Basic Study Open Access
Copyright ©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Clin Oncol. Oct 10, 2016; 7(5): 387-394
Published online Oct 10, 2016. doi: 10.5306/wjco.v7.i5.387
Tumor infiltrating lymphocytes in triple negative breast cancer receiving neoadjuvant chemotherapy
Carlos A Castaneda, Miluska Castillo, Patricia Arboleda, Ketty Dolores-Cerna, Carolina Belmar-Lopez, Department of Research, Instituto Nacional de Enfermedades Neoplasicas, Lima 15038, Peru
Carlos A Castaneda, Henry L Gomez, Tatiana Vidaurre, Department of Medical Oncology, Instituto Nacional de Enfermedades Neoplasicas, Lima 15038, Peru
Elizabeth Mittendorf, Department of Surgical Oncology, the University of Texas MD Anderson Cancer Center, Houston, TX 77030, United States
Sandro Casavilca, Patricia Arboleda, Teresa Nunez, Henry Guerra, Carlos Barrionuevo, Department of Pathology, Instituto Nacional de Enfermedades Neoplasicas, Lima 15038, Peru
Yun Wu, Department of Pathology, Division of Pathology, the University of Texas MD Anderson Cancer Center, Houston, TX 77030, United States
Julio Abugattas, Gabriela Calderon, Miguel De La Cruz, Manuel Cotrina, Jorge Dunstan, Department of Breast Cancer Surgery, Instituto Nacional de Enfermedades Neoplasicas, Lima 15038, Peru
Author contributions: Castaneda CA, Mittendorf E, Casavilca S and Wu Y contributed to the conception and design of the study and performed data analysis and interpretation; Castaneda CA, Casavilca S, Castillo M, Nunez T and Guerra H performed data acquisition, as well as providing administrative, technical, and material support; all authors drafted the article and made critical revisions related to the intellectual content of the manuscript, and approved the final version of the article to be published.
Institutional review board statement: This study was reviewed and approved by the Instituto Nacional de Enfermedades Neoplasicas Institutional Review Board.
Informed consent statement: Not needed due to the non-clinical study design.
Conflict-of-interest statement: To our knowledge, no conflict of interest exists.
Data sharing statement: No data were created so no data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Correspondence to: Carlos A Castaneda, MD, MSc, Department of Medical Oncology, Instituto Nacional de Enfermedades Neoplasicas, Av. Angamos Este 2520 Surquillo, Lima 15038, Peru.
Telephone: +51-1-2157220 Fax: +51-1-6204991
Received: April 18, 2016
Peer-review started: April 18, 2016
First decision: June 12, 2016
Revised: August 13, 2016
Accepted: September 21, 2016
Article in press: September 23, 2016
Published online: October 10, 2016


To determine influence of neoadjuvant-chemotherapy (NAC) over tumor-infiltrating-lymphocytes (TIL) in triple-negative-breast-cancer (TNBC).


TILs were evaluated in 98 TNBC cases who came to Instituto Nacional de Enfermedades Neoplasicas from 2005 to 2010. Immunohistochemistry staining for CD3, CD4, CD8 and FOXP3 was performed in tissue microarrays (TMA) sections. Evaluation of H/E in full-face and immunohistochemistry in TMA sections was performed in pre and post-NAC samples. STATA software was used and P value < 0.05 was considered statistically significant.


Higher TIL evaluated in full-face sections from pre-NAC tumors was associated to pathologic-complete-response (pCR) (P = 0.0251) and outcome (P = 0.0334). TIL evaluated in TMA sections showed low level of agreement with full-face sections (ICC = 0.017-0.20) and was not associated to pCR or outcome. TIL in post-NAC samples were not associated to response or outcome. Post-NAC lesions with pCR had similar TIL levels than those without pCR (P = 0.6331). NAC produced a TIL decrease in full-face sections (P < 0.0001). Percentage of TIL subpopulations was correlated with their absolute counts. Higher counts of CD3, CD4, CD8 and FOXP3 in pre-NAC samples had longer disease-free-survival (DFS). Higher counts of CD3 in pre-NAC samples had longer overall-survival. Higher ratio of CD8/CD4 counts in pre-NAC was associated with pCR. Higher ratio of CD4/FOXP3 counts in pre-NAC was associated with longer DFS. Higher counts of CD4 in post-NAC samples were associated with pCR.


TIL in pre-NAC full-face sections in TNBC are correlated to longer survival. TIL in full-face differ from TMA sections, absolute count and percentage analysis of TIL subpopulation closely related.

Key Words: Triple-negative breast cancer, Survival, Tumor-infiltrating lymphocytes, Neoadjuvant therapy

Core tip: We evaluated a series of 98 triple negative breast cancer cases who received neoadjuvant chemotherapy. Pre-neoadjuvant chemotherapy and post neoadjuvant chemotherapy samples were analyzed. We compared tumor-infiltrating lymphocytes evaluated in whole slides vs in tissue microarray slides. We also compared subsets of tumor-infiltrating-lymphocytes (TILs) evaluated through an absolute counting vs a percentage calculation. Our results confirm the predictive and prognostic role of tumor infiltrating lymphocytes, and evaluated the behavior of tumor infiltrating lymphocytes and lymphocyte subsets during chemotherapy in triple negative breast cancer. Our results increase the understanding of methodological issues for TIL evaluation as well as provide information about variation of the whole TIL population and TIL subpopulation during chemotherapy.

  • Citation: Castaneda CA, Mittendorf E, Casavilca S, Wu Y, Castillo M, Arboleda P, Nunez T, Guerra H, Barrionuevo C, Dolores-Cerna K, Belmar-Lopez C, Abugattas J, Calderon G, De La Cruz M, Cotrina M, Dunstan J, Gomez HL, Vidaurre T. Tumor infiltrating lymphocytes in triple negative breast cancer receiving neoadjuvant chemotherapy. World J Clin Oncol 2016; 7(5): 387-394
  • URL:
  • DOI:


Triple negative breast cancer (TNBC) represents 10% to 20% of breast cancer (BC) and is a biologically aggressive tumor with high response to Neoadjuvant chemotherapy (NAC) but poor outcome[1].

Prognostic and predictive role of tumor infiltrating lymphocytes (TILs) have been extensively studied in BC and different studies have found they are associated to better outcome in TNBC. Denkert et al[2] found that a high TIL percentage evaluated by Hematoxilin and Eosin (H and E) in full-face sections of pre-NAC samples can predict pathologic complete response (pCR) in a series of more than 1000 BC cases. Loi et al[3] evaluated the prognostic role of TIL percentage in more than 2000 node-positive BC and found that high TIL percentage are associated to better outcome only in the TNBC (P = 0.023). Adams et al[4] evaluated the role of TIL in two phase III adjuvant randomized BC trials and also found a better outcome only in the TNBC (P = 0.02)[5]. Finally, a recent meta-analysis and a retrospective analysis of 8897 TNBC confirm the prognostic value of TIL in TNBC[5,6]. Recent studies suggest that tumor infiltration by CD8 cytotoxic lymphocytes and absence of FOXP3 immunosuppressive regulatory cells could control tumor growth and carry a better prognosis[7-12].

Despite the international TILs working group defined harmonization criteria to evaluate TIL, there is some areas that still require a better understanding. One of these areas is the value of TILs in small pieces of tumor like tissue microarrays (TMA), as well as the value and the appropriate methodology to evaluate TIL subpopulations in TMA.

Finally, some recent studies also suggest a prognostic role of TIL in post-NAC samples, however, there is need for more information in this area because evaluation of these samples has special challenges to pathologists as neoadjuvant chemotherapy produces a spectrum of histopathologic changes including decrease in cancer cell number and changes in stroma composition that includes fibrosis, elastosis, collagenization, hyalinization, microcalcification, neovascularization, fibrinoid necrosis and mucinous changes[13].

This study aims to evaluate TIL variation during NAC through H and E in full-face and in TMA sections as well as the variation of TIL subpopulations through immunohistochemistry (IHC) staining in TMA sections.

Patients and sample selection

We retrospectively reviewed the files of all new BC cases which came to the Instituto Nacional de Enfermedades Neoplasicas between 2005 and 2010, and we selected 98 TNBC cases with Clinical Stage II-III who went to surgery of breast tumor and axilla after receiving NAC. All core biopsy prior to NAC and breast tumor excision specimens were fixed in 10% neutral buffered formalin and embedded in paraffin and stored at Institutes Pathology Department Archive. Institutional review board approved the protocol of this study.

Staining and quantification of H and E, CD3, CD4, CD8 and forkhead box protein 3 IHC staining

Tumor areas were selected and a 0.6 cm punch from the formalin-fixed paraffin-embedded specimens were obtained and organized in 8-10 cylinder cards (TMA). H and E staining was performed in the full-face and in the TMA sections, and TIL was evaluated as the percentage of the stroma area of tumor that contained lymphocytic infiltrate through a 10% increment system under 200 × - 400 × magnification.

Process of IHC preparation included cutting 4 μm sections from the TMA, deparaffinating, rehydrating and processing sections using an automatized stainer (Autostainer Link 48, DAKO, Carpinteria, CA, United States) through standard methods. The following antibodies: CD3 (IS503, Dako), CD4 (IS649, Dako), CD8 (IS623, Dako) and FOXP3 (clone: 236A/E7) were used for staining of TMA section. Lymphocyte subsets were calculated through the percentage between lymphocytes/ tumor cells in a 10% increment system, and through the absolute count of the lymphocytes in 5 high power fields under 200 × - 400 × magnification.

Clinical information and pathological response

We obtained clinical information from patient files archived at Instituto Nacional de Enfermedades Neoplasicas. The pathological therapeutic response of the surgically resected tumor was evaluated after NAC. The surgical specimens of breast lesions were cut into 5 mm slices and processed with H and E staining. A pCR was defined as the absence of all invasive cancer cells in breast and axillary lymph nodes, regardless of the presence of non-invasive cancer cells[11].

Statistical analysis

The mean percentage as well as the mean absolute number of immune cells was calculated, and those lesions above this mean were graded as increased. Analysis were also performed considering a high percentage of immune cells when were at least 50%. All statistical analyses were performed using STATA software version 12. Associations among variables were evaluated using Fisher’s exact test or the χ2 test. The Mann–Whitney U and Spearman’s correlation tests were used to compare groups. The measurement of agreements between TIL evaluated in whole slide and TMA was used intraclass correlation coefficient (ICC). Kaplan-Meier estimation curves disease free and overall survival was applied. All tests were two sided, and a P≤ 0.05 was considered statistically significant.

Assessment of TILs by H and E

The characteristics of 91 pre-NAC and 80 post-NAC TN BC cases are reported in Table 1. Most cases were ductal infiltrating carcinoma (96.9%), inflammatory (29.6%), clinical stage III (86.7%) and HGIII (75.5%). Most cases received neoadjuvant doxorubicin and paclitaxel (87.7%) and 29.6% obtained pCR. Sixty-six percent of the patients underwent mastectomy. After a median follow-up of 37.5 mo, there were 42% recurrences and 45% deaths. Pathologic complete response was associated to OS (P = 0.0071) but was not associated to DFS (P = 0.1050).

Table 1 Clinical and pathological general features.
Featuresn = 98 (%)
Median age49 ± 9
Clinical stage
II13 (13.3)
III85 (86.73)
Histologic grade
II23 (23.5)
III74 (75.5)
Subtype histologic
Ductal95 (96.9)
Lobular2 (2.1)
Neoadjuvant chemotherapy
AC9 (9.2)
AC-Taxane86 (87.7)
Taxane alone3 (3.1)
Tumorectomy29 (29.6)
Mastectomy65 (66.3)
Unknown4 (4.1)
No69 (70.4)
Yes29 (29.6)
No69 (70.4)
Yes29 (29.6)
No56 (57.1)
Yes42 (42.9)
No53 (54.1)
Yes45 (45.9)

Median Pre-NAC TIL percentage in the full-face (n = 91) and in the TMA section (n = 30) was 40 ± 20 and 20 ± 15, respectively. Median post-NAC TIL percentage in the full-face (n = 80) and in the TMA section (n = 58) was 20 ± 15 and 10 ± 5, respectively (Table 2).

Table 2 Tumor infiltrating lymphocytes in pre-neoadjuvant-chemotherapy and post-neoadjuvant-chemotherapy samples (median ± interquartile range).
TIL population(Me ± IQR)
Median percentage TIL H and E full-face (n = 91)40 ± 20
Median percentage TIL H and E TMA (n = 30)20 ± 15
Median counting TIL CD3 (n = 28)244.5 ± 253.8
Median counting TIL CD4 (n = 19)48 ± 107.5
Median counting TIL CD8 (n = 28)102 ± 98.8
Median counting TIL FOXP3 (n = 29)22 ± 35
Median percentage TIL H and E (n = 80)20 ± 15
Median percentage TIL H and E TMA (n = 58)10 ± 5
Median counting TIL CD3 (n = 68)156.5 ± 200.8
Median counting TIL CD4 (n = 54)12 ± 27.5
Median counting TIL CD8 (n = 70)75.5 ± 93.5
Median counting TIL FOXP3 (n = 70)7.5 ± 14.5

Pre-NAC TIL evaluated in full-face had low grade of agreement with TMA sections (n = 30) (ICC = 0.017). Post-NAC samples were larger and allowed to be divided in homogeneous (n = 26) and heterogeneous lesions (n = 26). Heterogeneous lesions had low level of agreement between full-face and TMA sections (ICC = 0.20), and homogeneous lesions had high level of agreement between full-face and TMA sections (ICC = 0.73).

Higher median pre-NAC TIL (n = 91) evaluated in full-face sections was associated to pCR (40% vs 30%, P = 0.0251), DFS (40% vs 20%, P = 0.0076) and OS (40% vs 30%, P = 0.0334); but not to age (P = 0.1427) nor inflammatory features (P = 0.6401), in the univariate analysis. Association between median TIL and pCR remained significant even with adjustment for age. Higher median pre-NAC TIL (n = 30) evaluated in TMA section was only associated to absence of inflammatory features (10% vs 30%, P = 0.0387).

Median post-NAC TIL evaluated in full-face (n = 80) or in TMA section (n = 58) was not associated to any of the previously mentioned features. Post-NAC median H and E TIL percentage evaluated in full-face section was similar in residual fibrous lesions (n = 17) (pCR) and in residual tumor lesions (n = 63) (no pCR) (20% vs 20%, P = 0.6331) (Table 3).

Table 3 Comparison between tumor-infiltrating-lymphocytes evaluated in full-face and tissue microarrays sections.
FeaturesH and E in the full-face section
H and E in TMA section
nMe ± IQDP-value2nMe ± IQDP-value3
≤ 495040 ± 201920 ± 15
> 494120 ± 151120 ± 25
No6330 ± 201530 ± 25
Yes2840 ± 17.51520 ± 10
Yes2740 ± 15810 ± 6.5
No6435 ± 202230 ± 15
< 32 mo4620 ± 202015 ± 13.8
≥ 32 mo4540 ± 201025 ± 25
< 41 mo5130 ± 202220 ± 15
≥ 41 mo4040 ± 20820 ± 15
≤ 494320 ± 153110 ± 15
> 493720 ± 152710 ± 5
Yes2615 ± 101610 ± 0
No5420 ± 154210 ± 15
No6320 ± 154410 ± 7.5
Yes1720 ± 101410 ± 7.5
< 32 mo3520 ± 152210 ± 0
≥ 32 mo2820 ± 152210 ± 15
< 41 mo4020 ± 152710 ± 10
≥ 41 mo2320 ± 151710 ± 5

Classification of TIL with a cut-off of 50% did not identify a population associated to any of the previously mentioned features (P = 0.16 for pCR, P = 0.14 for DFS and P = 0.64 for OS).

NAC produced a statistically significant decrease in median TIL percentage when evaluated in full-face section (n = 73 P < 0.0002), but not when evaluated in TMA sections (n = 16 P = 0.4321) (Table 4).

Table 4 Comparison of tumor-infiltrating-lymphocyte evaluated in pre- vs post-neoadjuvant-chemotherapy samples.
Population of lymphocytesnPre-NAC Me ± IQRPost-NAC Me ± IQRP-value2P corrected value3
H and E whole slide7340 ± 1520 ± 15< 0.001< 0.0021
H and E TMA1630 ± 17.515 ± 150.4321
Absolute counting
CD322244.5 ± 315.5255.5 ± 2670.8583
CD4714 ± 9432 ± 430.6721
CD821127 ± 193.5156 ± 90.50.7544
FOXP32118 ± 3112 ± 19.50.0917
Assessment of TIL subsets by IHC

Analysis of TIL subsets through IHC was calculated through percentage calculation and absolute counting methodology in TMA sections. Percentage calculation was significantly correlated with absolute counting for all markers in pre-NAC (CD3 n = 27 r = 0.7182, CD8 n = 27 r = 0.6064, FOXP3 n = 26 r = 0.7192) and in post-NAC (CD3 n = 55 r = 0.7733, CD4 n = 30 r = 0.6129, CD8 n = 55 r = 0.7338, FOXP3 n = 47 r = 0.5387) TMA sections that had enough material for both quantification methodologies (Table 5). The lymphocyte subset with highest absolute counts in the pre-NAC and post-NAC samples was CD8 (127 ± 193.5 and 156.5 ± 90.5) (Table 4).

Table 5 Relationship between percentage and absolute counting methodologies of tumor-infiltrating-lymphocyte subpopulations.
Population of lymphocytesnrho2P-valueP corrected value3
CD3270.7182< 0.001< 0.0041
FOXP3260.7192< 0.001< 0.0041
CD3550.7733< 0.001< 0.0041
CD8550.7338< 0.001< 0.0041

Higher absolute counts of CD3, CD4, CD8 and FOXP3 in pre-NAC samples were associated with longer DFS (n = 28 P = 0.003, n = 19 P = 0.0062, n = 28 P = 0.0096 and n = 29 P = 0.0019; respectively). Higher absolute counts of CD3 in pre-NAC samples had longer OS (n = 28 P = 0.0241).

Higher absolute counts of CD4 in post-NAC samples was associated with age (n = 54 P = 0.0393) and pCR (n = 54 P = 0.0095).

Higher ratio of absolute counts of CD8/CD4 in pre-NAC and post-NAC samples was associated with pCR (n = 17 P = 0.0343 and n = 43 P = 0.0086 respectively).

Higher ratio of absolute counts of CD4/FOXP3 in pre-NAC sample was associated with longer DFS (n = 16 P = 0.0389). Higher ratio of absolute counts of CD4/FOXP3 in post-NAC sample was associated with pCR (n = 30 P = 0.003).

Higher ratio of absolute counts of CD4/CD3 in post-NAC samples was associated with pCR (n = 48 P = 0.0095).


We have evaluated pre and post-NAC samples in an effort to produce a comprehensive analysis of the role of TIL variation during NAC in TNBC samples. Evaluation of pre-NAC H and E staining of full-face sections found that those tumors with higher TILs are associated to both pCR and better outcome. These results are similar to those found by Denkert et al[2] in the neoadjuvant setting and by Loi et al[3] and Adams et al[4] in the adjuvant setting, and confirm accuracy of our methodology.

We did not find an association between prognosis and TILs in post-NAC samples evaluated in full-face or in TMA sections. García-Martínez et al[14] evaluated 121 BC cases and found that high TIL level in pre-NAC samples was associated to pCR. TIL in pre-NAC and post-NAC were not associated to outcome. By other side, Dieci et al[15] evaluated 278 TNBC with residual disease after NAC and found that those residual lesions with high level of TIL had better prognosis.

We found that TIL percentage evaluated in full-sections were higher in pre-NAC than post-NAC samples. No association between TIL variation (pre- vs post-NAC) and response to NAC was found. Post-NAC samples of those cases who obtained pCR were similar to TIL levels in those cases who did not obtain pCR (residual cancer). By other side, Dieci evaluated 19 selected cases with high TIL level in post-NAC samples and found that lower TIL increased during NAC[15], and Demaria et al[16] found that those cases with higher response have an increase of TIL during NAC in a series of 25 BC.

Although lymphocytic infiltration has demonstrated to behave as a prognostic and predictive marker in breast cancer, there are some aspects without standardization. We evaluated TIL in the 0.6 cm TMA, and we found that the TIL percentages differ from those found in the full-face sections. TIL percentage in TMA was not associated to pCR nor prognosis. Breast tumors and especially TNBC are heterogeneous lesions and our findings indicate that TILs concentration is also heterogeneous inside the different tumor areas. The evaluation of only one region of the tumor through a TMA cylinder appears not to produce confident information about immune reaction against the whole tumor.

Different articles have evaluated the role of TIL subpopulations (IHC staining), however some of them have measured them by percentage (resembling methodology used for TIL evaluation with H and E)[2,3] and other have measured by an absolute counting[7,8,17-21].

We compared both methodologies in 0.6 cm TMA tumor samples for CD3, CD4, CD8 and FOXP3 lymphocyte subpopulations and we found a significant correlation between both methodologies.

We also evaluated the association between levels of CD3, CD4, CD8 and FOXP3 lymphocyte subpopulations (absolute counting) and clinical features. Although our sample size for evaluating lymphocyte subpopulations is small, we found that higher absolute counts of CD4 lymphocytes in post-NAC samples were associated with pCR. Higher absolute counts of CD3, CD4, CD8 and FOXP3 lymphocytes in pre-NAC samples were associated with DFS, and higher absolute counts of CD3 lymphocytes in pre-NAC samples had longer OS.

Finally, we also found an association of pCR with higher ratio of absolute counts of all CD8/CD4 in pre-NAC, and CD8/CD4, CD4/CD3, CD4/FOXP3 in post-NAC samples. Higher ratio of absolute counts of CD4/FOXP3 in pre-NAC sample was associated with longer DFS.

The role of one TIL subpopulation and the role of the relationship between two TIL subpopulations over tumor behavior have been previously described and some of authors confirm our findings. Rathore et al[9] found that higher levels of CD3+, CD4+ and CD8+ TILs was significantly associated with good prognosis in a series of 123 early BC cases. Kim et al[22] reported that lower number of CD8+ TILs in breast tumors were significantly associated with lymph node metastasis, higher stage and high proliferative index in a series of 72 early BC cases. Increased number of FOXP3 lymphocytes was associated to lymph node metastasis, high proliferative index and shorter DFS. Ladoire et al[10] evaluated 56 BC cases who went to NAC and found that high CD8 and absence of FOXP3 infiltration was associated to pCR. Miyashita et al[11] evaluated 131 TNBC patients treated with NAC and found that high CD8 TIL levels and high CD8/FOXP3 ratio in residual tumor had better outcome. García-Martínez et al[14] described a decrease of CD4, an increase of CD8 and an absence of changes of FOXP3 during NAC. High levels of CD3 and CD4 in pre-NAC were associated to pCR. A decrease of CD3 and CD4 during chemotherapy was associated to pCR. A decrease of CD3 during NAC was also associated to better DFS and OS. They also found that higher ratio of CD4/CD8 in pre-NAC was associated to a pCR. They also evaluated six public genomic datasets with around 1000 BC patients treated with NAC and found that higher CD4 count in post-NAC samples was associated to pCR. Finally, they found that high levels of CD3 in post NAC was associated to better DFS[14]. Other authors have also evaluated the role of TIL ratios, such as CD8/CD4[23] or FOXP3/CD3[12], as an alternative approach to better integrate the information provided by each TIL subpopulation[14].

Differences among mentioned authors and our findings could be explained by our small population size, analysis of a not representative area in the TMA samples or changes in CD4+ TILs phenotype from effectors to suppressors. Therefore, our results need to be validated in larger series. Remarkably, our work is the first to our knowledge to evaluate TIL in BC tumors from Latinoamerican women. And, it is the first to compare the evaluation of TIL percentage in full-sections and in TMA sections, as well as to compare the evaluation of TIL levels through percentage analysis and through absolute counting.

Identification of biomarkers and evaluation of therapy in the neoadjuvant setting has become a major challenge in BC since they could speed-up the development and approval of new drugs[24]. pCR is a validated surrogate for drug efficacy in the neoadjuvant setting but its specificity needs still to be improved. The finding of a biomarker related to host immunity in the pre or post-NAC samples could have the benefit to predict response not only to chemotherapy but also to immune checkpoint modulators.


The authors would like to express their gratitude to Maria del Rosario Villa and Vladimir Flores for their support in staining preparation of tumor sections.


Triple-negative-breast-cancer (TNBC) is associated to poor outcome and is highly prevalent among Latinoamerican women. Tumor-infiltrating-lymphocytes (TILs) have been associated to higher response to chemotherapy and better outcome in TNBC when evaluated in retrospective and prospective series as well as meta-analysis. An international TILs working group defined harmonization criteria to evaluate them in 2015, however, there is still some areas requiring a better understanding. There is not information describing TILs in small pieces of tumor, and the value and the appropriate methodology to evaluate TIL subpopulations in tissue microarrays (TMA). There is small information describing variation of TIL during chemotherapy.

Research frontiers

TNBC has higher prevalence in Latinoamerican women and is a poor prognostic malignancy without target therapy. Chemotherapy is the only available treatment for TNBC. TIL appears to identify prognosis in TNBC and some recent studies are evaluating if it predicts response to chemotherapy or immunotherapy. Therefore, information about TIL variation during chemotherapy is an important issue as it is the scenario we need to improve treatment efficacy.

Innovations and breakthroughs

As TNBC malignancy and role of TIL as biomarker are important issues, more research about relevance of TIL in TNBC is needed. Therefore, the authors revealed that TIL evaluated in a small area of tumor differs to those evaluated in full-face samples, and loss their prognostic and predictive value. The authors also found that evaluation of lymphocyte subsets can be equally performed through absolute counting or percentage calculating, and can provide prognostic information.


Based on the present study, the authors can suggest that full-face samples (and not core samples) are used for TIL evaluation in H and E. Absolute counting and percentage calculating could be considered appropriate for evaluation of TIL subsets.


TIL is a biomarker with current strong evaluation in different malignancies including TNBC. TIL is accepted as those stromal mononuclear cells inside the tumor but not in contact with cancer cells nor inside tumor niches. TMA is a technique allowing immunohistochemistry staining of many tumor samples at the same time.


The paper is very interesting and well written.


Manuscript source: Invited manuscript

Specialty Type: Oncology

Country of Origin: Peru

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P- Reviewer: Pan F, Tsikouras PPT, Yokoyama Y, Zafrakas M S- Editor: Qiu S L- Editor: A E- Editor: Lu YJ

1.  Curigliano G, Viale G, Ghioni M, Jungbluth AA, Bagnardi V, Spagnoli GC, Neville AM, Nolè F, Rotmensz N, Goldhirsch A. Cancer-testis antigen expression in triple-negative breast cancer. Ann Oncol. 2011;22:98-103.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 71]  [Cited by in F6Publishing: 77]  [Article Influence: 5.5]  [Reference Citation Analysis (0)]
2.  Denkert C, Loibl S, Noske A, Roller M, Müller BM, Komor M, Budczies J, Darb-Esfahani S, Kronenwett R, Hanusch C. Tumor-associated lymphocytes as an independent predictor of response to neoadjuvant chemotherapy in breast cancer. J Clin Oncol. 2010;28:105-113.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 1125]  [Cited by in F6Publishing: 1252]  [Article Influence: 83.5]  [Reference Citation Analysis (0)]
3.  Loi S, Sirtaine N, Piette F, Salgado R, Viale G, Van Eenoo F, Rouas G, Francis P, Crown JP, Hitre E. Prognostic and predictive value of tumor-infiltrating lymphocytes in a phase III randomized adjuvant breast cancer trial in node-positive breast cancer comparing the addition of docetaxel to doxorubicin with doxorubicin-based chemotherapy: BIG 02-98. J Clin Oncol. 2013;31:860-867.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 1020]  [Cited by in F6Publishing: 1158]  [Article Influence: 105.3]  [Reference Citation Analysis (0)]
4.  Adams S, Gray RJ, Demaria S, Goldstein L, Perez EA, Shulman LN, Martino S, Wang M, Jones VE, Saphner TJ. Prognostic value of tumor-infiltrating lymphocytes in triple-negative breast cancers from two phase III randomized adjuvant breast cancer trials: ECOG 2197 and ECOG 1199. J Clin Oncol. 2014;32:2959-2966.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 783]  [Cited by in F6Publishing: 917]  [Article Influence: 101.9]  [Reference Citation Analysis (0)]
5.  Salgado R, Denkert C, Demaria S, Sirtaine N, Klauschen F, Pruneri G, Wienert S, Van den Eynden G, Baehner FL, Penault-Llorca F. The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014. Ann Oncol. 2015;26:259-271.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 1473]  [Cited by in F6Publishing: 1879]  [Article Influence: 187.9]  [Reference Citation Analysis (0)]
6.  Ibrahim EM, Al-Foheidi ME, Al-Mansour MM, Kazkaz GA. The prognostic value of tumor-infiltrating lymphocytes in triple-negative breast cancer: a meta-analysis. Breast Cancer Res Treat. 2014;148:467-476.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 147]  [Cited by in F6Publishing: 168]  [Article Influence: 16.8]  [Reference Citation Analysis (0)]
7.  Liu S, Lachapelle J, Leung S, Gao D, Foulkes WD, Nielsen TO. CD8+ lymphocyte infiltration is an independent favorable prognostic indicator in basal-like breast cancer. Breast Cancer Res. 2012;14:R48.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 281]  [Cited by in F6Publishing: 312]  [Article Influence: 26.0]  [Reference Citation Analysis (0)]
8.  Baker K, Lachapelle J, Zlobec I, Bismar TA, Terracciano L, Foulkes WD. Prognostic significance of CD8+ T lymphocytes in breast cancer depends upon both oestrogen receptor status and histological grade. Histopathology. 2011;58:1107-1116.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 26]  [Cited by in F6Publishing: 59]  [Article Influence: 4.5]  [Reference Citation Analysis (0)]
9.  Rathore AS, Kumar S, Konwar R, Makker A, Negi MP, Goel MM. CD3+, CD4+ & amp; CD8+ tumour infiltrating lymphocytes (TILs) are predictors of favourable survival outcome in infiltrating ductal carcinoma of breast. Indian J Med Res. 2014;140:361-369.  [PubMed]  [DOI]  [Cited in This Article: ]
10.  Ladoire S, Arnould L, Apetoh L, Coudert B, Martin F, Chauffert B, Fumoleau P, Ghiringhelli F. Pathologic complete response to neoadjuvant chemotherapy of breast carcinoma is associated with the disappearance of tumor-infiltrating foxp3+ regulatory T cells. Clin Cancer Res. 2008;14:2413-2420.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 233]  [Cited by in F6Publishing: 237]  [Article Influence: 14.8]  [Reference Citation Analysis (0)]
11.  Miyashita M, Sasano H, Tamaki K, Hirakawa H, Takahashi Y, Nakagawa S, Watanabe G, Tada H, Suzuki A, Ohuchi N. Prognostic significance of tumor-infiltrating CD8+ and FOXP3+ lymphocytes in residual tumors and alterations in these parameters after neoadjuvant chemotherapy in triple-negative breast cancer: a retrospective multicenter study. Breast Cancer Res. 2015;17:124.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 155]  [Cited by in F6Publishing: 176]  [Article Influence: 19.6]  [Reference Citation Analysis (0)]
12.  Suzuki K, Kadota K, Sima CS, Nitadori J, Rusch VW, Travis WD, Sadelain M, Adusumilli PS. Clinical impact of immune microenvironment in stage I lung adenocarcinoma: tumor interleukin-12 receptor β2 (IL-12Rβ2), IL-7R, and stromal FoxP3/CD3 ratio are independent predictors of recurrence. J Clin Oncol. 2013;31:490-498.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 127]  [Cited by in F6Publishing: 157]  [Article Influence: 13.1]  [Reference Citation Analysis (0)]
13.  Sethi D, Sen R, Parshad S, Khetarpal S, Garg M, Sen J. Histopathologic changes following neoadjuvant chemotherapy in locally advanced breast cancer. Indian J Cancer. 2013;50:58-64.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 9]  [Cited by in F6Publishing: 13]  [Article Influence: 1.2]  [Reference Citation Analysis (0)]
14.  García-Martínez E, Gil GL, Benito AC, González-Billalabeitia E, Conesa MA, García García T, García-Garre E, Vicente V, Ayala de la Peña F. Tumor-infiltrating immune cell profiles and their change after neoadjuvant chemotherapy predict response and prognosis of breast cancer. Breast Cancer Res. 2014;16:488.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 159]  [Cited by in F6Publishing: 181]  [Article Influence: 18.1]  [Reference Citation Analysis (0)]
15.  Dieci MV, Criscitiello C, Goubar A, Viale G, Conte P, Guarneri V, Ficarra G, Mathieu MC, Delaloge S, Curigliano G. Prognostic value of tumor-infiltrating lymphocytes on residual disease after primary chemotherapy for triple-negative breast cancer: a retrospective multicenter study. Ann Oncol. 2014;25:611-618.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 319]  [Cited by in F6Publishing: 309]  [Article Influence: 30.9]  [Reference Citation Analysis (0)]
16.  Demaria S, Volm MD, Shapiro RL, Yee HT, Oratz R, Formenti SC, Muggia F, Symmans WF. Development of tumor-infiltrating lymphocytes in breast cancer after neoadjuvant paclitaxel chemotherapy. Clin Cancer Res. 2001;7:3025-3030.  [PubMed]  [DOI]  [Cited in This Article: ]
17.  Mahmoud SM, Paish EC, Powe DG, Macmillan RD, Grainge MJ, Lee AH, Ellis IO, Green AR. Tumor-infiltrating CD8+ lymphocytes predict clinical outcome in breast cancer. J Clin Oncol. 2011;29:1949-1955.  [PubMed]  [DOI]  [Cited in This Article: ]
18.  Hornychova H, Melichar B, Tomsova M, Mergancova J, Urminska H, Ryska A. Tumor-infiltrating lymphocytes predict response to neoadjuvant chemotherapy in patients with breast carcinoma. Cancer Invest. 2008;26:1024-1031.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 76]  [Cited by in F6Publishing: 71]  [Article Influence: 4.7]  [Reference Citation Analysis (0)]
19.  Seo AN, Lee HJ, Kim EJ, Kim HJ, Jang MH, Lee HE, Kim YJ, Kim JH, Park SY. Tumour-infiltrating CD8+ lymphocytes as an independent predictive factor for pathological complete response to primary systemic therapy in breast cancer. Br J Cancer. 2013;109:2705-2713.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 209]  [Cited by in F6Publishing: 229]  [Article Influence: 20.8]  [Reference Citation Analysis (0)]
20.  Klintrup K, Mäkinen JM, Kauppila S, Väre PO, Melkko J, Tuominen H, Tuppurainen K, Mäkelä J, Karttunen TJ, Mäkinen MJ. Inflammation and prognosis in colorectal cancer. Eur J Cancer. 2005;41:2645-2654.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 264]  [Cited by in F6Publishing: 288]  [Article Influence: 15.2]  [Reference Citation Analysis (0)]
21.  Ladoire S, Mignot G, Dabakuyo S, Arnould L, Apetoh L, Rébé C, Coudert B, Martin F, Bizollon MH, Vanoli A. In situ immune response after neoadjuvant chemotherapy for breast cancer predicts survival. J Pathol. 2011;224:389-400.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 175]  [Cited by in F6Publishing: 181]  [Article Influence: 13.9]  [Reference Citation Analysis (0)]
22.  Kim ST, Jeong H, Woo OH, Seo JH, Kim A, Lee ES, Shin SW, Kim YH, Kim JS, Park KH. Tumor-infiltrating lymphocytes, tumor characteristics, and recurrence in patients with early breast cancer. Am J Clin Oncol. 2013;36:224-231.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 83]  [Cited by in F6Publishing: 86]  [Article Influence: 7.8]  [Reference Citation Analysis (0)]
23.  DeNardo DG, Brennan DJ, Rexhepaj E, Ruffell B, Shiao SL, Madden SF, Gallagher WM, Wadhwani N, Keil SD, Junaid SA. Leukocyte complexity predicts breast cancer survival and functionally regulates response to chemotherapy. Cancer Discov. 2011;1:54-67.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 1184]  [Cited by in F6Publishing: 1297]  [Article Influence: 99.8]  [Reference Citation Analysis (0)]
24.  Food and Drug Administration. Guidance for Industry Pathological Complete Response in Neoadjuvant Treatment of High-Risk Early-Stage Breast Cancer: Use as an Endpoint to Support Accelerated Approval. U.S. Department of Health and Human Services, Center for Drug Evaluation and Research.  Available from: http//  [PubMed]  [DOI]  [Cited in This Article: ]