The Residual Tumor Autophagy Marker LC3B Serves as a Prognostic Marker in Local Advanced Breast Cancer after Neoadjuvant Chemotherapy

Neoadjuvant chemotherapy (NCT) directed to the breast and the axilla, followed by definitive surgical therapy, is the standard of care for locally advanced breast cancer (LABC). Achieving a pathologic complete response (pCR) is the ultimate goal of successful NCT treatment, as patients with pCR may have a relatively favorable outcome (1–3). The majority of patients with breast cancer undergoing NCT (ranging from 70% to 90% in different trials) who develop residual tumors in the breast or axilla (termed patients with non-pCR) have an increased risk of relapse and death even if they receive additional systemic therapy. Furthermore, residual cancer cells remaining after chemotherapy may have a decreased sensitivity to further systemic treatment and a more aggressive behavior that promotes metastasis (4). Various studies have indicated that the biologic characteristics of residual tumors, including their size, grade, and proliferative index (Ki-67 expression), and the number of involved lymph nodes is closely correlated with the outcome of patients with non-pCR (5–8). These factors can also be used to evaluate the risk of disease progression, which may affect the treatment strategy. Therefore, the study of prognostic markers for patients with non-pCR is of great interest for further understanding the biologic characteristics of residual tumors after chemotherapy.

In recent years, studies of a conserved catabolic process by which cells self-digest their organelles, known as autophagy, have led to an advanced understanding of nonapoptotic programmed cell death (9). Evidence supports the hypothesis that autophagy in cancer cells, which is triggered by starvation and environmental stress, may have two roles in cancer development, as a form of cell death but also as a protective pathway for the survival of cancer cells and resistance to antineoplastic treatment (10–12). During the process of autophagy, a lipid bilayer structure called an autophagosome is formed, which matures into an autolysosome following lysosome engulfment. The cytoplasmic materials in autolysosomes are resolved into amino acids and are redistributed for cellular viability under conditions of starvation or stress.

The membrane-bound microtubule–associated protein chain 3 (LC3) is one of the most specific biomarkers of autophagy. The synthesis and activation of LC3 are induced and regulated by autophagy-associated genes such as ATG3, ATG7, and ATG8, and this process involves transformation of the soluble form LC3-I to LC3-II, which is tightly bound to the membrane of the autophagosome (13, 14). In mammals, LC3 is expressed as three isoforms, A, B, and C. The B isoform, LC3B, has broad tissue specificities and is widely used in autophagy-related studies (15). Although Lazova and colleagues reported the common expression of LC3B in various malignancies and demonstrated that autophagy plays a significant role in cancer progression (16), the role of LC3 in primary tumors remains controversial because autophagy has also been shown to contribute to the suppression of tumorigenesis (17, 18).

In this study, we hypothesized that increased LC3B staining, which is commonly interpreted as evidence for autophagy activation in residual breast cancer cells after NCT, would be correlated with the risk of disease progression and death. We also investigated whether the expression of LC3B is concordant with the proliferation index of residual tumor cells. To our knowledge, this is the first study that demonstrates the prognostic role of autophagy in residual breast cancer cells after chemotherapy.

We collected data and surgical specimens from 303 consecutive patients who were diagnosed with local advanced breast cancer (T3/T4 and/or N2/N3 disease) and received NCT followed by radical surgery at the Shanghai Cancer Center (Shanghai, China) from 2003 to 2008. In total, 229 patients with residual invasive tumors in the operated breast were included in this study (39 patients were considered pCRs, with the absence of invasive carcinoma in both the breast and lymph node tissue of the surgical specimen, and 35 patients demonstrated only residual invasive carcinoma in the excision lymph nodes). Data on the medical history, patient characteristics, local and distant extent of disease (evaluated by chest CT, bone scan, abdominal ultrasound, bilateral mammography, breast ultrasound or breast MRI), and the pathologic assessment of morphologic and biologic features were collected. Core needle biopsy (CNB) was performed to confirm the diagnosis of invasive breast cancer before NCT, and fine needle aspiration of palpable or ultrasound-detected lymph nodes was performed at the time of diagnosis. Patients who had received any treatment before NCT or who had metastatic disease before surgery were not eligible for this study. Other exclusion criteria included bilateral breast cancer, male breast cancer, and inflammatory breast cancer.

All patients in this study underwent an NCT regimen consisting of NE (Navelbine 25 mg/m² on days 1 and 8 and epirubicin 60 mg/m² on day 1 every 3 weeks), CEF (cyclophosphamide 600 mg/m², day 1; epirubicin 60 mg/m², day 1; and 5-fluorouracil 600 mg/m², day 1; every 3 weeks) or PC (paclitaxel, 80 mg/m², and carboplatin area under the curve, 2 mg·· min/mL, on days 1, 8, and 15 of a 28-day cycle) for a median of four cycles (range 3–6 cycles). Mastectomy and axillary lymph node dissection were performed within 4 weeks of the completion of NCT, and additional cycles of chemotherapy were administered after the operation to complete a total of six cycles at the discretion of the treating physician. Radiotherapy was offered at the discretion of the treating radiation oncologist. Endocrine therapy was performed for patients with hormone receptor-positive. Trastuzumab was recommended for patients with human epidermal receptor-2 (HER-2)-positive in the adjuvant setting, but it was not included in any preoperative treatment. All patients were followed-up every 3 months for the first year and then every 6 months until death.

Each specimen obtained from CNB and surgery in this study was examined independently by 2 experienced pathologists at the Shanghai Cancer Center. All patients in this study were confirmed to have residual invasive ductal carcinoma in the breast after NCT following pathologic evaluation of the surgical specimens. Patients with residual disease in the lymph node only or with residual ductal carcinoma in situ (DCIS) were not included in this study. The Miller–Payne grading system was used to evaluate the pathologic response in the breast (19); no change or some alteration to individual malignant cells but no reduction in overall cellularity was classified as grade 1; up to a 30% loss of tumor cells was considered grade 2; an estimated 30% to 90% reduction in tumor cells was considered grade 3; more than 90% loss of tumor cells, such that only small clusters or widely dispersed individual cells remained, was considered grade 4; and no remaining invasive malignant cells was considered grade 5.

Data about expression of the estrogen receptor (ER), the progesterone receptor (PR), and HER-2 in the CNB and surgical specimens were collected from a database of the pathologic center in Shanghai Cancer Hospital (Shanghai, PR China). The cutoff value for ER positivity and PR positivity was 1% of tumor cells with positive nuclear staining. HER-2–positive status was defined as 3+ according to circumferential membrane-bound staining (HercepTest; Dako Cytomation) or amplification confirmed by florescent in situ hybridization. According to the 2011 St. Gallen consensus (20), we defined breast cancer subtypes as follows: luminal-A (ER and/or PR positive, HER-2 negative, and Ki-67 < 15%); luminal-B (ER and/or PR positive, HER-2 positive; ER and/or PR positive, HER-2 negative, and Ki-67 ≥ 15%); HER-2⁺ (ER and PR negative, HER-2 positive) or triple negative (TNBC; ER negative, PR negative, and HER-2 negative).

Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded tissue sections collected from residual tumor specimens using a two-step protocol (GTVision III). LC3B was detected using the rabbit anti-LC3B polyclonal antibody ab51520 (Abcam Inc.). Beclin-1 was detected using the rabbit anti-Beclin-1 polyclonal antibody ab51031 (Abcam Inc.). Ki-67 was detected using the mouse monoclonal antibody MIB-1 (Dako). Negative control samples were treated identically but with the primary antibodies omitted. Positive control samples were intratumoral breast cancer cells free of primary treatment, according to the antibody instruction. The density of positive staining for LC3B was measured using a computerized image system composed of a Leica CCD camera DFC420 connected to a Leica DM IRE2 microscope (Leica Microsystems Imaging Solutions Ltd.). Photographs of eight representative fields were captured using the Leica QWin Plus v3 software. To assess LC3B staining, a uniform setting was applied for all slides. The integrated optical density (IOD) of all positive LC3B staining in each photograph was measured, and its ratio to the total area of each photograph was calculated as the LC3B density. The density was calculated using Image-Pro Plus v6.2 software (Media Cybernetics) as described previously (21, 22). The positivity of Beclin-1 and Ki-67 expression was evaluated independently by two experienced pathologists on a case-by-case basis. Beclin-1 positivity was defined as greater than 10% of the tumor cells with positive staining. The Ki-67 value was expressed as the percentage of positive cells (at least 1,000) with nuclear staining.

For LC3B, the cutoff value for high and low density was the median value of the IOD. The relapse-free survival (RFS) was calculated from the date of surgery to the date of disease relapse (local or distant relapse). The overall survival (OS) was calculated from the date of diagnosis to the date of death or last follow-up. Patients without events or death were censored at the last follow-up. The Pearson χ² test was used to compare the qualitative variables (Fisher exact test was used when needed). The quantitative variables were analyzed with a Student t test (a paired sample t test was used when needed).

Survival curves were constructed using the Kaplan–Meier method, and the survival difference was determined with the log-rank test. Adjusted hazard ratios (HR) with 95% confidence intervals (CI) were calculated using Cox proportional hazards models. All reported P values were two-sided and were calculated at a significance level of 0.05. The statistical analysis was carried out using SPSS (version 13.0; SPSS).

Positive staining for LC3B protein was observed mainly in the cytoplasm of residual cancer cells. Most of the intratumoral or extratumoral stromal cells demonstrated negative staining, although sporadic positive staining in these cells was observed (Fig. 1A–D). The positively stained cells were classified as central tumor cells (from the tumor center to the midpoint of the tumor radius), peripheral tumor cells (from the midpoint of the tumor radius to the tumor margin), or extratumoral cells (normal cells outside of the tumor). The LC3B density was similar in the peripheral and central areas of the tumor (P = 0.905) but was significantly lower in the extratumoral area (P < 0.0001; Fig. 1E).

The overall pCR rates of the three NCT regimens were 9.1% (5 of 55) for CEF, 11.9% (20 of 168) for NE, and 17.5% (14 of 80) among 303 patients. There was no significant difference in pCR rate among the three regimens (P = 0.320, Fisher exact test). For all patients with non-pCR, 35 patients had only residual disease in the lymph nodes. The remaining 229 patients with residual disease in the breast were included in this study. The median age was 49 years, and 59.4% patients were premenopausal. The proportion of stage II and III disease was 36.2% and 63.8%, respectively. Most patients (94.3%) had residual invasive ductal carcinoma, 5 had residual invasive lobular carcinoma, 3 had mucinous adenocarcinoma, and 5 had mixed invasive carcinoma. The median IOD for LC3B protein was 0.019 (range, 0.007–0.076). An IOD of 0.007 to 0.019 was considered low LC3B density, whereas an IOD of 0.020 to 0.076 was considered high LC3B density. The relationship between LC3B expression and the patients' clinicopathologic features is shown in Table 1. High LC3B density was more commonly observed in patients with PR-negative staining at diagnosis (P = 0.033), Beclin-1–positive staining (P < 0.001) at surgery, and a higher Ki-67 index at surgery (P = 0.029). The breast cancer subtype was also correlated with LC3B expression (P = 0.003), in which a lower proportion of LC3B high-density staining was detected in patients with luminal-A in comparison with the other subtypes (32.4% in luminal-A, 59.4% in luminal-B, 65.4% in HER-2⁺, and 46.7% in triple negative).

The median follow-up time was 65 months. The 5-year RFS and OS times of all 229 patients were 67% and 79%, respectively. In the univariate analysis (Table 2), the number of residual involved nodes, Ki-67 status, LC3B status, and Beclin-1 expression were significantly associated with the RFS and OS. All significant factors in the univariate analyses were included in the multivariable model (Table 2); only LC3B and residual nodal status after NCT were significant predictors of both RFS (HR, 1.180, P = 0.012; and HR, 3.067, 6.103, P < 0.001, respectively) and OS (HR, 2.428, P = 0.008; and HR, 1.809, 4.283, P = 0.003, respectively). Ki-67 status also served as an independent predictor for RFS (HR, 1.122, P = 0.019). Because of their biologic relevance, we considered the ER status, HER-2 status, grade, and residual tumor size in the Cox model, although these factors were not statistically associated with recurrence or death in the univariate analysis. The inclusion of these parameters did not change the association between the three prognostic factors and survival. The survival curves of the patients with LC3-low and LC3-high are shown in Fig. 2A and B. The 5-year RFS and OS rates were significantly higher among patients with LC3B-low than those with LC3B-high (78% vs. 56% and 87% vs. 69%, respectively).

Using a combination of node status and LC3B expression, all patients were classified into four subgroups: node−/LC3B-low (n = 30); node−/LC3B-high (n = 19); node+/LC3B-low (n = 89); and node+/LC3B-high (n = 91). The survival curves of these four groups are shown in Fig. 3A and B. Differences in both RFS (P < 0.001) and OS (P = 0.003) were significant among the four groups. Patients with no positive residual node and low LC3B expression had a low risk of relapse and death, with both a 5-year RFS and OS of 97%, whereas the 5-year RFS and OS for patients with positive node status and high LC3B expression were 52% and 66%, respectively.

We also analyzed the relationship between LC3B expression and survival according to the different breast cancer subtypes (according to 2011 St. Gallen consensus; Fig. 4A–H). Among the four subtypes, the prognostic value of LC3B was most significant among patients with TNBC for both RFS and OS (P = 0.007 and 0.006, respectively). Furthermore, LC3B expression was able to help differentiate patients with low-risk TNBC from patients with high-risk TNBC with 5-year RFS values of 87% and 52%, respectively, and 5-year OS values of 93% and 60%, respectively.

The role of autophagy in cell-fate decisions remains controversial, as autophagy represents a mechanism of programmed cell death through which old and damaged cells are eliminated, as well as an indispensable survival pathway in response to unfavorable conditions. Recently, these two faces of autophagy in tumorigenesis and development have been widely discussed. Although autophagy suppresses tumorigenesis during the early stage of tumor initiation (23, 24), it also enables tumor cells to survive under stressful conditions, including hypoxia, nutrient deprivation, and antineoplastic therapies. Therefore, autophagic death of damaged cancer cells following antineoplastic therapy may contribute to the viability of the whole tumor cluster (25).

NCT, which is also known as preoperative, primary, or induction chemotherapy, is the standard of care for local advanced breast cancer. After NCT, the majority of patients with residual disease have a relatively higher risk of relapse and death even if they receive additional systemic therapy, and the biologic features of these chemoresistant cancer cells have drawn significant attention in recent studies. However, the role of autophagy in the neoadjuvant setting for breast cancer has not yet been discussed. In the present study, we focused on the role of autophagy in residual breast cancer cells by evaluating expression of the autophagy marker LC3B by IHC analysis. In the multivariate survival analysis, LC3B expression and residual lymph node metastasis showed independent prognostic value. Because treatment regimen, treatment cycle, and treatment response (evaluated in this study according to Miller–Payne grading) were not correlated with patient survival, our findings provide evidence that the properties, rather than the quantity, of the residual tumor may be responsible for unfavorable outcomes. In patients with non-pCR, the identification of more aggressive chemoresistant cancer cells may help to further tailor approaches to prevent disease relapse. In addition, autophagy may play an important role in identifying and understanding the mechanism responsible for the poor biologic behavior of residual tumors after NCT.

Previous studies have indicated that expression of the autophagy marker LC3B is a common feature of solid tumors and is associated with tumor proliferation and metastasis (16, 26, 27). There are two subtypes of LC3B protein, LC3B-I and LC3B-II; during autophagy, LC3B-I transforms into LC3B-II, which binds to the membrane of autophagosomes. Furthermore, the membrane-bound LC3B-II protein level has been proposed as a marker for the activation of autophagy (28). Although IHC detection of LC3B cannot distinguish LC3B-I and LC3B-II, this approach can still assess autophagic activity because LC3B-II is the dominant form of LC3B (26, 29). We found that the levels of LC3B and Beclin-1 protein in IHC were significantly correlated. Although autophagy can be regulated by other factors and Beclin-1 is not always overexpressed during the induction of autophagy, it still acts as an essential modifier of the autophagic process. Furthermore, IHC staining for LC3B is a reliable method to detect autophagy activity. However, it is important to note that due to insufficient collection of specimens through CNB, we were unable to obtain information about LC3B expression before NCT. Instead, we collected 30 tumor samples in patients with NCT-naïve LABC, and detected LC3B expression in a different part of tumor. We found that the LC3B expression was generally high, with an IOD range of 0.050 to 0.088. We failed to detect heterogeneity of LC3B in different patients and different parts of tumor. This may be because of the incomplete sampling of a large tumor; however, it may also indicate that autophagy is a ubiquity of process in development of primary breast cancer. Therefore, we hypothesized that primary LC3B expression before NCT would have limited impact on autophagy in residual tumors. With a sufficient sampling of the excision specimens of shrinked tumors, it may be possible to obtain accurate information about the level of autophagy in the residual tumor. In addition, we did not find heterogeneity in the intratumoral distribution of LC3B, which indicates that LC3B may serve as a reliable marker of autophagy in residual tumors after NCT.

The level of LC3B expression was not associated with the treatment regimen, primary disease stage (tumor size and node status), or treatment response. However, LC3B density was remarkably associated with the breast cancer subtype. A relatively lower LC3B expression was observed among patients with the luminal-A subtype, a less chemosensitive subtype, as compared with the luminal-B and HER-2⁺ subtypes in this study. Furthermore, the prognostic value of LC3B was most significant in patients with TNBC. TNBC is the biologic entity that lacks ER, PR, and HER-2 expression; although TNBC is initially chemosensitive to NCT, patients with non-pCR with residual TNBC disease generally have an unfavorable prognosis with a short RFS and OS (30). Furthermore, several reports have suggested that TNBC represents a heterogeneous group comprising subtypes with different outcomes (31–33), as TNBC with CK5/6⁺ and/or EGFR⁺ expression, which is referred to as a true basal subtype, has shown poorer survival than CK5/6⁻ and EGFR⁻ TNBC subtypes. TNBC can also be divided into aggressive and less aggressive clones. Our findings indicate that LC3B is a clinically applicable biologic marker for TNBC and can identify patients with a different risk of relapse or death. We also found a highly consistent correlation between LC3 protein and expression of the proliferation marker Ki-67, as residual cancer cells that expressed higher levels of LC3 protein also had a higher Ki-67 index. In patients with non-pCR after NCT, the Ki-67 index at surgery showed clear prognostic value, in agreement with previous investigators, suggesting that the Ki-67 index can be used to assess the proliferative capacity of micrometastases (6, 8). Recent studies also reported that in breast cancer, LC3B and Ki-67 showed strong correlations (P < 0.0001). High LC3B was associated with proliferation, invasion and metastasis, high nuclear grade, and worse outcome (16). Taken together, the expression and prognostic value of LC3B correlated with particular tumor clones, demonstrating more aggressive biologic behavior in comparison with the remaining residual cancer cells.

There may also be additional mechanisms to explain the prognostic role of LC3B in residual tumors. Tumorigenic breast cancer stem cells (BCSC) are considered to be resistant to chemotherapy and therefore responsible for cancer relapse. Li and colleagues reported that after NCT, the percentage of CD44⁺CD24^−/low BCSCs increased from 4.7% to 13.4% in the primary tumor, with increased mammosphere formation efficiency (34). Autophagy may play an indispensable role in the survival of BCSCs, as recent studies have shown that autophagy is mechanistically linked to the maintenance of BCSCs (35) and BCSC-induced tumor development (36).

Paradoxically, prolonged autophagic flux may lead to decreased LC3 expression, resulting in cancer cell death (29). The appearance of LC3 in residual tumors under chemotherapy reflects the widespread induction of autophagy, moderate autophagic activity, and sustained autophagy potential. However, although it is not possible to clarify whether residual tumor cells survive under NCT through the induction of autophagy, it is plausible to hypothesize that sustained autophagic potential of the surviving tumor cells may determine their resistance to further systemic treatment following surgery and therefore early disease progression.

In conclusion, the current study highlighted the utility of LC3B staining as a measure of autophagy in breast cancer and established LC3B as a potential prognostic marker in patients with non-pCR after NCT. Moreover, our findings may lead to further understanding of the importance of autophagy in the aggressive biologic behavior of chemoresistant cancer cells, as well as the therapeutic vulnerability of residual tumor cells after chemotherapy. Thus, the evaluation of autophagy in patients with non-pCR may help shape postoperative treatment strategies for the prevention of disease relapse, and it is also plausible to conceive of clinical trials employing drugs targeting autophagy in the neoadjuvant setting.

No potential conflicts of interest were disclosed.

Conception and design: S. Chen, Y.-Z. Jiang, K.-D. Yu, Y. Liu, Z.-M. Shao

Development of methodology: S. Chen, L. Huang, R.-J. Zhou, K.-D. Yu, Z.-M. Shao

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Chen, Y.-Z. Jiang, R.-J. Zhou, Y. Liu, Z.-M. Shao

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Chen, Y.-Z. Jiang, L. Huang, R.-J. Zhou, Y. Liu, Z.-M. Shao

Writing, review, and/or revision of the manuscript: S. Chen, Y.-Z. Jiang, L. Huang, K.-D. Yu, Z.-M. Shao

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Chen, L. Huang, K.-D. Yu, Y. Liu, Z.-M. Shao

Study supervision: S. Chen, K.-D. Yu, Z.-M. Shao

This research was supported by the Multidiscipline Comprehensive Treatment Cooperation Group Foundation of Fudan University Cancer Hospital, Shanghai, China (DXK200801), the Key Clinical Program of the Ministry of Health (2010–2012), the Shanghai United Developing Technology Project of Municipal Hospitals (SHDC12010116), and the National Natural Science Foundation of China (30971143, 30972936, and 81001169). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the article.

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