Cover Letter. Reviewer 1:

Transcription

1 Cover Letter Michael Yang, M.D., Ph.D. Managing Editor of Cancer Research Frontiers 1188 Willis Ave, #109, Albertson, NY 11507, USA Phone: Dear Dr. Yang, Thank you for your response to our manuscript. We found the comments to be very helpful to improve our content and have made most of the changes requested. We appreciate your consideration of our revised manuscript. Sincerely, Lori Millner, Ph.D., NRCC Reviewer 1: Major concerns: 1. I see a major conflict between the title and the content of the MS. The title does not specifies that the whole analysis was performed on breast cancer cells. This issue can be easily resolved by formulating the title more precisely. More importantly, however, the study analyzes breast cancer cell lines spiked into normal blood. This is handled as a surrogate for circulating tumor cells (CTC) throughout the MS. However, unlike the title suggests, these cells are not equal to CTCs. This issue is a week point of the study. The reviewer makes an excellent point regarding the title of the MS. We have changed the title to better reflect what was actually done in this study. 1. (continued) As a proof of principle, the authors should consider to verify their method on blood samples of breast cancer patients. Based on the fact that CTCs are not detectable in a very high percentage of metastatic breast cancer patients by the conventional EpCam capturing method, the comparative analysis of 2-3 cancer patients might be sufficient to proof the feasibility of the method in detecting non-epithelial CTCs. While we agree that providing patient sample data would further prove the feasibility of the method, the purpose of this manuscript is to introduce a novel method using data from a surrogate system, heterogeneous breast cancer cell lines. Some of the cell lines chosen do not express EpCAM (MDA-MB-231 and subset of SKBR-3) and serve as proof of concept that the method does capture these non-epithelial types of CTCs. This was stated and has been further clarified in the introduction found in lines

2 2. Authors should justify the clinical relevance of the number of breast cancer cells spiked into the blood before isolation (50 into 5 ml). Based on the very rare presence of CTCs in the blood, a sensitivity of 1 CTC per ml blood might be required for clinical application. Authors might want to show sensitivity of the system within this range or bring in data justifying the higher cell numbers they used. The reviewer correctly points out the need for extreme sensitivity. Although the gold standard (CellSearch) considers a positive test to be just 5 or greater cells in 7.5 mls of whole blood for patient samples, it is not ascertainable how many CTCs were actually present. Most spiking experiments use well above this using up to 100 [1], 200 [2], or 300 cells [3]. Additionally, there have been reports of up to hundreds of CTCs per patient sample [4]. 3. Authors should justify the relevance and sufficiency of the selected markers for the identification of breast CTCs, first of all for the positive identification of non-epithelial CTCs. In addition, they should comment on the possible presence of cells with NO positive marker detection (CD45-, EpCam-, HER2-, CD44-, Hoechst+), if they occurred in their analysis and how the method would handle these cells. Non-epithelial cells are retained in our enrichment step because we use a negative depletion method that removes unwanted leukocytes. We used a combination of EpCAM, HER2, and CD44 for positive identification. EpCAM was chosen for identification because it is a commonly used extracellular marker for epithelial CTC detection. In addition to epithelial markers we used 2 additional markers, HER2 and CD44, that are likely to be expressed on more aggressive or non-epithelial CTCs. HER2 was chosen because it s expression has been detected on CTCs in early and metastatic cancer and is not dependent on the primary tumor HER2 status [5, 6]. In several studies, HER2 expression was found more frequently on CTCs than the primary tumor, and expression on CTCs has been associated with poor clinical outcome of early breast cancer [6-8]. CD44 was chosen because it is involved in tumorigenesis, and it s presence on CTCs has been associated with lower overall survival than those with no CD44 expression [9]. CD44 has been described as a marker of tumor-initiating cells and high expression has been observed on a subset of metastasis-initiating CTCs [10, 11]. This explanation has been added in lines to further clarify the use of these markers. Other markers can easily be added to this method but for our feasibility studies, we used only the 3 mentioned. 4. Authors analyzed the heterogeneity of a single cell (SKBR3) line in re-isolated cells by staining for the markers EpCam, HER2 and CD44. Are these obtained percentages in accordance with a FACS analysis of this cell line using the same markers? A FACS analysis has confirmed the presence of SKBR-3 with low EpCAM expression but not the presence of the very rare subpopulation (EpCAM-, HER2-, CD44+) likely because there were so few cells, less than 10 out of 3,000 cells. It is difficult to differentiate these from false positive readings with cytometry. It is easier to positively identify these cells using microscopy because you can visualize the cell and confirm an intact nucleus as well as where the desired marker is expressed.

3 Minor concerns: 1. Abbreviations are not consequently introduced. Authors should check that abbreviations are introduced at first time use. Abbreviations FDA (p2, row48), PBMC (p2, row49), NGS (p3, row70) and WGA (p9, row197) are affected. List might be incomplete. The abbreviations were introduced at their first use as suggested in each line. 2. The authors should consider to reorganize the Introduction part and keep methodology and result description as brief as possible. E.g. last paragraph at p.3 is far too detailed, referring to result tables and figures should be avoided. Some detail in the last paragraph on page 3 lines was removed and added to the methods section (65-66) and the references to figures and tables were removed. To shorten, some detail was removed altogether (80-82). 3. Sentence in row is not clear, what the authors mean. This sentence has been clarified (now in row 92-93). 4. Figure 2 is not clear. According legend figure shows a comparison in the cell capture efficiency of EpCam pos selection vs. negative depletion. In text, the efficiencies for negative depletion are around 80%. What the % Negative on the diagram than stands for? The % negative on the figure refers to the amount of the desired cells (SKBR or MDA) that ended up in the negative (unexpected) fraction. Typically, analysis would only be performed on the positive fraction so we d lose any cells in the negative fraction. This has been clarified in the figure legend and in the text line 241 and There is a mistake in row 240. The marker should be CD44 and not CD45. This has been corrected. Reviewer 2: This is a very interesting process to identify circulating breast cancer tumor cells and agree it is in an interesting way to both identify and be able to deep sequence the cells identified. The technology to use single cell DEPArray to identify CTC is interesting and has not been done much in the literature. However, I do question that the markers selected would actually improve identification of CTCs as still using EPCAM and just adding HER2 and CD44. For particularly breast cancer which is so heterogenous, these markers would not be enough particularly to identify the most aggressive triple negative markers. There are many more EMT markers which could be tested and adding a few more stains may better identify heterogeneous targets. Our justification for the use of these markers of negative outcome and aggression has been clarified under reviewer 1 s comment 3.

4 Would have liked to see a study showing identification of a panel of different breast cancer cell lines and even discussion of looking at dissociated tumor tissue to see if could identify CTCs. This would speak to being able to extend this. We haven t looked at dissociated tumor tissue, but we have identified and differentiated a 4 cell line panel shown below. 4 cell line model MCF-7 HCC SKBR MDA The article however is well written and the technique is very interesting. Would show that it can identify a wider array of breast cancer cell lines or indeed tumor cells and then would be a more complete study. (END) References 1. Xu, L., et al., Optimization and Evaluation of a Novel Size Based Circulating Tumor Cell Isolation System. PLoS One, (9): p. e Ghazani, A.A., et al., Sensitive and direct detection of circulating tumor cells by multimarker micro-nuclear magnetic resonance. Neoplasia, (5): p Harb, W., et al., Mutational Analysis of Circulating Tumor Cells Using a Novel Microfluidic Collection Device and qpcr Assay. Transl Oncol, (5): p Uenosono, Y., et al., Clinical significance of circulating tumor cells in peripheral blood from patients with gastric cancer. Cancer, (22): p Kallergi, G., et al., Expression of truncated human epidermal growth factor receptor 2 on circulating tumor cells of breast cancer patients. Breast Cancer Res, : p. 113.

5 6. Fehm, T., et al., HER2 status of circulating tumor cells in patients with metastatic breast cancer: a prospective, multicenter trial. Breast Cancer Res Treat, (2): p Wulfing, P., et al., HER2-positive circulating tumor cells indicate poor clinical outcome in stage I to III breast cancer patients. Clin Cancer Res, (6): p Ignatiadis, M., et al., Prognostic value of the molecular detection of circulating tumor cells using a multimarker reverse transcription-pcr assay for cytokeratin 19, mammaglobin A, and HER2 in early breast cancer. Clin Cancer Res, (9): p Ascolani, G., A. Occhipinti, and P. Lio, Modelling circulating tumour cells for personalised survival prediction in metastatic breast cancer. PLoS Comput Biol, (5): p. e Zhang, L., et al., The identification and characterization of breast cancer CTCs competent for brain metastasis. Sci Transl Med, (180): p. 180ra Baccelli, I., et al., Identification of a population of blood circulating tumor cells from breast cancer patients that initiates metastasis in a xenograft assay. Nat Biotechnol, (6): p