In the present study we have found that epirubicin/docetaxel chemotherapy leads to a modulation of the complement cascade, as well as the acute phase proteins inter-α
-trypsin inhibitor, α
-1-antichymotrypsin and α
-2-HS glycoprotein. These effects were observed 24
h after the initial dose of therapy allowing for a very early evaluation of the body's response to treatment. Time course experiments revealed that most of these expression changes remained stable on day 2 and 3, but were undetectable at day 14 (data not shown). The complement system is affected by epirubicin/docetaxel chemotherapy at the level of different components of the cascade, as well as at the level of different isoforms of these components. This corroborates the significance of the results. It is important that a differential modulation of two C3 isoforms between responders and non-responders was observed in a training set of 12 patients which could be confirmed in an independent validation set of 13 patients. Our data highlight the role of complement as an important host response factor that could be used for early identification of non-responders to neoadjuvant chemotherapy in breast cancer patients.
Despite a growing body of evidence illustrating the complement system as an important factor in cancer patients, the complement system has so far achieved little attention in clinical oncology. Previous proteomic studies found increased levels of different components of the complement system in sera of patients with colorectal cancer (Habermann et al, 2006
; Ward et al, 2006
) and in plasma of patients with breast cancer (Nakshatri et al, 2009
). A study in patients with pancreatic cancer revealed that preoperative plasma levels of nine proteins, including complement factors C1q-B, C3 and C4A correlated with the 1-year disease-free survival (Lin et al, 2006
). Moreover, a panel of five serum proteins, including complement factor C3a was able to predict the 5-year metastasis-free survival in breast cancer patients (Goncalves et al, 2006
). However, complement component C3a was found to lack specificity for early diagnosis of breast cancer, limiting its utility as a stand-alone tumour marker (Li et al, 2005
Usually, complement is seen as an immunological guardian against pathogens and damaged cells. In this line complement is thought to promote antibody-dependent cytotoxicity in the cancer setting (Macor and Tedesco, 2007
). However, recent murine experiments have also indicated that the activation of C5a, which is activated by C3, leads to immunosuppression of tumour-infiltrating monocytes (Markiewski et al, 2008
). In this way C5a promotes murine tumour growth as tumours frequently modulate host response signals from the microenvironment for their own advantage. Furthermore, cancer cells have been found to be more resistant to complement-mediated lysis and they use this attribute to set up a locally immunosuppressive environment (Loveland and Cebon, 2008
In the present study, we describe for the first time a modulation of the complement cascade by a specific chemotherapy in breast cancer patients. We demonstrate a reduced level of total C3 and C4 that is suggestive of complement activation in response to chemotherapy, even though we did not find an increase of the complement activation marker C4d in our samples. We still favour the interpretation of our data as complement activation, as there is also evidence for several bypass pathways of complement activation (Li et al, 2007
). Activation might be induced by chemotherapy-induced apoptosis of tumour cells. Corroboration for this hypothesis comes from the fact that complement factors are involved in the removal of apoptotic cells. Complement factors C1q, C3 and C4 have been described to bind specifically to apoptotic cells (Paidassi et al, 2008
). Such marked cells are then recognised and eliminated by macrophages in a process involving C1q binding to cell surface expressed calreticulin (Vandivier et al, 2002
). Calreticulin is normally found in the cytoplasma and appears on the cell surface in response to different apoptotic stimuli, such as chemotherapeutic agents (Apetoh et al, 2007
). Apoptosis-induced complement activation differs from the classical antibody-induced activation cascade that could explain the lacking increase in C4d levels. Further in vitro
and in vivo
studies will be necessary to prove this assumption. Recent reports indicated that tumour cells acquire a resistance to complement-induced cell death by increasing expression of membrane-bound complement inhibitory proteins (Gorter and Meri, 1999
) and/or soluble inhibitors, such as factor H (Ajona et al, 2007
). This might further explain why complement differentially contributes to the response to chemotherapy.
Previously, it was shown that part of the cytotoxic effect of chemotherapy is mediated by the innate immune system, such as toll-like receptors (Zitvogel et al, 2008
). Our data now suggest that the complement cascade and acute phase proteins may also be involved in this process. However, in contrast to the substantial modulation of the complement cascade and selected acute phase proteins, we found only little modulation of cytokines in the early phase of chemotherapy in our patients. Most studies in humans compared the plasma levels before therapy with the ones after its completion and hence, there are only few data available about the immediate effect of the administration of the initial dose on patients. In this study, we found minimal elevation of the interleukins IL-6, IL-10 and sICAM3 suggesting only a poor systemic cytokine response in humans. This is in contrast to several murine experiments where mice treated with etoposide or cyclophosphamide showed a rapid increase in blood levels of IL-6 that peaked at 3–6
h after administration (Elsea et al, 2008
). Similarly, splenocytes collected from mice treated with cytarabine, cisplatin, etoposide or melphalan displayed an increase in the synthesis of several cytokines, including tumour necrosis factor-α
h (de Vries and Singer, 2000
). This provides further evidence that the chemotherapy-induced modulation of complement is not part of a classical inflammatory response, but rather an atypical stimulation.
Aside from gaining a better understanding of the immunological consequences of chemotherapy in humans, we identified early predictive markers that could enable more individualised therapy in the future. We identified two protein spots of the complement component C3 that were differentially modulated by epirubicin/docetaxel-based chemotherapy. Spot 195 is likely to represent a still non-cleaved full transcript of C3, whereas spot 529 is an isoform of its proteolytic product C3 α-chain. This further supports our assumption that epirubicin/docetaxel-based therapy results in activation of C3. Responders seem to show higher activation in comparison to non-responders. To date, the isoforms that we have detected can only be identified by 2D-gel electrophoresis, which renders diagnosis for a larger set of patients not possible yet. The generation of isoform-specific antibodies could change this technical obstacle. Moreover, analysis of further complement-specific or -associated markers might improve a complement-based prediction assay.
It is well established that corticosteroids can affect acute phase proteins and the complement system. With the exception of only one patient all received the glucocorticoid dexamethason-21-dihydrogenphosphate together with epirubicin and docetaxel. However, in a still ongoing study with colon carcinoma patients receiving dexamethason-21-dihydrogenphosphate, but combination with other chemotherapeutics we could not see the same changes in the plasma proteome (data not shown). Therefore, it is unlikely that this drug contributes substantially to the effects on the complement cascade described above.
It is important that our study has followed a new concept in response prediction as two different points in time were taken into consideration, allowing for the assessment of dynamic changes during chemotherapy. Previous efforts to predict chemotherapy efficiency largely relied on the assessment of predictive markers or marker sets in pre-treatment biopsies that then formed the basis to predict treatment outcome. However, assessing dynamic changes throughout the early course of neoadjuvant chemotherapy could offer the advantage that the ‘actual' efficacy of chemotherapy is monitored in vivo that could provide more reliable predictive information.
In conclusion, our data suggest a significant role of the complement cascade and acute phase proteins in the host response to neoadjuvant chemotherapy with epirubicin and docetaxel in breast cancer patients. If validated in a larger series of patients, the complement system might, therefore, serve as an important future surrogate marker for therapeutic response and allow for early identification of breast cancer patients who will not profit from epirubicin/docetaxel-based neoadjuvant chemotherapy.