PCR

262-266. As above. What was analyzed: the amount of cfDNA or specific mutations in cfDNA? when "total cfDNA levels" is mentioned, no particular mutations are taken into account, only concentration of free nucleic acid Correlation was associated with better o worse overall survival?. the word "poorer" was added to clarify

Line 337-341. Repetition of a notion already discussed elsewhere. It seems out of context here. We have rewritten the paragraph to highlight the idea about combination of both biomarkers, CTCs and cfDNA, despite the fact that the latter is more studied than the former

Line 359. Please specify that mutations were found in liquid biopsy The sentence "(most of which were missense producing non-functional proteins)" was added for clarification

Line 373. please define dPCR The definition "digital PCR" was added

Lines 390-391. An important concept. Should be described in a clearer way. Two sentences (lines 423 to 428) were added to better describe the study by Cohen, J., et al (2018).

Round 2

Reviewer 4 Report

I think authors have addressed the most critical issues and the text is now improved in clarity.

I have just a few additional suggestions and a minor peeve:

Line 29. A targeted treatment is hardly done against a gene, especially if the gene is mutated. Authors should consider rephrasing the sentence in the abstract

Line 166-167. There is a leftover of the original sentence that has been moved at the end of paragraph

Lines 182-230. The blue text is identical to original version, except for the sentence 187-190 which is the only new part. By the way, check line 189 since I think there is a missing word.

Also lines 287-310 and lines 325-342. All the blue text is identical to original version except for reference numbers. I found this confusing and a little tricky.

Author Response

We thank reviewer 4 for taking the time of reassessing the new version. Here we have some answers for his/her considerations together with the modifications we have made:

Line 29: There is actually a wide variety of p53 targeted therapies some of which were reviewed by Levine, A.,J., (2018). In the case of TP53 mutations, it is important to identify whether they are gain-of-function (GoF) mutations (in which case, GoF inhibitors might be used) or loss-of-function (LoF) (in which case structural correctors might be used).

Reference: Arnold J. Levine. (2018). Targeting Therapies for the p53 Protein in Cancer Treatments. Annual Review of Cancer Biology 2019 3:1, 21-34

Lines 166-167. We believe the sentence is the original one and no leftover has been moved

Lines 182-230: Some words were added and sentences rewritten to clarify:

- The word "for" was added in line 187 and the word "mutations" was added in line 191 as we believe these might be the "missing word" the reviewer suggested in line 189.

- We rewrote the sentence on lines 205-208 to make it more clear.

- The word "and" was removed between references 40 and 50 in line 216.

- The word "it" was added to line 221 as subject of the sentence.

- The words "among different studies" were added in line 222

- The words "suggesting good negative predictive value" were added in line 227

- The word "or" was removed from line 237

- The sentence in lines 236-239 was rewritten for clarification

- The sentence in lines 247-250 was split into to.

Lines 287-310 and 325-342 were not rewritten as no previous comment was made regarding that need. Only paragraphs 2.1 and 2.2 were somehow rewritten to facilitate the readers understanding. Also, the fact that reference numbers changed was due to the addition, in the reviewed version, of references 31 in line 129 and 41 in line 192 to clarify the limit of detection of CTCs and cfDNA respectively.