Disseminated tumor cells as selection marker and monitoring tool for secondary adjuvant treatment in early breast cancer. Descriptive results from an intervention study

The introduction of systemic adjuvant therapy has improved the survival of patients with early breast cancer. However, there is a lack of established tools to measure the direct effect of a given systemic treatment on minimal residual disease/micrometastases after primary surgery.

Techniques for identification and characterization of disseminated tumor cells may open possibilities for prediction of treatment response and tailored treatment decisions. Immunocytochemical detection (ICC) of disseminated tumor cells (DTCs) in the bone marrow (BM) and further analyses of these cells have been introduced as means to meet these needs [1‐9]. Moreover, presence of DTCs during relapse-free follow-up (+/- tamoxifen) has been found to be a strong predictor of systemic relapse and breast cancer death [10‐12]. Similar results were also reported in two smaller studies analyzing DTC status in very high-risk breast cancer patients early after completion of chemotherapy [1, 13]. The presence of DTCs after chemotherapy clearly indicates a rationale for testing of alternative (secondary) treatment approaches. Detection of DTCs following treatment intervention should also be further tested for potential value as a surrogate marker for future relapse/treatment effect. During the last decade, docetaxel has been established as a highly active treatment against breast cancer. Response rates of 40-50% have regularly been reported in the metastatic setting [14, 15]. Prior to the initiation of this study, results from several trials indicated that use of docetaxel in addition to anthracycline could improve the outcome of patients compared to non-taxane regimens [16‐18].

In the current study, DTC-status was monitored after completion of anthracycline-containing adjuvant chemotherapy and used to identify high-risk patients as candidates for secondary treatment with docetaxel. The BM-status was analyzed 2-3 months (BM1) and 8-9 months after chemotherapy (BM2). To reduce inclusion of patients who might be in the process of gradual eradication of DTCs caused by an effective standard treatment, BM2 was chosen as the time point for decision about docetaxel treatment. There was also some support from previous studies to assess BM-status between 6 and 12 months after chemotherapy [13, 19]. DTC-status was also explored as a surrogate marker for response by monitoring changes in DTCs after the docetaxel treatment. The clinical follow-up is still ongoing. Here, we present the descriptive data from the study.

The present study is, to our knowledge, the first reported study to use DTC-status to select for and monitor secondary adjuvant chemotherapy intervention in breast cancer. The identification of high-risk patients for future relapse, at a time point where otherwise no additional prognostic information can be achieved from standard histopathological/clinical assessment, is attractive. This opens for testing of alternative treatment strategies in a “window of opportunity” for potential eradication of minimal residual disease. The results show that persistent DTCs 8-9 months after 6 courses of FEC chemotherapy are changed to DTC-negativity in 79.2% of the cases following secondary treatment with docetaxel. This indicates a potential for docetaxel to eradicate minimal residual disease burden in high-risk patients. It cannot be excluded that presence of 1 DTC can be followed, by chance, by a negative result in the next test (Poisson distribution and/or methodological limitations). However, persistent negativity at two time points, and especially the fact that ¾ of the patients with ≥3 DTCs at BM2 turned negative at BM3/4, suggest a change in the tumor cell load after the intervention. The recent meta-analysis of 14 randomized clinical trials by Jean-Philippe Jacquin et al [24], support a clear additional effect of docetaxel-containing adjuvant chemotherapy to a non-taxane-containing regimen in patients with early stage breast cancer (HR 0.84 (95% CI 0.78-0.89; P < 0.001) for DFS and 0.86 (0.78-0.94; P < 0.001) for OS). The benefit is consistent across all patient subgroups, although proliferation status was not analyzed. These results may support an association between the docetaxel secondary adjuvant treatment and the reduction in DTC-positivity in our study.

The subgroup analysis shown in Table 4 reveals a significant difference in the fate of DTCs after docetaxel treatment according to histological tumor subtype. Half of the patients with lobular carcinoma had persistent DTCs, as compared to 15% of the ductal carcinoma patients. This observation is in line with a reported relative chemotherapy resistance for lobular carcinoma [25‐27], and adds further support to the possibility of docetaxel-induced changes in the observed DTC-status. Furthermore, a higher fraction of patients with DTC-positive status at BM1 seemed to have persistence of DTCs after the treatment, although statistical significance was not reached (p = 0.11). It may be speculated whether a proportion of these patients have a more resistant disease (i.e. less fluctuations of DTCs despite chemotherapy). It is known from several studies that patients with primary resistance to first line chemotherapy also have a higher risk of not responding to second line treatment [28, 29]. The clinical outcome of the patients included in the present study needs to be awaited, before further interpretation of the results.

The analysis of BM2 showed that DTC-status was associated with pN-status and pT-status (Table 1). The same was reported in a different study from our group, analyzing DTC-status 3 years after diagnosis [12]. In this previous study it was also observed that DTC-positivity was positively associated with lobular carcinoma, which can be explained by a relative resistance also to the anthracycline-containing chemotherapy [25, 30]. Our study supports the selection of higher stage patients into future DTC intervention trials (Table 1), in order to select those with both the traditionally highest risk of relapse and the highest frequency of DTC-positivity. A consideration of the histological type may also be of importance, for selection to the proper type of systemic treatment to the right tumor subtype.

The testing of novel therapeutical principles or drugs is highly resource demanding. In addition, the effect of a new adjuvant treatment can only be evaluated when a relapse occurs, often several years later. The need for surrogate/intermediate markers to predict and monitor the therapeutic effect is obvious, but needs to be thoroughly validated. The present study is an initial step to explore the possibility to use DTCs in BM as a monitoring tool. Other possible approaches could be monitoring of circulating tumor cells (CTCs) or, as very recently reported, analysis of circulating tumor DNA in plasma/serum [31]. Detection of CTCs was not a part of the current study, because no standardized CTC-method was available at the time of study start. The performed repeated BM-analyses, however, were feasible and acceptable for the large majority of the patients.

The observed frequency of DTCs in the BM was markedly lower than what was expected prior to the study. This might be due to the assay sensitivity, but may have several additional explanations. The previously reported studies of DTCs have mostly been performed on BM-aspirates at the time of primary surgery. The subsequent administration of adjuvant chemotherapy might give a reduction in DTC-positivity. Furthermore, recent studies, using more standardized criteria, generally have shown lower DTC-positivity rates [12, 20, 22, 32, 33] than those reported in older studies. Additionally, there has been a stage migration after introduction of organized mammography screening (which is established in Norway), which probably results in less patients with micrometastatic disease. In our study, all the patients were screened for metastases before inclusion, which also might have affected the frequency of DTC presence. Finally, we used a conservative approach for inclusion of patients to docetaxel treatment in the current study. Doubtful cases were concluded as DTC-negative. It is possible to increase the sensitivity by analyzing larger number of cells (higher BM-volume). However, the clinical significance of DTC-status at primary surgery was not increased by analyzing more cells in our previous study [34]. Use of larger volumes of BM, or larger numbers of BM MNC, might require additional characterization of the detected DTCs, in order to identify markers of DTC aggressiveness and to secure both sensitivity and specificity. Accordingly, available FISH, CGH and multi-marker analyses may improve the utility of DTCs as a surrogate marker for response [33, 35‐39]. Characterization of the DTCs also opens for studies of tumor dormancy, EMT, stemness and/or identification of treatment targets.

The present study does not allow a direct interpretation of the effect of docetaxel on DTC-status. Although a randomized approach would have been the optimal design for this purpose, we chose the current design to explore the clinical potential for DTC-directed intervention. A randomized trial would raise several concerns, if performed un-blinded to the DTC-status. To inform the patients about a DTC-positivity without intervention (in one arm) was considered ethically difficult. A blinded study (blinded randomization of both DTC-negative and DTC-positive patients to no additional versus docetaxel treatment) was found to be premature without supporting data and would have needed a very large and expensive study. Recently a randomized trial was reported for DTC positive early breast cancer patients at diagnosis, where patients received chemotherapy +/- zoledronic acid. The results showed improved elimination of DTCs in patients treated with zoledronic acid [40]. In another study in locally advanced breast cancer, DTC status was also affected by the administration of zoledronic acid [41]. Although clinical outcome results have not yet been reported, these data support the potential use of DTCs as a monitoring tool. In our study, comparison to clinical end points has to await completion of the follow-up.

We chose 8-9 months after the standard adjuvant chemotherapy as time point for the DTC-analysis decisive for secondary adjuvant treatment. This was partially based on the results of the SBG study [13], where a positive DTC-status 6 months after chemotherapy identified patients with very poor prognosis. Furthermore, Slade et al performed repetitive BM-analyses at follow-up and found that the frequency of DTC-positive events was highest at 12 months after surgery [19]. Considering the increasing support for a detrimental outcome of patients with a positive DTC-status at later time points during follow-up [11], it might be an even more optimal approach to perform serial BM-aspirations during the first follow-up years, and to test secondary intervention whenever a DTC-positive status appear. This might be a reasonable consideration for future studies.

DTC-analysis can be a useful tool for identifying patients who do not respond to a chosen standard adjuvant therapy and accordingly should be tested for benefit of additional secondary adjuvant therapy. Elimination of DTCs after docetaxel treatment was observed in the majority of the patients. Although the clinical significance of these results awaits mature follow-up data, the current study presents a novel potential approach for optimized adjuvant treatment of breast cancer, supporting further exploration of this intervention principle.