Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Authors in the current study tried to demonstrate availability to isolate EpCAM+ and cell surface vimentin (CSV)+ breast cancer cells. It’s well known in the field that the mesenchymal marker vimentin is an intracellular intermediate filament protein, attaching to the nucleus, ER and mitochondria to play a role in cell directional movement, orientation, cell sheet organization and Golgi complex polarization at the cell migration front. There might be a little amount of vimentin on cell surface, but I don’t understand why authors only focus on the rare cell surface vimentin, instead of intracellular vimentin to identify and characterize CTCs. What exactly specific clone of the mAb against CSV was applied in the current study? How this antibody was fully characterized? Disclosure of the relevant information is necessary. It is highly recommended that a parallel comparing study with both anti-CSV and intracellular vimentin need to be performed to provide convincing data. Moreover, in Table 2, authors are required to describe in detail how exactly number (from 1 to 5) of cells be precisely and repeatedly spiked in their recovery study, via serial dilution?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

There are only 2 FDA approved CTC isolation platforms. Cell Search isolates CTCs by CD45 cell exclusion and EpCAM positivity while Parsortix isolates CTCs based on size. However, more mesenchymal transitioned CTCs, particularly those which may have lost EpCAM expression may be lost for further analysis. The intermediate filament vimentin has long been accepted as an EMT marker and more recent studies have indicated that cell surface vimentin (CSV) correlates with an EMT. Per the manuscript title, this study was performed to determine the diagnostic and prognostic ability of enumerating CSV positive CTCs using an automated negative selection platform.

This is a stepwise, well performed and explained study. Below are some comments for enhanced clarity.

1. The study revolves around the Chiline CATCH CTC Enrichment System. It would help if the manuscript incorporated a more detailed description of the system besides simply mentioning “a negative selection technique to enrich EpCAM+-CTCS and CSV+-CTCs”. Plus, the picture of the system (Figure 1A) is small and of low quality, and the text highlighted with purple boxes is illegible.
2. Please consider including the information about the antibody (name/clone) used for positive selection in both the epithelial and mesenchymal CTC kits.
3. The NB4 cells are used as a WBC background, but please mention what type of cell line they are in the methods and the rationale for using them. The assumption is that these cells are CD45 positive.
4. Add in the fixing and staining procedure for the CTCs after isolation. Is staining completed after each isolation to verify the surface marker or is it assumed that the CTCs captured are EpCAM or CSV positive?
5. The high number of CTCs isolated from 2ml of patient blood is surprising. Is this a peripheral venous bleed or a central arterial bleed? This should be stated. Can you report on how long after surgery CTCs are collected, whether the patients have had chemotherapy and what type? Surgery and different therapies can alter CTC numbers which may confound your results.
6. It is mentioned that 13 patients were considered low risk because they were at stage 0-II or had no recurrence for 5 years. Does that mean the high-risk patients already have reoccurrence? If so, how is the PFS determined?
7. Is it possible to include a chart with the 29 individuals with stage, breast cancer type (ER+, HER2+, TNBC), number of EpCAM+ CTCs, number of CSV+ CTCs, and time to first metastasis detection, even if it is in a supplement? Although unlikely because of low patient number, you may possibly be able to determine that the number of CSV+ CTCs is a better prognostic indicator than the >5 CTCs /7.5ml of blood.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Though existence of CSV on CTC was reported by one article, it is UNLIKELY that all the vimentin are expressed only on the surface of CTC, it's not difficult at all to confirm it via searching literatures published by many others elsewhere.  Authors are encouraged to disclose and discuss limitations of CSV-based strategy. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf