Locally advanced breast cancer (LABC) is a generalized diagnosis that includes all stage III disease and a subset of stage IIB disease 1. The clinical definition of LABC continues to evolve and differ among physicians, and now includes nonmetastatic T3 or T4 tumors as well as N2/N3 disease involving limited metastasis 2, thus broadening the already diverse spectrum of LABC presentations. According to the American College of Surgeons Data Base, approximately 6% of all US breast cancer cases present as stage III 3. This number has declined dramatically over the past decade due to improved screening and detection practices. The 5-year relative survival rate for stage III breast cancer is approximately 50%, compared with 87% for stage I. The median survival for women with stage III disease is 4.9 years 1.

Neoadjuvant chemotherapy (NAC), the delivery of systemic chemotherapy prior to surgical resection, has emerged as the preferred initial component of therapy for patients diagnosed with LABC in an effort to enhance the prospect of breast-conserving surgery and to render inoperable tumors resectable 45. NAC offers the theoretical advantages of early initiation of systemic therapy, delivery of drugs through intact vasculature, in vivo assessment of therapy response, and the opportunity to study the biological effects of chemotherapy 6. Preoperative systemic chemotherapy may also eradicate distant micrometastases and thus improve the overall effectiveness of treatment 7. Several studies comparing NAC with more traditional adjuvant chemotherapy have found similar survival rates between the two options 589, and the use of NAC is further supported by findings that delaying surgery for the administration of chemotherapy does not adversely affect treatment outcome when compared with adjuvant chemotherapy 1011.

A pathologic complete response (pCR) following NAC implies the absence of residual invasive or in situ disease and correlates strongly with both prolonged disease-free survival and overall survival 1213. A recent review of several randomized clinical trials of NAC for operable breast cancer reported a response rate of 49% to 94% with a pCR rate of 4% to 34% 12. In patients treated with NAC, 60% to 80% demonstrate some clinical response with 10% to 20% achieving a clinical complete response (cCR) 4. Clinical response, however, often does not correlate with pathologic response as a full one-third of patients achieving a cCR are found to have pathologic evidence of residual disease 914. Despite these difficulties in assessing response, patients demonstrating complete clinical or pathologic responses to NAC generally achieve improved outcomes to overall treatment 1415.

Accurate modalities for assessing chemotherapy response are critical to the evaluation and expansion of the use of NAC for breast cancer. Conventional methods including clinical examination, mammogram, and breast ultrasound are incorrect in identifying pCR patients in nearly one-half of all cases 2. Several groups have reported promising results in their attempts to predict treatment response utilizing unconventional techniques such as diffuse optical spectroscopy and magnetic resonance imaging 1617. A growing number of investigators have begun to utilize the preoperative nature of NAC to conduct intreatment analyses of molecular markers that may predict response to therapy. The emergence of new technologies such as transcriptional and proteomic profiling has greatly aided such investigations 1819. For instance, it has been reported that mutations in p53 are associated with a lower response rate following NAC 2021, while coexpression of HER-2/Neu and topoisomerse II is associated with greater response rates 22. Measurements of the traditional breast cancer markers CA15-3 and HER-2/Neu, however, have demonstrated only limited predictive value in the NAC setting 2324.

In the present study we examine a diverse panel of serum biomarkers in order to identify individual biomarkers and combinations that may be useful in predicting treatment response early in the course of NAC for the treatment of LABC.

The heterogeneity displayed by patients diagnosed with LABC runs counter to the rationale for generalized treatment regimens. A wide array of treatment options exist for the treatment of breast cancer – including adjuvant and NAC, hormone therapy, radiotherapy, and surgery – and the vast majority of these options have been well researched. The ability to dynamically tailor the components of a particular treatment regimen on a patient by patient basis would be invaluable. Such an accomplishment will require the identification and development of improved prognostic factors on which to base therapeutic decisions, since currently used measurements of clinical and radiological response lack the necessary precision. In the present article we demonstrate the predictive value of serum biomarkers for the response to NAC for LABC. The diverse nature of the biomarker relationships identified in the study underscores the diversity of the disease characteristics present in the patient population.

Previous studies have examined the value of biomarkers such as carcinoembryonic antigen, CA 15-3, MMP-2, MMP-9, tissue polypeptide antigen (TPA), tissue polypeptide-specific antigen (TPS), EGFR, and HER-2/neu in predicting response to NAC for breast cancer 33343536. The results of these investigations have been mixed. To our knowledge, the panel of serum biomarkers examined in the present study is the largest and most diverse to date and the majority of the relationships we identify have not been described previously.

Our results indicate that elevated serum levels of IL-6, IL-8, and MMP-9 prior to the initiation of treatment correlate with improved clinical response. The observed increases in IL-6 and IL-8 serum levels of 2.74 and 3.13 pg/ml, respectively (Figure 1a), were not significant in our study given the limited 23-patient enrollment. Utilizing these data, we predict that an enrollment of 59 patients would be sufficient to confer significance (P < 0.05) upon these observations with a power of 0.9. Increased serum levels of the inflammatory cytokines IL-6 and IL-8 have been associated with poor prognosis in women with breast cancer 3738. These cytokines have also been implicated in the blood-borne response to paclitaxel treatment 39. The prognostic value of MMP-9 serum levels is currently unclear 33, but several studies have examined extensively the role of matrix metalloproteinases in breast cancer 4041.

Our results also demonstrate a correlation between lower pretreatment serum levels of tPAI-1 and improved clinical response. In fact, tPAI-1 levels alone were able to discriminate responders from nonresponders with 75% sensitivity and 77% specificity prior to the start of NAC. tPAI-1 is a major physiological inhibitor of tissue-type plasminogen activators and has been implicated in tumor growth, invasion, and angiogenesis. The function of tPAI-1 as a regulator of plasminogen activation places it in a position to modulate degradation of the extracellular matrix and antiangiogenic effects mediated by plasmin and angiostatin, respectively. Several groups have illustrated experimentally the role of tPAI-1 in tumor progression and its negative prognostic value in breast cancer 424344.

In sera collected after patients had received the initial cycle of chemotherapy we observed that increased levels of IL-4, IL-8, and adrenocorticotropic hormone and decreased levels of insulin-like growth factor binding protein 1 all correlated with improved clinical response at differing levels of significance. The increase in levels of inflammatory cytokines following chemotherapy is in line with clinical expectations and is evidence that the therapeutic agents are active in the patient's system. The roles of the insulin-like growth factor system and hormones in the response to chemotherapy have not been significantly evaluated; however, both groups have known roles in the development of breast cancer 4546.

With regards to pathologic response, we observed a correlation between increased levels of soluble Fas ligand, MMP-2, MIF, and EGFR and improved response. A proapoptotic response following chemotherapy, as evidenced by increased soluble Fas ligand in the sera, is to be expected. Our observation of increased levels of MMP-2 in the sera of responders supports the notion of matrix metalloproteinase involvement in breast cancer. The precise role of macrophage MIF in breast cancer development and treatment response remains unknown, but MIF has been implicated in tumor cell survival pathways 47. The value of EGFR serum and tissue levels in predicting response to chemotherapy has been examined previously with inconclusive results 4348. Increased expression of EGFR has been demonstrated elsewhere to suggest a poor prognosis in breast cancer 49.

It is noteworthy to mention that our present analysis did not identify any significant relationships between CA 15-3 or HER2/Neu and response to NAC. This observation adds to several sparse and conflicting reports in the literature 235051. It would appear from this uncertainty that, although these particular biomarkers have shown the most promise in terms of diagnosis and prognosis of breast cancer, they may not offer predictive power for chemotherapy response.

In addition to the individual serum biomarkers found to be significant at each timepoint, our multivariate analysis of the data identified several multimarker panels with predictive value for both clinical response and pathologic response prior to or early in the course of treatment. These multimarker panels demonstrated greater predictive power than any single biomarker and illustrate the potential clinical utility of this type of approach to the design and maintenance of treatment regiments. Previous studies from our group have demonstrated the usefulness of multimarker panels in the early detection of ovarian cancer, endometrial cancer, and head and neck cancers 275253.

Our relatively small study population limits the predictive power of the panels presented here, but the benefits of a serum biomarker and multimarker approach are clearly illustrated and further studies utilizing larger clinical cohorts, as discussed above, are well warranted. As we increase our knowledge of the biomarkers involved in these panels and continue to identify additional players, our ability to utilize the information we receive from exploratory investigations such as this will increase immensely. Continuing efforts in line with those presented here should bring us closer to providing effective and efficient individualized treatment to women diagnosed with this challenging disease.

cCR = clinical complete response; EGFR = epidermal growth factor receptor; IL = interleukin; LABC = locally advanced breast cancer; MIF = migration inhibitory factor; MMP = matrix metalloproteinase; NAC = neoadjuvant chemotherapy; pCR = pathologic complete response; TNF = tumor necrosis factor; tPAI-1 = tissue plasminogen activator inhibitor 1.

The authors declare that they have no competing interests.

BMN participated in the Luminex assays, oversaw the data analysis, and drafted the manuscript. JRM and KB conceived the study and coordination the transfer of patient samples. ST, AR, TML, and YW carried out the statistical analysis. AEL conceived the study, participated in the study design, and helped to draft the manuscript.