|Year : 2017 | Volume
| Issue : 4 | Page : 1168-1177
Predictive significance of stromal-associated lymphocytes in response to neoadjuvant chemotherapy in breast cancer
Afaf T Ibrahiem1, Hala A Mouhamed2, Abeer H Anter3
1 Department of Pathology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
2 Department of Pathology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
3 Department of Clinical Oncology and Nuclear Medicine, Faculty of Medicine, Mansoura University, Mansoura, Egypt
|Date of Submission||13-Aug-2017|
|Date of Acceptance||08-Oct-2017|
|Date of Web Publication||04-Apr-2018|
Afaf T Ibrahiem
Department of Pathology, Faculty of Medicine, Mansoura University, Mansoura
Source of Support: None, Conflict of Interest: None
In this work, we aimed to assess whether the high stromal-associated lymphocytes and their subtypes in pretreatment core biopsies from patients with invasive breast carcinoma have a predictive role in pathologic response to neoadjuvant chemotherapy (NACT).
Tumor-infiltrating lymphocytes including stromal-associated lymphocytes in breast cancer are considered a form of the host immune response to malignancy. Several studies suggested that tumor-infiltrating lymphocytes could predict better response to NACT, but their roles in predicting complete pathologic response (pCR) rate after NACT still needs further evaluation.
Patients and methods
Stromal-associated lymphocytes were evaluated in 100 pretreatment core biopsies from primary breast cancer patients eligible for NACT. Cases were defined as lymphocyte-rich if the percentage of lymphocytes was more than 50% and also the lymphocytes were evaluated for CD20, CD3, CD8, and CD4. Type of pathological response to NACT was determined in post-therapy surgical specimens of the same patients, and we correlated between type of response to stromal-associated lymphocytes and their subtypes.
pCR was detected in 22 (22%) breast cancer patients, partial pathologic response in 54 (54%), and no pathologic response in 24 (24%) patients. There was a significant association between percentage of stromal-associated lymphocytes and achievement of pCR rate (P = 0.006). In addition, high stromal-associated lymphocytes are significantly associated with advanced histological grade (P = 0.004), Ki-67 (P = 0.028), and negative estrogen receptor state (P = 0.015).
High stromal-associated lymphocytes and high percentage of CD8-positive T cells in stromal-associated lymphocytes could be reliable predictors for pCR rate after NACT.
Keywords: breast cancer, neoadjuvant chemotherapy, stromal-associated lymphocytes, tumor-infiltrating lymphocytes
|How to cite this article:|
Ibrahiem AT, Mouhamed HA, Anter AH. Predictive significance of stromal-associated lymphocytes in response to neoadjuvant chemotherapy in breast cancer. Menoufia Med J 2017;30:1168-77
|How to cite this URL:|
Ibrahiem AT, Mouhamed HA, Anter AH. Predictive significance of stromal-associated lymphocytes in response to neoadjuvant chemotherapy in breast cancer. Menoufia Med J [serial online] 2017 [cited 2020 Jun 6];30:1168-77. Available from: http://www.mmj.eg.net/text.asp?2017/30/4/1168/229229
| Introduction|| |
Neoadjuvant chemotherapy (NACT) has made a tremendous impact on the management of locally advanced breast cancer and has increased the chance of breast-conserving surgery. NACT of early breast cancer leads to high clinical response rates in 70–90% of patients; however, complete pathologic response (pCR) is observed only in 10–25%.
Several studies considered that pCR is a predictor of better survival in breast cancer and could be a potential surrogate of clinical outcome. The analysis of predictive biomarkers for pCR on pretreatment core biopsies may help to optimize therapy decisions and clinical benefits from NACT.
Immunotherapies aimed to increase and support immune responses against tumors. Chemotherapy is considered as a form of immunotherapy. Tumor-infiltrating lymphocytes (TILs) are considered a form of the host immune response to malignancy. Several studies suggested that TILs could predict better response to NACT in breast cancer, but their roles in predicting pCR rate after NACT still need further evaluation. Understanding the function of TILs and their specific subtypes within breast cancer might help in selection of patients who are most likely to benefit from immunotherapy.
This study aimed to determine whether the stromal-associated lymphocytes in pretreatment core biopsies from invasive breast cancer have a predictive role in the response to NACT and the correlation of high stromal-associated lymphocytes to pCR.
| Patients and Methods|| |
In the period between June 2014 and June 2016, 100 female patients with cancer breast were investigated. These patients have primary invasive breast carcinoma and were planned to receive NACT followed by definitive surgical resection. Patient's clinicopathological data [age, clinical size, histological type, grade, axillary lymph node (LN) state] and paraffin blocks were retrieved from archives of Pathology Lab and Oncology Unit, OCMU, Mansoura University, Egypt.
This study included 100 female patients with breast cancer; 24 (24%) cases received anthracycline-based regimens (4–6 cycles of adriamycin 60 mg/m 2 and cyclophosphamide 600 mg/m 2), 56 (56%) cases received anthracycline-and-taxane-based regimens [four cycles of Adriamycin and cyclophosphamide (AC) followed by paclitaxel (80 mg/m 2) weekly for 12 weeks or four cycles of AC followed by four cycles of docetaxel (75 mg/m 2)], and 20 (20%) cases received Herceptin-based NACT regimens (Roche/Genentech's, NY.USA). Four cycles of Herceptin consisting of Herceptin 6 mg/kg plus docetaxel or weekly Herceptin 2 mg/kg for 12 weeks were given for patients with human epidermal growth factor receptor 2 (Her2)/neu overexpression tumors. The surgery was performed 3–4 weeks after the last chemotherapy cycle. Patients with Her2-positive tumors completed 1 year of Herceptin, chest wall irradiation if indicated, and received adjuvant hormonal therapy if the tumors were estrogen receptor (ER) and/or progesterone receptor (PR) positive.
Histopathological examination and assessment
Core biopsies of all patients were examined for histopathological type, histologic grade, and percentage of stromal-associated lymphocytes. ER, PR, and Her2/neu immunoreactivity of the tumors were reviewed and confirmed.
According to the recommendation of Salgado et al. for evaluating TILs, we evaluated only stromal-associated lymphocytes within the tumor. Areas with necrosis, inflammation, and crush artifacts in biopsies were not scored. Stromal-associated lymphocytes were recorded as a percentage of lymphocytes in relation to surface area of stroma. Fifty percent was used as a cutoff value to divide the tumor into stroma-rich (>50%) and stroma-poor (<50%). Intratumoral lymphocytes were assessed by observing lymphocytes that were in contact with tumor cells and divided into tumors with intratumoral lymphocytes and tumors without.
The resection specimens from the patients were sliced considering the pretreatment area of involvement with 10 blocks that were taken. Assessment of pathologic response to NACT was done according to Chevallier et al.; the pathological response to NACT was categorized into the following:
- Complete pathologic response: complete absence of residual tumor in breast and axillary LNs, or only in-situ carcinoma
- Partial pathologic response: residual tumor with evidence of therapy effect
- No pathologic response (pNR): tumor showed no modification or evident therapy effect.
Formalin-fixed, paraffin-embedded 4-μm-thick sections of core biopsies are mounted on charged slides and subjected to immunohistochemical (IHC) studies. The detection kit used was Dako Cytomation EnVision + Dual Link System (code K4061) using DAB as chromogene. Antigen retrieval required pretreatment in 0.01 M citrate buffer (pH 6.0) in a microwave. The primary antibodies were monoclonal antibodies against CD4 (catalog #MA1-145, clone 4B12; BioGenex) (DCS, Hamburg, Germany), CD8 (catalog #AN740-10M, clone SP16; BioGenex), CD3 (clone AN846-10M, dilution 1: 100; Novocastra), and CD20 (clone L26 M0755, 6 ml ready to use; BioGenex). The sections were counterstained for 3 min with Meyer's hematoxylin. Negative control was stained without applying primary antibody. Normal human tonsil was used as an external positive control.
Immunostaining for ER, PR, Her2, and Ki-67 was done following the methodology described by Han et al..
Interpretation of immunohistochemical staining
Allred score system was used for assessment of ER and PR. For Her2/neu status, first we assessed Her2/neu status by IHC. If IHC was 2+, tumor blocks underwent confirmatory FISH test. Tumors with a score of 2+ or 3+ were considered Her2-overexpressing tumors. Assessment of Ki-67 was done by identification of hotspots in cancerous regions. Finally, using ×40 magnifications over the hotspot, the number of Ki-67-positive tumor cells from at least 500 tumor cells was calculated. The percentages of positive cells were calculated and 14% was used as cutoff value.
CD20 or CD3 was evaluated in cases with stroma-rich lymphocytes and the cases with predominant CD3-positive T cells were determined. To evaluate CD8-positive and CD4-positive lymphocyte tumor infiltration, we selected four nonoverlapping stromal fields (hematoxylin and eosin, low-power magnification) with CD3-positive rich stromal-associated lymphocytes within the invasive tumor; then, in the four fields, the relative percentage of CD8-positive and CD4-positive lymphocytes was determined under ×400 magnification. The examination of all slides was done by two pathologists blindly and independently to all clinical and pathological data.
Statistical analysis was performed by IBM SPSS Statistics software, version 19.0 (SPSS Inc., Chicago, Illinois, USA). All parameters including age, sex, tumor size, pathologic type, histological grade and node metastasis, hormonal receptor state, Her2 status, type of given NACT regimen, tumor-associated lymphocytes, CD3-rich, and CD8 predominance were evaluated by statistical analysis in relation to response to therapy and tumor-associated lymphocytes. P values of less than 0.05 were considered significant.
| Results|| |
The clinicopathological characteristics of the 100 breast cancer patients were recorded as shown in [Table 1]. The patient's age ranged from 38 to 76 years with a median of 55.62 ± 11.56 years. Most of the patients (90%) had invasive duct carcinoma type, whereas only 10% were of invasive lobular. Fifty-two percent of cases had grade II carcinoma and 48% had grade III carcinoma. Fifty percent of patients were of clinical tumor size T2, 40% of patients were of T3, and only 10% were of T4. Fifty-two percent of cases had positive axillary LN metastasis. ER was negative in 60%, PR was positive in 60%, and Her2 was positive in 24%, and Ki-67 was 14 or more in 64% of patients. Rich stromal-associated lymphocytes [Figure 1] were present in 54% of cases [Figure 1]a,[Figure 1]b,[Figure 1]c,[Figure 1]d, whereas 46% of cases had poor stromal-associated after lymphocytes [Figure 1]e and [Figure 1]f. Among rich stroma-associated lymphocytes, 44 (81.5%) cases were CD3 T lymphocyte predominant and 10 cases were of CD20 B immunophenotype. CD8 T lymphocyte predominance was recorded in 27 (61.4%), whereas CD4 was recorded in 12 (27.4%) of cases with mixed population of CD4 and CD8 in 5 (11.4%) cases.
|Figure 1: Histological patterns of stromal lymphocyte infiltration in breast cancer tissue relative to the response to neoadjuvant chemotherapy. (a and b) Invasive ductal carcinoma (IDC): group II showed dense infiltration of tumor stroma by lymphocytes in stroma-rich [hematoxylin and eosin (H and E), (a) ×100, (b) ×400]. (c and d) IDC, group III with (stroma-rich) lymphocytes and intratumoral lymphocytes [H and E, (c) ×100, (d) ×200]. (e and f) IDC, group II showed scattered lymphocytes (stroma-poor) lymphocytes [H and E, (e) ×200, (f) ×100]. (g) Post-NAC (Neo-adjuvant chemotherapy) resection specimens showing residual invasive carcinoma with extensive stromal lymphocyte infiltration (H and E, ×400). (h and i) A case of complete pathologic response showed no residual invasive component, but there are lymphohistocytic aggregates and sclerosed stroma [H and E, (h) ×100, (i) ×200].|
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Regarding pathologic response, we recorded pCR in 22 (22%) breast cancer patients [Figure 1]e and [Figure 1]h and partial pathologic response in 54 (54%) cases [Figure 1]g. Twenty-four (24%) patients showed pNR [Table 1].
The association between type of response to therapy and patients' characteristics is summarized in [Table 2]. Results of our work revealed a significant association between type of response and invasive ductal carcinoma type (P = 0.008) and advanced tumor size (P = 0.003); patients with T2 and T3 appeared to achieve pCR more than T1. There was also a significant association between pCR and positive axillary LNs (P = 0.014), negative ER (P< 0.001), and Her2 overexpression (P = 0.027). In addition, lymphocyte phenotype affected the therapy response as CD3 (T)-rich stromal-associated lymphocytes [Figure 2] and predominant CD8 T-cell type [Figure 3] in tumors were significantly associated with pCR (P = 0.006 and 0.004, respectively). However, no significant association was found in relation to patient age, affected side, histological grade, PR state, and Ki-67 index.
|Table 2: Relation between type of response to therapy and patients' characteristics|
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|Figure 2: (a and b) Immunohistochemical staining for CD20 and CD3 showed mixed CD20 B cells and CD3 T cells in stromal lymphocytes in IDC [CD20 immunostaining, (a) ×200; CD3 immunostaining, (b) ×200]. (c-f) Another case of invasive ductal carcinoma stroma-rich with predominance of CD3-positive T cells than CD20-positive B lymphocytes [CD20 immunostaining, (c) ×100, (f) ×200; CD3 immunostaining, (d) ×100, (e) ×200].|
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|Figure 3: Immunohistochemical staining for CD4 and CD8. T lymphocytes stroma-rich cases of invasive ductal carcinoma showed predominance of CD8 cytotoxic T cells than CD4 helper T cells. CD8 immunostaining, (a and c) ×200; CD4 immunostaining, (b and d) ×200.|
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The association between stromal-associated lymphocytes and clinicopathological characteristics is summarized in [Table 3]. Statistically positive association was observed between rich stromal-associated lymphocyte values and high-grade tumors (P = 0.004), tumors with negative ER (P = 0.015), and tumors with high Ki-67 index (P = 0.028).
|Table 3: Correlation between stromal-associated lymphocytes and clinicopathological patients' characteristics|
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[Table 4] shows comparison between cases with predominant CD3-positive T cells and those with predominant CD20-positive B cells. There was no significant difference related to the recorded histopathological parameters except Ki-67 (P = 0.054). A total of 36.4% of CD3-positive stroma-rich associated lymphocytes exhibited pCR in comparison with 20% of CD20-positive stroma-rich associated lymphocytes, but this was statistically insignificant (P = 0.098).
|Table 4: Comparison between CD3-positive stroma-rich lymphocytes and CD20-positive stroma-rich lymphocytes in breast cancer studied cases|
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Further immunophenotyping of the T-cell predominant stroma-rich cases using CD4 and CD8 found that 27 cases were of CD8, whereas 12 cases were of rich CD4 and five cases were mixed with no predominance. [Table 5] shows the results of comparing CD8-rich cases with CD4-rich cases, which reveals a significant difference in relation to response to therapy (P = 0.046) as 66% of cases with dominant CD8 in stroma achieved pCR compared with 16.7% of those with dominant CD4 cells; also, there was a significant difference in relation to Her2/neu expression (P = 0.048) and LN state (P = 0.020).
|Table 5: Comparison between CD8-positive stroma-rich lymphocytes and CD4-positive stroma-rich T lymphocytes in breast cancer studied cases|
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| Discussion|| |
Breast cancer is the most common cancer among females in Egypt and worldwide. It represents 38.8% of the total cancer incidence locally and 22.9% globally. In Egypt (2013), the estimated number of cases was 17 905 and by year 2050 it is expected to triple.
NACT was used in patients with locally advanced breast cancer. However, its use was expanded to include patients with large, operable tumors to decrease tumor size and increase the possibility of breast-conserving surgery.
Achievement of pCR after NACT was associated with favorable patient outcomes, and became a suitable surrogate endpoint for patients with cancer breast. Choi et al. reported that assessment of prognostic markers should be performed on core biopsy before NACT, as the treatment may influence expression of these markers and treatment decision.
The presence of several definitions of pCR made interpretation of results in previous studies confusing; Choi et al. studied the prognostic effects of different definitions of pCR on patient outcomes. They concluded that the most preferable definition of pCR related to prognosis is complete disappearance of invasive tumor tissue in the breast and LN,.
In this study, 22 (22%) cases experienced pCR, a residual disease in 54 (54%), and pNR in 24 (24%) cases. These results cope with results obtained by Hegmane and Karele as they reported pCR in 12 (17%) patients.
It is important to identify predictors for chemotherapy sensitivity and pCR, as this may allow for tailoring of chemotherapy and single out patients who will benefit from this therapy and spare others from toxicity of ineffective treatment. In this study, there was a significant association between pCR and many pathologic parameters including ductal histological subtype, early clinical stage, negative LN state, rich stromal-associated lymphocytes, intratumoral lymphocytes, negative ER, Her2/neu overexpression, T-cell rich, and CD8 T phenotype.
Tumor size is a powerful prognostic factor in breast cancer and is used to make adjuvant treatment decisions. Previous studies suggested that advanced size was associated with less sensitivity to NACT. This is in agreement with our results; we recorded significant association between clinical tumor size and pCR (P = 0.003), as 63.6% of patients who achieved PCR were of T3 compared with 18.2% who were of T4. However, Tan et al. documented that there was no significant association between clinical tumor size and pCR in his series; this could be explained by the difference in the studied population as 30% of cases had a clinical T3, whereas 39% of them had a clinical T4. We reported no significant association between pCR and histological grade. These results cope with results of Fisher et al. and Luangdilok et al..
Axillary LN state is a significant prognostic factor for breast cancer related to the risk for distant recurrence and adjuvant therapy decision. We reported that negative LN state (P = 0.014) is a significant predictor for pCR; 38.5% compared with 4.2% in LN-positive state achieved pCR. This is consistent with the finding of Tan et al. and Luangdilok et al.. However, Jiayu et al. reported no significant association between pCR and axillary LN state.
Positive ER status has been described as a good prognostic factor in breast cancer associated with better patient outcomes. However, some studies reported that ER-negative receptor states tumors are more likely to experience a pCR compared with positive ER tumors. These results are in a concordance with our results as pCR is achieved in 90.9% of ER-negative tumors in contrast to 9.1% in ER-positive tumors (P< 0.001). Buzdar et al. suggested that ER signaling can induce therapy resistance through overexpression of antiapoptotic protein bcl-2. Although PR status is an independent predictor of good response for adjuvant endocrine therapy, there was no reported significant association between PR state of the tumor and response to NACT. These findings are consistent with our results (P = 0.063).
Her2/neu gene amplification and protein expression in breast carcinoma has been considered a prognostic factor that may have therapeutic implications and aggressive biological behavior. This is probably owing to Her2-driven proliferation and angiogenesis.
The predictive role of Her2/neu gene amplification or protein overexpression in response of breast cancer to NACT is still controversial. In our study, overexpression of Her2/neu protein was significantly associated with pCR (P = 0.027); this finding is in agreement with that of Nwaogu et al., and this supports that Her2/neu overexpression is a predictor of chemosensitivity and likelihood of pCR. However, other studies showed no significant difference between carcinomas overexpressing Her2/neu protein and others without in the rate of pCR.
Although triple-negative breast cancer (TNBC) tends to be particularly aggressive compared with other subtypes, TNBC showed chemosensitivity to anthracycline-and-taxane-based NACT and exhibited higher rates of pCR than non-TNBC. In our study, there was no significant difference in pCR between TNBC and non-TNBC (P = 0.186).
The Ki-67 index has been studied extensively as a prognostic predictor for breast cancer. In this study, we did not detect a significant correlation between therapy response and Ki-67 index. This agreed with the study by Ingolf et al.; however, other studies found that the pCR rate was higher in patients with a high Ki-67 expression than in patients with low Ki-67 expression.
Special attention has been focused on the effect of the immune system on tumor prognosis. Both innate and adaptive immune cells orchestrate and function to either stimulate or inhibit cancer. Lymphocytic infiltration in melanoma has been demonstrated to be of better prognostic significance suggesting that TILs have a role in antitumor immunity.
The immune system has a complex and integral role in breast cancer biology, but the relationship between TILs and breast carcinoma is still controversial. Medullary breast carcinomas are characterized by extensive lymphoplasmacytic infiltrate carrying a favorable prognosis. Some studies address that high TILs predicted better response to NACT and higher survival rates. However, St Gallen Consensus meeting in 2015 did not accept TIL as a prognostic marker. These conflicting results need further evaluation.
TILs are formed of stromal-associated lymphocytes and TILs. In this study, we focused on evaluation of stromal-associated lymphocytes and its correlation to therapy response, as TIL working groups (2014) recommended the evaluation of stromal-associated lymphocytes as a principal parameter in future studies.
In this study, 46 (46%) of cases showed stroma-rich lymphocytes (>50%) and 24 (24%) had intratumoral lymphocytes. We reported a significant association between stroma-rich lymphocytes in core biopsies and pCR (P = 0.004). pCR rates were significantly increased from 8.7% in tumors with stroma-poor lymphocytes to 33.3% in tumors with stromal-rich lymphocytes. These results are similar to those of Yamaguchi et al., who reported pCR rates of 42% with lymphocyte predominant compared with 3% in tumors poor in lymphocytes. West et al. reported pCR in 74% of patients with high TIL compared with 31% of patients with low TIL.
The composition of stoma-associated lymphocytes has a prognostic impact on response to NACT. Several subtypes of immune cells could infiltrate the tumor tissue. These cells include T cells, macrophages, NK cells, and dendritic cells. Conventional chemotherapy can stimulate the immune system through triggering cellular rearrangements that help recognition of tumor cells by T cells and NK cells. Other drugs induce depletion of regulatory T cells. In addition, other chemotherapeutic drugs can stimulate cytotoxic T cells. B cells also contribute to antitumor immune responses through activating and presenting antigens to T cells.
In this study, we tried to clarify the importance of lymphocytes subtypes in sensitivity to NACT; we screened cases with lymphocyte-rich stroma for CD20 and CD3, and then CD4 and CD8.
In our work, 81.5% of stroma-rich tumors showed predominance of CD3 T to therapy (P = 0.004). Our results are in concordance with previous studies, as Rathore et al. found that stromal CD3 T lymphocytes were associated with better overall survival in the adjuvant setting; however, Heys et al. found that CD3-positive lymphocytes were not prognostic markers in the neoadjuvant setting.
The tumor-specific CD4 T and CD8 T cells also influence the predictive role of stromal-associated lymphocytes; it was suggested that the presence of CD8 cells in stroma lymphocyte infiltrate was associated with better patient survival in colorectal and ovarian cancers. In breast cancer, CD8-positive lymphocytes in pre-NACT core biopsy predicted pCR, and better survival. The results of our studies were in agreement with these studies; we recorded that CD8 predominance in the stromal lymphocytes was significantly (P = 0.004) associated with higher rate of pCR. This could be possibly explained by the fact that chemotherapy induced cancer cell death, releasing tumor antigens that are engulfed and presented by antigen-presenting cells (APCs) to cytotoxic CD8-positive T cells.
In this study, higher percentage of stromal-associated lymphocytes infiltrates in core biopsy was significantly associated with high histological grade (P = 0.004), high Ki-67 (P = 0.028), negative ER state (P = 0.015), and this is in agreement with other studies,.
| Conclusion|| |
From this study, we concluded that stromal-associated lymphocytes in breast cancer could be a reliable predictor of pCR to NACT. In addition, composition and subtypes of lymphocytes could affect the response. Higher percentage of CD8-positive T cells in stromal-associated lymphocytes in breast cancer is associated with higher pCR after NACT. Therefore, evaluation of stromal-associated lymphocytes and its specific subtypes may be able to predict prognosis, thereby guiding future therapy decisions in breast cancer patients. We recommend incorporating an 'immunoscore' into traditional pathology report as one of the future prognostic and predictive markers in breast cancer.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]