Author's response to reviews Title: Persistent tumor cells in bone marrow of early breast cancer patients after primary surgery are associated with inferior outcome Authors: Kjersti Tjensvoll (ktje@sus.no) Satu Oltedal (olts@sus.no) Reino Heikkilä (reino.heikkila@roche.com) Jan T Kvaloy (jan.t.kvaloy@uis.no) Bjørnar Gilje (gibj@sus.no) James M Reuben (jreuben@mdanderson.org) Rune Smaaland (rusm@sus.no) Oddmund Nordgard (nood@sus.no) Version: 2 Date: 26 January 2012 Author's response to reviews: see over
Dear editor Thank you for considering our manuscript entitled "Persistent tumor cells in bone marrow of non-metastatic breast cancer patients after primary surgery are associated with inferior outcome" for publication in BMC Cancer. As requested, we have addressed the reviewer s comments and incorporated them fully into a revised version of our manuscript. I have also indicated the changes point-by-point (underlined in red) in this cover letter, and the changes have been highlighted in the revised version of our manuscript. The Kaplan-Meier plots (figure 1 and 2) have also been corrected to incorporate the number of patients at risk. Moreover, the name of the ethical committee has been provided. We hope you find our revised paper suitable for publication in BMC Cancer. I declare that none of the authors have any competing interests. Yours sincerely, Kjersti Tjensvoll Laboratory for Molecular Biology, Department of Haematology and Oncology, Stavanger University Hospital, N-4016 Stavanger, Norway. E- mail: ktje@sus.no/tjensvollk@gmail.com Phone: +47 47809206
Reviewer's report Title: Persistent tumor cells in bone marrow of early breast cancer patients after primary surgery are associated with inferior outcome Version: 1 Date: 16 November 2011 Reviewer: Gro Wiedswang Reviewer's report: The paper Persistent tumor cells in bone marrow of early breast cancer patients after primary surgery are associated with inferior outcome from Tjensvoll et al is an important paper bringing new information to the fate and prognostic value of DTC in BM after surgery. Unlike the other published studies addressing this item, they have included a very early postoperative BM sample in their study, only 3 weeks after surgery. This is completed with another aspiration 6 months after surgery, and they show even at this early time point positive DTC detection is associated with impaired outcome. Patients being persistent positive, both pre and postoperatively, do worse. Their methods are well described and appropriate, the data are consistent and their results are well presented. The following remarks are discretionary and minor essential revisions, just to improve the presentation. 1. In the title they use early breast cancer, but in table1 1/3 of the patients have pn1-2 tumor and 1/3 tumor size T2-4. All patients are M0 however, so the term localized or non-metastatic breast cancer is more appropriate. Author response: We have now replaced the term early breast cancer with nonmetastatic breast cancer in the title, and throughout the manuscript. 2. In the method part in the abstract, it is not mentioned that the patients also had a preoperative BM examination. As this is referred in the result part in the abstract, it should be included. There is also a complete lack of presentation of the patient cohort. Author response: The following has been included in the method part of the abstract on page 2 Patients with non-metastatic breast cancer were consecutively recruited to this project during the years 1998-2000. Furthermore, the following has also been included The results were compared to previously published data from pre-operative BM analyses for the same patients. 3. In the background part of the paper, the reference 21 should be cited. The ref 20 is only an abstract on the same patient cohort and should be omitted. Author response: Janni et al. (2011) have been cited in the background part of the paper on page 4 as follows DTCs have also been found in the BM after surgery, both before and after adjuvant treatment [6-12]. The previous ref 20 (Janni et al. (2006)) have been deleted.
4. The authors hypothesize that repeated BM sampling; in particular following administration of adjuvant treatment may improve prediction of disease recurrence. This information in the present patient cohort would be interesting, but are not included in the result part. This analysis has the potential to be really interesting, as a multimarker panel is used and in table 2 the positivity of marker expression changes during follow-up. Is there a co variation with the adjuvant therapy offered? Author response: Of the 44 patients receiving adjuvant treatment only six had evidence of DTCs in BM2, and one of those still had DTCs in BM3. However, in the group not receiving adjuvant treatment the five patients with DTC-positive BM2 also had DTC-negative BM3 findings. Thus, due to the small number of patients included in this study it is not possible to conclude on the potential for monitoring or prediction of adjuvant treatment efficiency. This has been discussed on page 14. The small sample size, in this respect, makes it also impossible to conclude on the co-variation between marker expression and the adjuvant therapy that was offered. 5. In the ref 6, the extra strong negative predictive value of double-positive BM, both pre- and postoperatively is demonstrated (fig 3). Author response: This article has been referred to on page 14. 6. Under the methods subheading, a more detailed presentation of the 154 patient cohort is mandatory. This could be performed by simply referring to table 1. Details of the surgical procedure should be included, how many had a breast conserving procedure, how many were included in the SN diagnostics of the axils etc. Author response: We have now referred to table 1 for more details on the 154 included patients on page 6 as follows In total, BM2 and/or BM3 aspirates were obtained from 154 patients (for more details see table 1), and 99 patients allowed aspirations at all three time points. Unfortunately, we have not registered how many of the patients had a breast conserving procedure in our database. SLN diagnostics were, however, not performed at our hospital at the time of the patient recruitment. 7. Is really only the 99 pts with a complete data set included in the response evaluation to adjuvant therapy? Is this referring to the Cox regression analyses presented in table 3? There are performed analyses for potential associations with chemo- and endocrine therapy and DTC status in table 1 including the complete cohort. What are the numbers at risk in the Kaplan Meier plots? Author response: The Cox regression analyses performed in table 3 are based on the total cohort (n=154). The BM2 and BM3 analysis were, however, performed on those samples available as described in table 2 and in the text on page 6. Nevertheless, we have clarified this a bit more by adding an asterisk after BM2 and BM3 in table 3 with the following explanation *only patients with this sample available were included in the analysis.
In addition to this evaluation, we did also want to investigate the response to adjuvant treatment by looking into the patients DTC status before and after adjuvant treatment. For this analysis we only included the 99 patients which had undergone BM aspirations at all three time points (BM1, BM2 and BM3). Unfortunately, the number of patients included in this study was too small to conclude on the potential for monitoring or prediction of adjuvant treatment efficiency as discussed on page 14. The numbers at risk has been included in the Kaplan Meier plots. We have also deleted the following sentence on page 6 With regard to the evaluation of response to adjuvant treatment only the 99 patients with BM aspirations obtained at all three time points (BM1, BM2 and BM3) were included. 8. The FU procedure should be presented in more detail, how often do the patients see a doctor and what examinations are performed? How is recurrence of the disease stated? And how is the cause of death established? Author response: The FU procedure has been presented in more details on page 7. Follow-up data for all patients were collected from the hospital records and from their primary physician s records. The control program for the patients was according to the routines of the institution, with one to two visits per year, depending on patient age, stage, breast conserving treatment versus mastectomy, time from primary treatment and method of diagnosis (screening-detected vs. not). Blood tests were performed 1-2 times per year, in addition to mammography examination once a year. Information on time of death was obtained from the Hospital records, through an automatic update from the National Registry in Norway. The cause of death was determined from the medical files at the hospital, or by information from the patients primary physicians. 9. In the qrt-pcr part, the sensitivity for hmam mrna detection is increased. Is this only performed for the postoperative BM examinations? What is the reason for this chance in protocol? Author response: Due to a small number of hmam positive patients in Tjensvoll et al. (2008), and the fact that the hmam level was barely detectable in some cases, we wanted to increase the sensitivity of the hmam assay. Hence, we increased the cdna concentration from 20 to 50 ng, and re-analysed all the pre-operative BM samples included in Tjensvoll et al. (2008). In this re-analyses we obtained similar results as already presented in Tjensvoll et al. (2008). After the pre-operative BM results had been confirmed, we analysed all the post-operative samples using the new hmam reaction conditions as described in this manuscript. 10. CP value should be written without abbreviation. Author response: The CP abbreviation has been explained at its first use (on page 8) as follows: mrna concentrations, based on mean crossing point (CP) values, were normalized against BCR mrna level and expressed relative to a calibrator sample as previously described.
11. In the result part there is referred to table 2 to confirm that only 5/154 patients had elevated levels of more than one marker. According to the line BM2 and/or BM3 in the table there are 27 positive samples in 23 patients, which corresponds to 4/154 pts had more than one marker positive? Author response: We are sorry, this has now been corrected (page 11). 12. Under the subheading Prognostic significance of persistent DTCs, 5 th line, in the sentence beginning with In this patient cohort. it is unclear which of the patients cohorts is referred or if it is the whole patient cohort. Author response: We agree that this was a bit confusing, and it has now been clarified by deleting this sentence and only include the following Eight of the 10 (80%) patients with recurrent disease have subsequently died from breast cancer (see page 11). 13. In the discussion part, page 14 the abbreviation CTC is introduced without explication (=circulating tumor cells). In the following passage there is a discussion between immunocytochemical and molecular methods for detection of tumor cells. Bringing CTC into this discussion can be omitted, or the role of CTC should be discussed in a more extensive way. Author response: The CTCs have now been omitted from this discussion (page 14). 14. Page 15 comparing the present positive frequency of positive postoperative BM with other studies where BM aspiration is performed after surgery, it is surprising that the present frequency is not higher because of the earlier time point. This can of course be secondary to the methological differences, but one would assume the present study included those with an early relapse, excluded in the other studies. Author response: We agree with the reviewer that the same positive frequency (15%) between several studies might be a consequence of the methodological differences. We have therefore incorporated the following on page 15: In the present study we demonstrate that 15% of the patients with non-metastatic breast cancer have DTCs detected in BM after primary surgery (Table 4). Although the use of different methodologies complicates a direct comparison between the studies, our results seem to correspond with Wiedswang et al. (2004) also showing that 15% of the non-metastatic breast cancer patients had detectable DTCs in BM at a median 66 months from diagnosis [6]. Janni et al. (2005) detected DTCs in 13% of the patients after surgery [7]. 15. References: Exclude ref 20. Author response: Previous ref 20 (Janni et al. (2006)) has now been deleted from the manuscript.
16. Table 1: Remove the sentence None of the breast cancer patients had evidence of distant metastasis. Include in the text that numbers in brackets are%. Replace DTC status with postoperatively DTC status. Details of the operative treatment should be included? Author response: The suggested sentence has been removed, and the following has been added to the table 1 heading Table 1 Comparison of the clinicopathological parameters of the patients according to DTC status in bone marrow after primary surgery. Numbers in brackets represent the number of patients in percent. As already mentioned, we have not registered the data from the operative treatment (breast conserving procedure etc.) in our database unfortunately. 17. Figure 1 &2: Numbers at risk should be included. Months should be noted without decimals. The figure legends describe fig 1A and B, fig 2 A and B. This corresponds to the information in the text. The Kaplan Meier plots are labeled fig 1-4. Author response: Numbers at risk have been included in figure 1 and 2, and the months are indicated without decimals. The figures are labelled as figure 1 and 2.