Neoadjuvant chemotherapy reduces the levels of HMGB1 and E-cadherin in patients with breast cancer

This study investigated the changes in serum tumor marker levels in patients with breast cancer (BC) after neoadjuvant chemotherapy (NACT) and their potential as prognostic factors in NACT. A total of 134 consecutive patients with BC treated at our hospital between January 2019 and December 2021 were retrospectively analyzed. Patients were treated with NACT based on the docetaxel, epirubicin, and cyclophosphamide (TEC) regimen and assessed for marker levels, T cell subsets, and therapeutic outcomes. Receiver operating characteristic (ROC) curves were constructed to evaluate the predictive performance of the markers. Outcome assessments showed that NACT effectively reduced the tumor size, leading to increased complete remission, partial remission, stable disease, and significantly reduced disease progression. Improved immune function has also been observed after NACT. The levels of two (E-cadherin and HMGB1) out of five markers (CA153, CK19, CEA, E-cadherin, and HMGB1) were significantly reduced after NACT before surgery compared with those at admission, suggesting that NACT modulates the levels of biomarkers. ROC analysis revealed that the area under the curve (AUC) of HMGB1 and E-cadherin combination was 0.87 for discrimination of therapeutic response with a sensitivity and specificity of 91.3% and 88.4%, respectively. Serum tumor marker levels were reduced after NACT in patients with BC. The reduction was most prominent for HMGB1, followed by E-cadherin. These biomarkers can be used to predict the therapeutic response to NACT with an AUC of 0.87, thus offering a new tool to monitor treatment progress in NACT for patients with BC.


Subjects.
In this retrospective study, we consecutively recruited 134 patients with BC treated at our hospital between January 2019 and December 2021.Patients were included if they were female, aged between 30 and 75 years, diagnosed with primary BC based on the eighth edition of the AJCC Cancer Staging Manual 20 , did not receive any previous BC-related therapy, such as radiotherapy and endocrine therapy, and completed NACT with survival time > 6 months.Patients were excluded if they had a mental disease, were comorbided with severe heart, liver, kidney, and other organ disorders/diseases, had cachexia or very poor health condition.Had infectious diseases, blood system diseases, metastatic tumors, or other gynecological tumors; or they were allergic to chemotherapy drugs, or had other contraindications preventing the use of NACT.Pregnant and lactating women were excluded from the study.To minimize potential bias in patient selection, all patient data were independently collected from the hospital data bases and from clinician records by two investigators.The data were cross-verified in case of possible inconsistence.Patients with incomplete/inconsistent data set were excluded.This study was approved by the institutional ethics committee of Liaocheng People's Hospital, and informed consent was obtained from all the patients.This investigation was conducted in accordance with the guidelines outlined in the Declaration of Helsinki and with relevant guidelines and regulations.

Treatment.
After baseline assessment at admission, patients were treated with NACT using an intravenous drip of docetaxel (75 mg/m 2 ) on day one, epirubicin (80 mg/m 2 ), and cyclophosphamide (500 mg/m 2 ) on day two (TEC) regimen 21 for a 3-weekly cycle.After six chemotherapy cycles, patients were subjected to radical surgery if the treatment was effective.All patients received colony-stimulating factor as a supportive therapeutic from day 2.

Blood sample analysis.
Fasting peripheral blood was collected in tubes containing ethylenediaminetetraacetic acid before and after NACT.The serum was separated by centrifugation at 3000 rpm for 10 min at room temperature, aliquoted, and stored at − 80 °C.The HMGB1 concentration was measured using the Sandwich ELISA of Shino-Test (Tokyo, Japan).The samples were added to the wells of microtiter dishes coated with anti-HMGB1 antibody.After incubation for 24 h at 4 °C, the plates were washed and incubated with a second enzyme-labelled antibody for 1 h at room temperature.A colored solution was added for 30 min, and the HMGB1 concentration was spectrophotometrically determined at 450 nm using a standard curve prepared with the kit.CEA, CK19, CA15-3, and E-cadherin levels were measured by enzymatic chemiluminescent immunoassay on an automatic immunoassay analyzer (Cobas e601 module, Roche, Sweden) using enzyme-linked immunosorbent assay (ELISA) kits from CanAg Diagnostics, Sweden and Roche Diagnostics, Sweden, according to the manufacturer's instructions.Th17, CD4 + CD25 + Tregs, and Treg cells were assessed using a flow cytometer (FACSymphony A1, BD Biosciences, USA).All samples were assayed in triplicates.
Evaluation of therapeutic effect.All lesions, except bone metastases, were measured using CT, MRI or ultrasonography, and bony lesions were evaluated using bone scintigraphy.Therapeutic outcomes were evaluated according to The Response Evaluation Criteria in Solid Tumors (RECIST) 22 .The responses were classified Statistical analysis.All statistical analyses were performed using the IBM SPSS software (version 18.0; IBM, USA).Normality of the data was determined using the Kolmogorov-Smirnov (K-S) test.Normally distributed data are expressed as mean ± standard deviation (SD).Otherwise, the data were represented as M (P25, P75).Numerical data were compared using paired t-tests.Counting data were expressed as percentages and compared using Chi-square or Fisher's exact tests.To evaluate the ability of serum tumor markers levels to predict therapeutic outcomes following NACT in patients with BC, receiver operating characteristic (ROC) curves, which are based on true-and false positive rates (sensitivity and 1-specificity) of actual therapeutic response and predicted response, were generated as described 23 .Areas under the ROC curve (AUC) were calculated to measure the predictive performance.For single and combination of biomarkers, binary logistic regression was used to obtain the probability, which was used as the predictive value for ROC curve.The confidence intervals (95% CI) for AUC were calculated are described previously 24 .The higher the AUC value, the higher the accuracy of prediction of markers.To plot the ROC curve, data regarding the therapeutic outcomes and predicated responses were imported into SPSS, converted into binary outcomes (positive or negative) to calculate the sensitivity and specificity at each threshold value and then the ROC curves were plotted using the true positive rate against the false positive rate.P-value < 0.05 was considered statistically significant.
Ethical approval.This study was approved by the institutional ethics committee of Liaocheng People's Hospital.

Results
Baseline clinicopathological characteristics.A total of 134 patients were included in this study from 237 eligible patients (Fig. 1).The mean age 41.90 ± 4.43 years.The LD of the lesions was 3.80 ± 1.54 cm, ranging from 0.8 to 8.7 cm.The histological types of tumors included ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), and invasive lobular carcinoma (ILC), and the cancer stage was between TNM stage II and IV, with over 70% being stage II (Table 1).Other baseline data, such as BMI, blood pressure, and renal and liver function tests, are shown in Table 1.

NACT effectively reduces tumor size.
The outcomes of patients undergoing NACT were assessed at the end of the therapy.The results showed that NACT significantly reduced tumor size (3.80 ± 1.54 vs 1.60 ± 0.64, P < 0.01), resulting in a CR of 37.3% and a total response of 70%.PR and SD were 32.9% and 25.7%, respectively (Table 2), demonstrating that NACT is effective in treating BC.

NACT reduces the level of serum tumor markers.
The levels of the five circulating biomarkers were assessed at admission and after NACT.After NACT, there were significant reductions in the levels of HMGB1 and E-cadherin.Compared to that at admission, the overall levels of HMGB1 and E-cadherin were reduced by 62.2% and 55.1%, respectively, at the end of NACT (Table 3, P < 0.01).The levels were reduced more remarkably in patients with CR and PR outcomes, and less or not reduced in patients with SD and PD outcomes (Table 3).
HMGB1 and E-cadherin combination has excellent predictive performance on therapeutic response to NACT .Since the levels of HMGB1 and E-cadherin were significantly reduced after NACT, we analyzed their predictive performance for therapeutic response to NACT.ROC curve analysis showed that the AUCs of HMGB1 and E-cadherin alone were 0.78 and 0.71, slightly lower than 0.80, a value recognized for acceptable predictive marker.On other hand, when the two makers were combined, the AUC reached 0.87 for discrimination of therapeutic response (Fig. 2), indicating that HMGB1 and E-cadherin combination has excellent predictive performance.Furthermore, the sensitivity and specificity of the marker combination were 91%

Discussion
In the present study, we retrospectively analyzed changes in the levels of serum tumor markers before and after NACT and determined the prognostic significance of these markers in patients with BC.Our results showed that NACT had a significant therapeutic effect on BC; the levels of HMGB1 and E-cadherin were reduced after NACT; and the combined use of the two markers could accurately predict the therapeutic response to NACT.Our data suggest that these markers can be used to monitor NACT outcomes in individual patients.
Although NACT has become a routine treatment method for locally advanced BC and could effectively improve the overall efficacy of breast cancer treatment 8 , the assessment of therapeutic response to NACT is mainly based on imaging studies, such as chest and abdominal computed tomography (CT) and bone scintigraphy, which are relatively costly, time consuming, and insensitive.These techniques show only macroscopic changes in tumor volume after several therapy cycles.Serum tumor markers offer promising alternatives for evaluating the therapeutic effects of various methods, such as surgery, chemotherapy, and radiotherapy 25,26 .In this study, we found that TEC regimen-based NACT is effective in BC patients with high CR, PR, and low DP, confirming that NACT is an effective option for downsizing locally advanced BC as anticipated 27 .The CR and PR rates observed in our study were similar to those observed in previous works 28 .
To evaluate the potential of serum biomarkers to measure therapeutic responses, we assessed the levels of the five markers before and after treatment.A significant reduction in the levels was observed in two out of the five markers assessed after NACT, suggesting that NACT modulates the expression and accumulation of these markers.Among the markers evaluated, CA153 is widely used clinically as a serum biomarker for the diagnosis and prognosis of BC.It is used to evaluate chemotherapy efficacy and postoperative outcome 29 .In this study, CA153 levels changed slightly, but not significantly, before and after NACT, suggesting that it could not be used as a predictor of therapeutic response to TEC regimen-based NACT in the treated patient population.
CEA is a broadly proven biomarker for cancers such as colorectal, lung, pancreatic, rectal, and breast cancers 30,31 .Its levels are elevated with aging, cancer, and non-cancer diseases 31 .In the present study, CEA levels were slightly downregulated following NACT.However, although CEA levels were significantly reduced after treatment with taxane and anthracycline-based NACT drugs 32 , it was not useful in predicting BC relapse after curative surgery 33 .Based on the present study, CEA is not a suitable marker for prognosing TEC regimen-based NACT.
CKs are the major structural proteins in epithelial cells, and cells become cancerous when their expression is abnormal 34 .However, the change in CK19 protein is not well documented after NACT.Our data showed that the CK19 levels did not change significantly after NACT.This is consistent with earlier results that CK19 expression did not change significantly in tumor and node biopsies before and after treatment 35 .
HMGB1 has recently been identified as a potential diagnostic biomarker for breast cancer because it may be involved in its development and progression of breast cancer.The sensitivity and specificity of HMGB1 level for the diagnosis of breast cancer were 73.2% and 84.0%, respectively 18 .However, the response to NACT remains controversial.Our assessment showed that serum HMGB1 levels were reduced by over 60% after NACT, suggesting that HMGB1 is likely suppressed by NACT.It is likely that the dynamics of HMGB1 may vary with NACT regimes and cancer subtypes and more studies are needed to elucidate the correlation in various NACT regimens.Previously, HMGB1 levels were found to be higher in non-responders and lower in metastatic breast cancer patients with CR or PR undergoing NACT 36 .However, increased HMGB1 after the initial dose of neoadjuvant www.nature.com/scientificreports/epirubicin/docetaxel combination chemotherapy appeared to be associated with better disease-free survival, although the association did not reach significance 37 .E-cadherin is a transmembrane glycoprotein that functions in calcium-dependent cell-to-cell adhesion 38 .Increased levels of serum E-cadherin are an indication of serious dysfunction of cell surface molecules.Our assessment showed that E-cadherin levels were significantly reduced after NACT, confirming the usefulness of E-cadherin as a predictive marker for NACT.This result is consistent with previous studies in BC 39,40 , but it is in contrast to results obtained in colorectal cancer where E-cadherin is inversely associated with tumor stage 41 .
Although NACT has become a standard clinical option to downsize the tumor for BC-conserving surgery, some BC patients do not respond well to the treatment or even have adverse reactions, resulting in missed surgery due to tumor heterogeneity that causes insensitivity to NACT drugs.Therefore, timely and accurate evaluation of the effect of NACT is important for patients.In this regard, traditional methods such as physical examination, B ultrasound, and X-rays are often not sensitive enough to detect an early response to NACT treatment.The significant changes in the levels of these markers after NACT suggest that they could be used to reflect the therapeutic response in a timely and sensitive manner.ROC analysis showed that the AUCs of HMGB1 and E-cadherin alone were < 0.80, indicating that the use of single markers may not provide adequate predictive accuracy for therapeutic outcomes.However, when the two markers were combined, the AUC was close to 0.9, indicating that the combination had excellent discrimination power for favorable and unfavorable therapeutic responses.To our knowledge, this is the first report demonstrating that the combined use of HMGB1 and E-cadherin is able to provide better predict the treatment response of NACT.For clinical application, HMGB1 and E-cadherin in patients subjected to NACT could be analyzed over the course of treatment in a regular base, for instance, in a weekly basis, as along as the NACT is initiated, and the changes of the two markers be followed along with other pathological response.If the levels of the two markers trend to reduce, this is an indication that the current treatment plant is effective, otherwise, increased or decreased dosing or even ceasing of drugs may be considered.
Although our study demonstrated changes of serum tumor marker level after NACT, the changes, however, are influenced by many factors.Different patient population and breast cancer subtypes are likely response to NACT differently and different biomarkers may be applied to patients with hormone receptor (HR)-positive/human epidermal growth factor 2 (HER2)-negative BC 7 .NACT regimens would also be a major factor influencing the therapeutic outcome as well as the changes in the serum tumor marker level.Although the major chemotherapy regimens include anthracyclines and taxanes, additional drugs such as trastuzumab, pertuzumab and Dual Her2 blockade have been added to NACT 42 .Therefore, the changes in tumor marker levels for various regimens and treatment plans need to be defined since the drugs have different model of actions and will have different impact on the regulation of expression of biomarker.
Our study has some limitations.This was a retrospective investigation at a single center with a relatively small number of patients and heterogeneous subtypes of BC.The NACT in this study was based only on the TEC regimen, and the results obtained might not be applicable to NACT based on other drugs/regimens.In addition, the patients were enrolled mainly based on the availability and were not classified and grouped based on BC subtypes and other demographics such as age.The evaluation of NACT outcomes were made immediately after the completion of NACT and long-term follow-up was not included in the analysis.Analysis of biomarker levels was made only before and after NACT.Prospective studies should include more patients, BC cancer subtypes, NACT regimens and plan, and measurement timepoints to validate the usefulness of these biomarkers, particularly the HMGB1 and E-cadherin combination.

Conclusions
We examined changes in several serum tumor markers after NACT and the therapeutic response to NACT in patients with BC.We found that HMGB1 and E-cadherin levels were significantly reduced in patients after NACT treatment.This reduction was the greatest in HMGB1 followed by E-cadherin.ROC analysis showed that these biomarkers had prognostic value for NACT and combined use of HMGB1 and E-cadherin had an AUC of 0.87 for discrimination of therapeutic response with the sensitivity and specificity of 91.3% and 88.4%, respectively.Therefore, the combined use of HMGB1 and E-cadherin may offer a new tool for monitoring treatment progress in NACT.

Figure 1 .
Figure 1.Diagramic chart of patient selection and analysis.

Figure 2 .
Figure 2. Receiver operating characteristic (ROC) curves of HMGB1 and E-cadherin for the discrimination of a BC with and without favorable therapeutic responses to NACT.

Table 2 .
Therapeutic response of patients with breast cancer after neoadjuvant chemotherapy (NACT) in patients with breast cancer.NACT, neoadjuvant chemotherapy.**P < 0.01 compared with at admission.