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Tumor Infiltrating Lymphocytes and Tertiary Lymphoid Structure as Prognostic and Predictive Factor for Neoadjuvant Chemotherapy in Stage II & III Breast Cancer

Mohamed L1*, Elsaka A2, Zamzam Y2 and Elkady A2

1Department of Clinical Oncology, Tanta University, Egypt

2Department of Pathology, Tanta University, Egypt

*Corresponding Author:
Mohamed L
Assistant Professor
Department of Clinical Oncology, Tanta University, Egypt
Tel: 201224351806
E-mail: lamissmohamed2@yahoo.com

Received date: October 15, 2018; Accepted date: October 31, 2018; Published date: November 05, 2018

Citation: Mohamed L, Elsaka A, Zamzam Y, Elkady A (2018) Tumor Infiltrating Lymphocytes and Tertiary Lymphoid Structure as Prognostic and Predictive Factor for Neoadjuvant Chemotherapy in Stage II & III Breast Cancer. Arch Can Res Vol.6 No.3:17. doi:10.21767/2254-6081.100178

 
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Abstract

Purpose: Tumor-infiltrating lymphocytes (TILs) have a strong prognostic and predictive value in triple-negative breast cancer (TNBC) and HER2 enriched subtype but no other breast subtypes but no studies have evaluated tertiary lymphoid structure (TLS).

Materials and Methods: Eighty patients with stage II and III breast cancer in Tanta oncology and pathology department with luminal A, B and her 2 enriched and TNBC diagnosed with core needle biopsy treated with neoadjuvant chemotherapy (4 AC followed by 12-week taxol + herceptin). TIL and TLS were evaluated histopathologically using hematoxylin and eosin–stained slides. The immune cell aggregates which were TLS positive showed the presence of CD20+ B lymphocytes within the follicles, with areas of CD3+, CD4+ T lymphocytes mainly in the periphery [T-cell zone] resembling the highly organized structures of secondary lymphoid organs.

Results: TLS were detected in 53.7% of the tumors in whole breast groups, 83.7% in triple negative subgroup. Increased number of tumor infiltrating lymphocytes and tertiary lymphoid structure are associated with longer OS and DFS for TNBC and HER2-positive breast cancer.

Conclusion: For predicting treatment response in patients with TNBC and HER2 enriched subtype, TILs and TLS may be prognostic and predictive marker triplenegative breast cancer (TNBC) and HER2 enriched subtype but no other breast subtypes.

Keywords

TNBC; HER2neu; TIL; TLS; Prognostic; Predictive; Molecular; DFS; OS

Introduction

Invasive breast cancers (IBC) are heterogeneous with variable tumor biology and outcome [1]. The tumor grade, staging and receptor status are among the many prognostic factors that affect breast cancer outcome, but they are insufficient to give precise prognostication [2]. To improve outcome searching for new prognostic factors are required. Tumor microenvironment has been found to promote cancer growth. The importance of tumor microenvironment in promoting cancer growth has been increasingly recognized [3]. Immune system was found to play a role on oncogensis and tumor progression [4]. The prognostic and predictive role of tumor infiltrating lymphocytes (TILs) have been demonstrated in many solid tumors with its anti tumor activity [5,6]. Among breast cancer subtypes, triple negative breast cancer had the worst prognosis, but high TIL is associated with favorable prognosis and pathologic complete response in neoadjuvant setting [7,8]. The presence of ectopic lymphoid tissue known as tertiary lymphoid structures (TLS) resembling secondary lymphoid organs (SLO) have been described in many cancers including breast cancer [9-11]. TLS is formed of T and B cells, the former composed of germinal centre and small number of follicular dendritic cells while the latter is formed from mature dendritic cells and endothelial venules respectively. So, it is the site of immune response responsible for triggering, recruiting and activating TIL and thus improves antitumor response and prolongs survival [12-16].

The TLS and its correlation with different prognostic factors in invasive breast cancer with different molecular subtypes have been studied in very little trials [12-16]. But its correlation with type of chemotherapy was not discussed.

Patient and Methods

In this study, Eighty patients with stage II and III breast cancer according to AJCC TNM 8TH edition [17] in Tanta oncology and pathology department with luminal A, B and her 2 enriched and TNBC diagnosed with core needle biopsy and properly staged in the period from June 2012 to June 2018 received standard neoadjuvant protocol formed of AC (doxorubicin 60 mgm/m2 plus cyclophosphamide 600 mgm/m2 on day 1 every 3 weeks for 4 cycles followed by paclitaxel in dose of 80 mgm/m2 for 12 weeks). Patients with HER2 positive tumor received trastuzumab 6 mgm/kg (loading dose 8 mgm/kg) once every 3 weeks from the start of taxol start.

Prechemotherapy formalin fixed paraffin embedded core biopsies were collected. Hormone receptor positivity was defined as 1% or more for estrogen and progesterone receptors. The intensity of HER2 membrane staining was scored as 0, 1+, 2+ or 3+. Tumors with 3+ scores were classified as positive for HER2 over expression, whereas tumors with 0 or 1+ scores were considered as negative. FISH was done for her 2 neu +2 to determine whether they are negative or positive. Tumors were classified as ‘not amplified’ (FISH negative) if the calculated ratio was less than 2 and ‘amplified’ (FISH positive) if the ratio was 2 or greater. Ki 67 labeling index was recorded as high if more than 14%.

Assessment of tumor immune cell infiltrate

Immunohistochemistry: For Immunohistochemical staining, prechemotherapy 4μm sections of formalin fixed paraffin embedded core biopsies blocks were collected. The avidin-biotinperoxidase complex method was used for the immunostaining of monoclonal antibodies to CD3, CD4 (pan T cell marker), and CD20 (pan B cell marker) to label T and B cells [Dako, Glostrup, Denmark]. In brief, after dewaxing, inactivating endogenous peroxidase activity and blocking cross-reactivity with normal serum (Vectastain Elite Kit; Vector, Burlingame, CA, USA), the sections were incubated overnight at 4°C with a diluted solution of the primary antibodies. Location of the primary antibodies was achieved by subsequent application of a biotinylated antiprimary antibody, an avidin-biotin complex conjugated to horseradish peroxidase, and diaminobenzidine (Vectastain Elite Kit, Vector, Burlingame, CA). The slides were counter-stained by hematoxylin. Human lymph node was used as positive control. Negative controls were performed by omitting the primary antibody.

Immunohistochemical analysis of tumor infiltrating lymphocytes in tumor stroma was performed by two pathologists independently performed the categorizing and had no knowledge of the patients’ background history. The number of TILs in stroma surrounding the stained cancer cells was quantitatively measured in each field under 400x magnification. Areas of in situ carcinoma and crush artifacts were not included. Score 0 and 1 for <20% and >20% respectively and score one is considered positive (Figure 1) A case of Infiltrating duct carcinoma showed severe form of TLIs >20% (score 1) in direct contact to tumor cells (H&E 400x). The immune cell aggregates which were TLS positive showed the presence of CD20+ B lymphocytes within the follicles, with areas of CD3+, CD4+ T lymphocytes mainly in the periphery [T cell zone] resembling the highly organized structures of secondary lymphoid organs (Figure 2). Tertiary lymphoid structures with germinal center formation (2A and 2C) were detected by CD20+ B lymphocytes and CD3+ (2B) and CD4+ (2D) T lymphocytes in the T cell zone (streptavidin biotin 400x). Only TLS located in the adjacent stroma (in direct contact or within 0.5 mm of the tumor) were scored, with TLS positive 1% and TLS-negative tumors by their absence.

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Figure 1: A case of Infiltrating duct carcinoma showed severe form of TLIs >20% (Score 1) in direct contact to tumor cells (H&E 400x), 1B, A case of breast carcinoma showed organic tertiary lymphoid structure (TLS) resembling secondary lymphoid organs within the tumor area (H& E 100x).

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Figure 2: Characterization of tertiary lymphoid structures in breast carcinoma. Immunohistochemical detection of the indicated antigens in serial sections of breast carcinoma with extensive immune cell infiltration. Tertiary lymphoid structures with germinal center formation (2A and 2C) were detected by CD20+ B lymphocytes and CD3+ (2B) and CD4+ (2D) T lymphocytes in the T cell zone (streptavidin biotin 400x).

Response: Response evaluation was done according to RECIST 1.1 [18]. The surgery was done after 4 weeks from last cycle. Pathological complete response was defined as no residual disease in the breast on axillary lymph nodes or residual carcinoma in situ. After neoadjuvant chemotherapy breast conserving or modified radical mastectomy. Radiotherapy was given to all patients with breast conserving surgery and in patients for which modified radical mastectomy radiotherapy with 4 or more positive lymph nodes or tumor large than 5 cm with negative lymph nodes or close surgical margin. Overall survival (OS) time was defiened as the period from the start of neoadjuvant to the time of death from any cause or last follows up. Disease free survival (DFS) was defined as freedom from locoregional, and distant recurrence. Follow up by physical examination every 4- 6 months during the first 5 years. Mammogram is done annually US every 6 months, and CT and bone scintigraphy annually. The study protocol was approved by the Ethics Committee. Informed written consent was obtained from patients enrolled in the study.

Statistical Methods

SPSS version 19.0 was used for statistical analysis. The associations between TILs, tertiary lymphocyte structure and different clinicpathologic factors were done. Analysis of pCR was carried out using a binary logistic regression model. The Kaplan–Meier method was used to estimate DFS, and OS, with p-value<0.05 was considered statistically significant [19]. Cutoff values for different biomarkers included in this study were chosen before statistical analysis. Use Cox regression hazard model for multivariate analysis [20].

Results

Intrinsic subtypes among eighty patients studied from June 2012 to June 2018 with breast cancer histopathologically examined in pathology department, Tanta university hospital who received neoadjuvant chemotherapy in oncology department, Tanta university hospital were distributed as following hormone positive breast cancer (HRBC) in 32 (52.5% of whom 24(30%) and 18 (22.5%) were luminal A and B respectively), HER2 enriched subtype in 14 (17.5%), and TNBC in 23 (30%) patients.

According to tumor infiltrating lymphocyte, TNBC (83.3% p-value<0.0001) and HER2 enriched (78.6%, p-value<0.0001) had high tumor infiltrating lymphocyte as seen in Table 1. Also, the factors with statistical significance were age, menopausal status, histology, type of surgery, pathological CR. Thirty-three patients (41.2%) and 47(58.8%) achieved pCR and non-pCR in respectively. According to intrinsic subtype, pCR was achieved in 4 (16.7%), 8(44.4%), 8 (57.1%), 13(54.2%) patients with luminal A, B, HER2 enriched and triple negative breast cancer respectively which was statistically significant (p-value 0.027). Tertiary lymphoid structure is present in 43/80 (53.7%) for which pCR was reported in 62.8% versus 16.2% in patients with present and absent tertiary lymphocyte structure respectively which was statistically significant (p-value=0.001) (Table 2). In patients with her 2 enriched subtype overall survival was higher in TIL more than>20% versus low (p-value=0.032) and in present TLS than absent (p-value=0.117). DFS were significantly longer in high TIL than in low one and in present TLS versus absent respectively and this was statistically significant (p-value=0.027, 0.013 respectively) (Figure 3) survival according to tumor infiltrating lymphocyte and tertiary lymphocyte structure for HER2 enriched subtype). Also, in patients with triple negative overall survival was higher in TIL more than>20% versus low (p-value=0.001) and in present TLS than absent (p-value=0.001). DFS was significantly longer in high TIL than in low one and in present TLS versus absent respectively (p-value=0.003, 0.057 respectively) (Figure 4) (Survival according to tumor infiltrating lymphocyte and tertiary lymphocyte aggregate for triple negative breast cancer subtype). As regards, hormone positive subtypes and different breast subtypes no statistically, significant difference between TIL and TLS and OS and disease-free survival except for So with TIL in different breast subtypes (p=0.008) (Figures 5 and 6) Survival according to tumor infiltrating lymphocyte and tertiary lymphocyte structure for hormone enriched subtype and all breast subtypes respectively. The multivariate analysis maintains statistical difference between DFS with TIL and TLS with both TNBC and HER2 enriched breast subtype but not with hormone positive and all breast subtypes (Table 3).

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Figure 3: Survival according to tumor infiltrating lymphocyte and tertiary lymphocyte structure for her2 enriched subtype.

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Figure 4:Survival according to tumor infiltrating lymphocyte and tertiary lymphocyte aggregate for triple negative breast cancer subtype.

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Figure 5: Survival according to tumor infiltrating lymphocyte and tertiary lymphocyte structure for hormone enriched subtype.

acanceresearch-breast

Figure 6:Survival according to tumor infiltrating lymphocyte and tertiary lymphocyte structure for all breast subtypes.

Clinicopathological characteristics TIL<20% TIL>20% p-value
N % N %  
Age
< 55 9 23.70% 32 76.20% 0.000*
>55 29 67.30% 10 23.80%  --
Menopausal 3 --   -- --  -- 
premenopausal 6 15.80% 26 61.90% 0.000*
postmenopausal 30 78.90% 8 19%  --
NOS 2 5.30% 8 19%  --
Sex 
Female 36 94.70% 34 81% 0.062
Male 2 5.30% 8 19  
Performance status 
    0-1 28 73.70% 24 57.10% 0.121
2 10 26.30% 1 42.90%  
Histology 
IDC 36 94.70% 30 71.40% 0.006*
ILC 2 5.30% 12 28.60%  
Stage          
IIb 2 5.30% 4 9.50%  
IIIa 24 63.20% 14 33.30%  
IIIb 10 26.30% 22 52.40% 0.055
IIIc 2 5.30% 2 4.80%  
Type of surgery 
MRM 28 73.70% 6 14.30% 0.000*
BCS 10 26.30% 36 85.70%  
Ki 67 
<  14% 17 44.70% 27 64.30% 0.079
>14% 21 55.30% 15 35.70%  
LVSI
present 22 57.90% 28 66.70% 0.418
absent 16 42.10% 14 33.30%  
Intrinsic subtype
Her 2 enriched 3 7.90% 11 26.20% 0.032*
Non her2 enriched 35 92.10% 31 73.80%  
Intrinsic subtype 
TNBC 4 10.50% 20 47.60% 0.000*
Non TNBC 34 89.50% 22 52.40%  
Intrinsic subtype 
HRBC 32 84.20% 10 23.80% 0.000*
Non HRBC 6 15.80% 32 76.20%  
Response 
Pathological CR 11 28.90% 22 52.40% 0.033*
Non pCR 27 71.10% 20 47.60%  

Table 1: Correlation between different clinicopathological and tumor infiltrating lymphocytes in different breast cancer intrinsic subtypes.

  Response Subtypes p-value
luminal A luminal B her2enriched TNBC
N % N % N % N %
pCR 4 16.7% 8 44.4% 8 57.1% 13 54.2% 0.027
Non pCR 20 83.3% 10 55.6% 6 42.9% 11 45.8%  

Table 2a: Pathological complete response.

Tertiary lymphocyte structure Response Total p-value
pCR Non pCR
Present Count 27 16 43     0.001*
% within TLS 62.8% 37.2% 100.0%
% within response 81.8% 34.0% 53.8%
% of Total 33.8% 20.0% 53.8%
Absent Count 6 31 37
% within TLS 16.2% 83.8% 100.0%
% within response 18.2% 66.0% 46.3%
% of Total 7.5% 38.8% 46.3%
Total Count 33 47 80
% within TLS 41.3% 58.8% 100.0%
% within response 100.0% 100.0% 100.0%
% of Total 41.3% 58.8% 100.0%

Table 2b: Breast cancer intrinsic subtypes and TLS.

Parameters Univariate analysis Multivariate analysis
Sig Sig
All breast subtypes
Menopause 0.153 0.08
Histology 0.08 0.015*
PS 0.151 0.012*
Stage 0.191 0.16
Grade 0.834 0.402
LVSI 0.16 0.576
ki67per 0.121 0.355
Surgery 0.263 0.37
TIL 0.501 0.915
TLS 0.834 0.359
Pathological response
TNBC 0.589 0.116
Menopause 0.902 0.397
Histology 0.098 0.045
PS 0.359 0.712
Stage 0.635 0.723
Grade 0.596 0.799
LVSI 0.682 0.726
ki67per 0.49 0.89
Surgery 0.829 0.639
TIL 0.007* 0.002*
TLS 0.046 0.004*
Pathological CR 0.22 0.397
HRBC Menopause 0.221 0.752
Histology 0.865 0.939
PS 0.423 0.232
Stage 0.71 0;606
grade 0.245 0.82
LVSI 0.306 0.019*
Ki67 0.906 0.176
Surgery 0.186 0.154
TIL 0.802 0.915
TLS 0.142 0.793
Pathological compete response
Her2 enriched subtype 0.202 0.904
Menopause 0.306 -
Histology 0.617 0.829
PS 0.517 0.897
Stage 0.284 0.817
grade 0.699 0.983
LVSI 0.862 0.526
Ki67 0.1 0.671
Surgery 0.063 -
TIL 0.014* 0.002*
TLS 0.004* 0.003*
Pathological CR 0.354 0.524

Table 3: Univariate and multivariate analysis with disease free survival in relation to different breast subtypes.

Discussion

The emerging role of new cancer immunotherapeutic drugs highlights the importance of tumor immune response. The detailed characteristics of tumor immune environment in breast cancer will help to identify patients who will benefit from these new drugs. Different immune response interacts to produce immunological parameters including tumor infiltrating lymphocytes which are considered predictors of response to neoadjuvant chemotherapy in breast cancer and tumor lymphocyte structure are considered the site of active immunological response [21]. TIL were found to be high in HER2 and TNBC and correlated with better response to neoadjuvant chemotherapy [6-8,20-24]. In the present study, we found 47.6%, 26.2% of our TNBC and HER2 enriched patients had TIL more than other breast subtypes, which is similar to that reported by Stanton et al. [25].

In this study we noted that high tumor infiltrate are associated with formation of tertiary lymphoid structure composed of B follicle with germinal center nearby or surrounded by T cell zone similar to secondary lymphoid follicles [26]. In this study, TLS were detected in 53.7% of the tumors in whole breast groups, 83.7% in triple negative subgroup which is lower than lee et al. who reported that TLS were 96.7% because they scored it depending on H&E which makes the differentiation between TLS and lymphoid aggregates very difficult. Also, in our study we use dual CD3/CD20 IHC to identify the B cell follicles and T cell zone [27].

Again, we reported a high TLS (71.4%) in HER2 enriched which is similar to that reported by Liu et al. 12]. One of the main problems of chemotherapeutic agents is immune escape of cancer cell [28]. The present study included patients treated with NAC (AC followed by paclitaxel with/without trastuzumab). Paclitaxel improve cytotoxic and regulatory T-lymphocytes sensitivity [29]. Immunogenic death is caused by cyclophosphamide and anthracycline [30].

Tipple negative and HER2 enriched breast subtypes are associated with high pathological CR than other breast subtypes (p=0.027) as reported by Prat et al. [31,32]. Higher pathological CR was reported with high tumor infiltrating lymphocytes (0.027) and positive lymphoid structure (p=0.001) as reported by other authors [20-23,33]. Increased number of tumor infiltrating lymphocytes and tertiary lymphoid structure are associated with longer OS and DFS for TNBC and HER2-positive breast cancer [34]. In univariate, longer DFS are associated with high tumor infiltrating lymphocytes and positive lymphoid structure in both TNBC and her 2 enriched and is maintained in multivariate analysis [27].

Conclusion

In conclusion, for predicting treatment response in patients with TNBC and HER2 enriched subtype, TILs and TLS may be prognostic and predictive marker but this is not true for hormone receptorpositive subtypes of breast cancer. Limitation of our study: More studies are important to evaluate the role of these immune markers in tumor relapse. But as shown in our study the number of recurrences is very low so larger trials with large number of patients are needed to confirm this conclusion.

Ethics Approval and Consent to Participate

• Written consent was taken from patients and hospital approval of university hospital.

• Consent for publication from all authors and university hospital.

• Data and material are available.

Conflicts of Interest

Authors have no conflicts of interest to disclose.

Funding

Funding by authors, no support

Author’s Contributions

Author 1: The idea of research, collection of the data and analysing.

Author 2: Pathological interpretation.

Author 3: Discussion and comparing with others.

Acknowledgements

Thanks to staff of Department of Oncology.

Author’s information refers to Lamiss Mohamed or Lamiss Sad at Pubmed.

• Prognostic and predictive role of ERCC1 protein expression in locally advanced stage III non-small cell lung cancer. Medical Oncology in 2014.

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• Genetic variations of selected genes using target deep sequencing in colorectal cancer patients Oct 2017, Journal of Cancer Science and Therapy.

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