healthmedicinet

Primary systemic NACT in BC is considered to convert inoperable tumors to operable
primary tumors and is undertaken to improve surgical options. Furthermore, NACT reflects
the tumor response to treatment and allows a more individual therapeutic concept.
Response to NACT has been associated with improved PFS and OS. The OS rate in our
sample was 89 %, as compared with an overall OS rate of 87 % in Germany 62]. Taking into account that we exclusively had a high-risk patient cohort undergoing
NACT, we documented a better OS than the overall OS rate of patients with BC in Germany,
containing high- as well as low-risk patients. Furthermore, we showed a significant
correlation of response to NACT with regard to PFS when patients with pCR or pPR were
compared with nonresponders. For OS, no significant associations were found, probably
due to the fact that the observation period was still too short. However, some patients
experienced relapse, even those having achieved a pCR, as recently published 63]. This indicates that the disease is able to persist in secondary organs such as the
BM, which might have spread tumor cells into the circulation. We demonstrate that,
although CTCs were eradicated more effectively than DTCs, CTCs detected after treatment
seemed to be associated with tumor cells showing tumor stem cell characteristics as
well as resistant tumor cell populations, which might indicate worse outcome in the
future. These findings underline our assumption that CTCs, probably circulating from
reservoirs in the lung or liver, might be a high-risk indicator for already ongoing
metastasis not limited to bone metastasis.

Thus, these patients might benefit from additional second-line treatment protocols
for the eradication of minimal residual disease. However, after a median follow-up
time of nearly 5 years, no significant correlations were found for DTCs, CTCs, and
slCTCs as well as for their changes before and after therapy with regard to PFS and
OS.

Disseminated tumor cells

DTCs have been analyzed mainly after NACT, with a detection rate of 40–50 % but demonstrating
that 30 % of the detected cells were apoptotic 21], 64]. In only one study have researchers analyzed these cells before and after NACT, showing
positivity rates of 21 % and 16 %, respectively, which is quite in accord with our
data showing 27 % positivity before and 19 % after NACT, respectively 11]. Interestingly, 14 of 87 patients with a negative DTC status before therapy switched
to a positive DTC status after chemotherapy. We can only speculate and hypothesize
that these patients probably were DTC-positive before therapy and that DTCs probably
had undergone EMT and lost their epithelial character, which allowed them to travel
to metastatic sites without being affected by conventional treatment 65].

Furthermore, DTCs in our patients were significantly associated with nodal status
before and after NACT as well as with the histology- and IHC-determined subtype before
NACT, while no significant correlations could be documented for PFS and OS, which
is in contrast to the results obtained in the above-mentioned studies. The fact that
DTCs persist after treatment and are associated with worse outcome has already been
described and is explained by a clinically significant biological heterogeneity, most
likely due to phenotypical changes, EMT and stem cell characteristics, and altered
genomic characteristics between DTCs, CTCs, and the primary tumor 9]–11], 32], 35], 66]–70]. One treatment option for DTCs has been demonstrated by Naume et al., who administered
six cycles of docetaxel as secondary treatment in DTC-positive patients after first-line
therapy with fluorouracil, epirubicin, and cyclophosphamide, resulting in better survival
of these patients 71]. We did not further characterize DTCs; thus, we can only speculate that residual
cells might have stem cell characteristics in some patients. However, we did not see
any negative prognostic effect of DTCs before and/or after NACT with regard to PFS
and OS after a median follow-up of nearly 5 years, which is in accord with our other
published adjuvant BC studies, where we demonstrated that the intake of clodronate
in case of DTC positivity was able to eradicate DTCs even years after the first diagnosis
26], 72]. Thus, clodronate intake was also offered to DTC-positive patients in this study.
However, it is still unknown whether bisphosphonates are also able to eradicate stem
cell–like DTCs present among DTCs in the BM. The fact that stem cell–like and EMT-like
cells, probably circulating from reservoirs other than the BM (e.g. liver, lung),
were present in blood samples before and after therapy makes the hypothesis that DTCs
with stem cell–like characteristics are eradicated by bisphosphonates more or less
unlikely.

Circulating tumor cells

CTCs have been suggested to be potential surrogate markers for minimal residual disease,
the precursor of metastatic disease. There is increasing evidence that CTCs could
be a strong predictive biomarker of response to NACT because BM is too invasive and
painful for monitoring purposes. Unfortunately, a recently published meta-analysis
confirmed that, although CTCs before NACT significantly correlated with reduced PFS
in some studies, the change (decrease or increase) in CTC numbers during NACT in patients
with locally advanced BC was not associated with pCR, and a decrease in CTC counts
after NACT did not indicate that patients had an improved response 44].

The main problem with using CTCs as a so-called liquid biopsy is the fact that, at
present, there is no standard definition for the identification of CTCs 73]. Currently, the CELLSEARCH system, based on immunomagnetic EpCAM capturing, is the
only system for CTC enumeration in BC approved by the U.S. Food and Drug Administration
7]. However, despite the prognostic impact of CTC counts in BC, it has been shown that
this procedure is not able to detect the entire, highly heterogeneous population of
CTCs, including slCTCs and CTCs in EMT 74], 75]. Despite the prognostic impact of CTC counts, it is indispensable to characterize
these cells, which might complement these studies by improving the overall detection
rate as well as sensitivity and thus permit the assessment of genomic markers in CTCs
of patients with BC, as recently published 45].

Although we and others, by using molecular methods, have already characterized the
heterogeneous CTC populations in primary BC before adjuvant therapy 7], 33], 37], 38], 76], 77], in few studies have researchers evaluated the presence, not the characteristics,
of CTCs after adjuvant therapy, resulting in a positivity rate of 22–34 % 17], 20], 78]. Furthermore, in the neoadjuvant setting, the number of circulating epithelial cells
was mostly used for monitoring the effect of chemotherapy after every cycle of treatment
23], 24]. To the best of our knowledge, our present study is the first in which CTCs have
been characterized so comprehensively before and after NACT. In contrast to DTCs,
most of the CTCs before therapy, present in about 24 % of the patients and reflecting
the heterogeneous CTC population, were eliminated by the given therapy. Although we
cannot definitively prove that all residual CTCs were stem cell–like and EMT-like,
molecular marker expression might allow characterization of them as stem cell–like.
In addition, 72 % of the residual cells were characterized as ERCC1-positive, indicating
therapy-resistant tumor cell populations. Interestingly, seven of these eight patients
had been treated with taxanes or anthracyclines, which were shown to eradicate DTCs
in a Norwegian study 13]. The fact that ERCC1-positive CTCs were present after these therapies might indicate
that these cells survived treatment. This knowledge might help clinicians to decide
more precisely about further secondary treatment options in the future. However, CTCs
and slCTCs before as well as after therapy did not significantly correlate with decreased
PFS or OS, as described in the above-mentioned studies. On one hand, follow-up time
might have been too short to see an effect on outcome; on the other hand, factors
determining if single tumor cells form metastasis have not been identified yet. In
this regard, it has been demonstrated that CTCs can be detected after more than 20 years
in patients with BC without any sign of relapse 79].

Secondary treatment options

Tumor stem cells are well known to be resistant to various chemotherapeutic agents
and radiotherapy 80]. In this context, Creighton et al. reported supportive evidence that the residual
breast tumor tissue cell populations surviving after letrozole or docetaxel were enriched
for subpopulations of cells with both tumor-initiating and mesenchymal features 41]. Thus, additional therapeutic strategies are urgently needed to prevent relapse of
the disease. In this regard, signaling pathways that maintain cancer stem cells are
attractive targets for these therapies. One example is everolimus (RAD001), an oral
inhibitor of mammalian target of rapamycin acting downstream of the PI3K/AKT pathway,
which was shown to have effective inhibitory effects on cancer stem cells in vitro
and in vivo, and combination treatment with RAD001 and docetaxel or trastuzumab has
been reported to be effective in refractory metastatic BC 81]. Li et al. demonstrated that remaining tumorigenic cells after chemotherapy had unique
properties of enhanced self-renewal as demonstrated by formation of mammospheres and
increased propensity for tumor formation. In addition, lapatinib did not lead to an
increase in these tumorigenic cells; thus, in combination with conventional therapy,
specific pathway inhibitors may provide a therapeutic strategy for eliminating these
cells to decrease recurrence and improve long-term survival 82]. Concerning HER2, a recently published study suggested that the clinical efficacy
of adjuvant trastuzumab may relate to the ability of this agent to target the cancer
stem cell population in a process that does not require HER2 gene amplification 83]. These results are partly comparable with ours because we observed clearance of slCTCs
after NACT in some patients who received trastuzumab, lapatinib, or bevacizumab, whereas
patients who did not receive these combinations had slCTCs left after therapy (data
not shown). These observations have to be interpreted with caution, and more patients
have to be followed to prove whether this observation holds true. Further promising
agents that are thought to attack BC stem cells are salinomycin, where treatment resulted
in the loss of expression of BC stem cells 84], and a new synthetic curcumin analogue against ALDH1 and glycogen synthase kinase-3?
85]. In the future, targeting the tumor microenvironment, such as by interrupting the
immune cells (e.g., myeloid-derived suppressor cells) and cytokines [e.g., interleukin
(IL)-6, IL-8] as well as the immune checkpoints [programmed cell death protein 1/programmed
cell death ligand 1 (PD1/PDL1)], may provide additional new tools for immunological
targeting of cancer stem cells 86].