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Tumor infiltrating lymphocytes profile and response in neoadjuvant chemotherapy-treated triple-negative breast carcinoma patients


1 Department of Pathology, Medical College and Hospital, Kolkata, West Bengal, India
2 Department of Hepatology, PGIMER, Chandigarh, India

Date of Submission23-Jul-2020
Date of Decision09-Sep-2020
Date of Acceptance21-Dec-2020
Date of Web Publication24-Jul-2021

Correspondence Address:
Debadrita Ray,
House No 1031, Sector 40B, Chandigarh - 160 036
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_997_20

 > Abstract 


Background: Triple negative breast carcinoma (TNBC) has the highest mortality among all the breast carcinoma subtypes, but paradoxically, it shows the best response to neoadjuvant chemotherapy (NACT). Tumor infiltrating lymphocytes (TIL) density has been shown to have prognostic significance in TNBC. However, there are limited data on TIL subpopulation and their association with response to NACT in TNBC.
Materials and Methods: The study included 80 consecutive patients with TNBC prospectively diagnosed for two and half years, who underwent tru-cut biopsy before NACT, followed by subsequent definite surgical procedures. Global TIL profile and immunohistochemistry (IHC) analysis of CD3, CD4, CD8, CD20, and CD56 were done on all baseline tru-cut biopsies and post-NACT surgical specimens.
Results: Almost half the patients were postmenopausal with a mean age of 45.89 ± 4.62 years. The majority had low CD3, low CD4, low CD56, low CD20, and high CD8 positivity in both pre- and post-NACT specimens. On multivariate analysis, low CD3, CD4, CD56 and CD 20 were established as independent predictor of poor pathologic response (PR). Low CD4 (adjusted odds ratio [OR]: 228.46) was associated with the highest OR for poor PR. Low CD8 was associated with significantly decreased odds of poor PR on univariate analysis (OR: 0.26), but it was not been established as an independent predictor of PR on multivariate logistic regression. NACT did not significantly alter the profile of TILs.
Conclusions: TIL profile with low CD3, CD4, CD20, and CD56 expression predicts PR to NACT in TNBC and may thus help in prognostication of these patients.

Keywords: Neo-adjuvant chemotherapy, pathologic response, triple-negative breast carcinoma, tumor infiltrating lymphocytes



How to cite this URL:
Ray D, Gupta SD, De A, Jain P, Bhattacharya NK, Biswas PK. Tumor infiltrating lymphocytes profile and response in neoadjuvant chemotherapy-treated triple-negative breast carcinoma patients. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 5]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=322276




 > Introduction Top


Breast cancer is the leading cause of cancer-related death in females.[1] Tumor infiltrating lymphocytes (TILs) are emerging as potential prognostic markers in breast cancers, particularly in HER2-positive and triple-negative breast cancers (TNBC). Immune infiltrates are detectable in 75% of early-stage HER2-positive and TNBC with a dense infiltrate being found in up to 20% of tumors.[2] Neo-adjuvant chemotherapy (NACT) followed by surgery is the standard of care for locally advanced breast cancer and is increasingly being used in earlier stages with the aim of achieving tumor downstaging and potential breast conservation.[3] Emerging evidence suggests that the stromal TIL density is predictive of response to NACT.[4],[5]

Immune regulated pathways influence multiple aspects of cancer development.[6] T cells account for 75% of TILs. Natural killer cells (NK) which comprise 5% of lymphocytes also serve as the first line of defence.[7] It is plausible that not only the density of TILs but also their composition have a bearing on the prognosis and response to treatment in breast cancer. However, there are scanty data on the relationship between TIL subpopulation and response to NACT in breast cancer, particularly in TNBC.[8] TNBC precludes targeted therapy and is associated with higher mortality as compared to other subtypes of breast cancer. Counterintuitively, pathological response to NACT is better in TNBC.[9] Thus, we designed this study to evaluate the stromal global TIL profile and its composition using CD3, CD4, CD8, CD20, and CD56 markers in pre-NACT tru-cut biopsies and subsequent post-NACT surgical specimens in patients with TNBC.


 > Materials and Methods Top


We evaluated the tissue samples of 80 prospectively diagnosed, consecutive cases of TNBC patients who received NACT followed by definite surgical procedures at a tertiary hospital over a period of two and half years. Patients who were immunocompromised or on immunosuppressant therapy (other than chemotherapy), who did not receive NACT or definite surgical procedure after NACT and those who refused to give informed consent were excluded from the study. The flow-diagram of the study is depicted in [Supplementary Figure 1]. NACT regimen used was at the discretion of the clinical team. The study was approved by the Institutional Ethical Committee. Our primary objective was to evaluate the association between the profile of TIL subpopulation and pathologic response (PR) to NACT. Our secondary objective was to assess changes in TIL subpopulation induced by NACT.



Clinical records of the patients were reviewed. Histopathology and immunohistochemical (IHC) evaluation of pre-NACT tru-cut biopsies and post-NACT definite surgical specimens were reported by two pathologists with experience in scoring TILs. Disputes were resolved by consensus.

Global tumor infiltrating lymphocyte density assessment

Global TIL assessment was made on Hematoxylin and Eosin (H and E)-stained sections of pre-NACT tru-cut biopsy and post-NACT definite surgical specimens following International Guidelines. Briefly, TILs were assessed within the boundaries of the tumor area and the invasive edge was included in the assessment. Care was taken to avoid hot-spots and only mononuclear cells (lymphocytes and plasma cells) were counted. Only stromal TILs (% of the stromal area occupied by mononuclear inflammatory cells) were evaluated at a high-magnification (×400) and were estimated as an averaged percentage of the examined stromal area. For the assessment of global TIL in residual disease in post-NACT tissue samples, residual tumor bed was defined as the largest cross-sectional area between residual invasive tumor cells. Global TIL density was then graded as low (0%–10% stromal TILs), intermediate (10%–40% stromal TILs), or high (>40% stromal TILs).[4],[7]

IHC analysis of tumor infiltrating lymphocyte profile and scoring

CD3, CD4, CD8, CD20, and CD56 markers were studied on both pre-NACT tru-cut biopsy and post-NACT definite surgical specimens using standard techniques.[10] Clonal and control details of IHC markers are included in [Supplementary Table 1]. For each subtype, TILs were counted in five randomly selected high-power fields (HPFs) at ×40 magnification and the counts were averaged. Initially, the TIL count was graded as + (1–25 TIL/HPF), ++ (26–50 TIL/HPF) and +++ (>51 TIL/HPF). Only stromal TILs within the boundaries of the tumor were considered, as discussed previously. TIL subtype count of ≤25 cells TIL/HPF (+) was considered as “low” count while >25 cells TIL/HPF (++ and +++) were considered as “high” count.[11]



Histopathologic assessment of pathologic response following neoadjuvant chemotherapy

The PR after NACT was graded on gross and microscopic examination of H and E stained sections of surgical specimens using the system of Chevallier et al.[12] Good PR was defined as a Grade 1 response (disappearance of all tumor on macroscopic and microscopic assessment). Poor PR was defined as Grade 2 (cancer in-situ), Grade 3 (invasive cancer with stromal changes) or Grade 4 response (no or negligible change).[12]

Sample size calculation

Previous studies have reported good PR in 28%–36% patients of TNBC. To estimate a proportion of 32% with precision of the estimate of 0.1 and confidence level of 0.95, the required sample size is 80 patients.

Statistical analysis

Statistical analysis was performed using Microsoft Excel 365 (Microsoft, USA) MedCalc Statistical Software version 19.3 (MedCalc Software Ltd, Belgium). Data were reported as mean ± standard deviation or median with range. Quantitative data were compared using the Mann–Whitney test for independent data and Wilcoxon test for paired data, respectively. Chi-square test (Fisher exact test when applicable) and McNemar test were used for comparing categorical unpaired and paired data, respectively. Interobserver agreement was analyzed using Cohen's kappa statistics and 95% confidence intervals for the same were estimated using Fleiss technique. Multivariate logistic regression analysis for prediction of PR was done by incorporating variables in a step-wise model where variables were entered into the model if P < 0.05 and removed if P > 0.1. All statistical tests were performed two-sided and P value < 0.05 was considered significant.


 > Results Top


Patients' demographic and risk factor profiles

Most patients (45%) presented in the age group of 45–50 years, with the mean age of presentation being 45.89 ± 4.62 years. Half (n = 40) of the patients were postmenopausal. Twelve patients (15%) had a positive family history in first-degree relatives. Other demographic and risk factor profiles are mentioned in [Supplementary Table 2].



Baseline tumor and lymph node characteristics

Average tumor size was 4.8 (2.2–7.8) cm. Thirty-seven (46%) patients presented with tumors fixed either to underlying structures or to the skin (skin ulceration and peau d'orange). Lymph-nodal involvement was present in 54 (67.5%) patients. On mammography, all patients had BIRADS 4 (n = 36, 45%) and BIRADS 5 (n = 44, 55%) [Table 1].
Table 1: Baseline tumour characteristics

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Most of the patients (n = 62, 77.5%) received only anthracycline-based chemotherapy, while 18 (22.5%) patients received anthracycline and taxane-based regimen. None of them received platinum-based chemotherapy. Fifty-eight (72.5%) patients underwent post-NACT breast conservative surgery while modified radical mastectomy was performed on 22 (27.5%) patients.

Preneoadjuvant chemotherapy histopathology examination and tumor infiltrating lymphocyte profile

Invasive breast carcinoma of no special type was the most common histological type encountered in 62 (77.5%) patients. Other histological types found were medullary carcinoma, infiltrating lobular carcinoma, invasive mucinous carcinoma. According to modified Bloom-Richardson grading, 53 (66.25%) patients had Grade III tumor.

Most of the patients had pre-NACT intermediate global TIL (n = 38, 47.5%), while high and low global was observed in 7 (8.75%) and 35 (43.75%) patients only.

On IHC high CD3, CD4, CD8, CD20, CD56 were seen in 27 (33.8%), 25 (31.2%), 48 (60%), 35 (43.8%), and 25 (31.2%), respectively. Mean TIL count of CD3, CD4, CD8, CD20, CD56 were 23 ± 7.56, 22.6 ± 9.1, 27.48 ± 10.01, 25.5 ± 8.07, 22.4 ± 8.62, respectively [Table 2].
Table 2: Preneoadjuvant chemotherapy histopathology and tumour infiltrating lymphocytes profile

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Pathologic response and correlation with pre-neo-adjuvant chemotherapy tumor infiltrating lymphocyte profile

Majority of our patients had poor PR to NACT (n = 54, 67.5%). There was no difference in PR rates between patients who received only anthracycline-based chemotherapy and those who received anthracycline-taxane-based regimens (poor response: 69.3% vs. 61.1%; P = 0.51). Most of the patients with poor pathological response had low pre-NACT global TIL (n = 30, 55.5%), while most of the good PR patients showed intermediate global TIL (n = 15, 57.7%). Poor PR was significantly more common in patients with lower global TILs.

The difference between the patients who had good and poor PR is shown in [Table 3]. Poor PR was associated with low CD3 (odds ratio [OR]: 33.6, 95% confidence interval [CI]: 9.3–125.8, P < 0.001), CD4 (OR: 6, 95% CI: 2.1–17.5, P < 0.001), CD8 (OR: 0.26, 95% CI: 0.1–0.69, P = 0.008), CD56 (OR: 3.5, 95% CI: 1.2–9, P = 0.02), and CD20 (OR: 2.9, 95% CI: 1.2–7.3, P = 0.03). On multivariate analysis, low CD3 (adjusted OR: 177.59), CD4 (adjusted OR: 228.46), CD56 (adjusted OR: 24.35 and CD20 (adjusted OR: 132.60) were associated with poor PR independent of tumor stage or lymph nodal involvement [Supplementary Table 3].
Table 3: Univariate predictors of pathological response

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Post neo-adjuvant chemotherapy histopathology examination and tumor infiltrating lymphocytes profile

In post-NACT surgical specimens, the mean tumor size is 2.6 ± 0.79 cm. Post-NACT, the microscopic lymphovascular invasion was seen in fifty (62.5%) patients. NACT induced elastosis was found both macroscopically and microscopically in twenty-two (27.5%) of the study population [Table 4].
Table 4: Postneoadjuvant chemotherapy tumour characteristics and tumour infiltrating lymphocytes profile

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Most of the patients had post-NACT intermediate global TIL (n = 34, 42.5%) while high and low global TIL were observed in 14 (17.5%) and 32 (40%) patients only. NACT did not induce any change in global TIL profile.

On IHC mean TIL count of CD3, CD4, CD8, CD20, CD56 were 24.6 ± 8.52, 23.3 ± 7.8, 32.5 ± 9.04, and 24.4 ± 9.12, 23.7 ± 7.04, respectively.

Post NACT IHC revealed predominantly low CD3 (n = 55, 68.8%), low CD4 (n = 55, 68.8%), high CD8 (n = 48, 60%), low CD20 (n = 48, 60%), and low CD56 (n = 54, 67.5%).

NACT did not induce any change in post-NACT TIL subpopulation profile as compared to pre-NACT TIL profile, irrespective of the type of regimen used [Figure 1]a, [Figure 1]b, [Figure 1]c, [Figure 1]d, [Figure 1]e and [Supplementary Table 4], [Supplementary Table 5], [Supplementary Table 6], [Supplementary Table 7], [Supplementary Table 8], [Supplementary Table 9], [Supplementary Table 10], [Supplementary Table 11], [Supplementary Table 12], [Supplementary Table 13], [Supplementary Table 14], [Supplementary Table 15], [Supplementary Table 16], [Supplementary Table 17], [Supplementary Table 18].
Figure 1: Preneoadjuvant chemotherapy versus postneoadjuvant chemotherapy CD3 (a), CD4 (b), CD8 (c), CD20 (d), CD56 (e)

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 > Discussion Top


Targeting the immune system is an evolving paradigm in the management of different breast cancers. Current evidence suggests that global TIL counts and its change induced by NACT are predictors of PR and outcomes.[13],[14],[15],[16],[17],[18] However, the role of TIL subpopulations in the biological behavior of breast carcinoma and its response to NACT remains largely unexplored. Moreover, previous studies have included patients with breast carcinoma of different sub-types, resulting in heterogeneity.[8] It is well known that the various subtypes of breast carcinoma exhibit distinct biologic behavior and are associated with different responses to chemotherapy, outcomes, and mortality. TNBC is associated with high mortality but a paradoxical better response to NACT as compared to other subtypes. We thus looked at the profile of TIL subpopulations and its association with PR in a well-defined cohort of TNBC patients.

Neoadjuvant regimens used in the management of breast cancer include anthracyclines, taxanes, cyclophosphamide and platinum-based therapies. The standard of care in the neoadjuvant setting for TNBC is sequential anthracycline–taxane-based chemotherapy.[19],[20] The pathologic complete response rate of these regimens ranges from 28% to 36%.[21],[22],[23] Although, the addition of carboplatin to anthracycline and taxane has been shown to increase the pathologic complete response rate, this is still not recommended by current guidelines.[9],[24] Most of the patients (n = 62, 77.5%) in our study received only anthracycline-based regimen and a good PR was observed in 32.5% patients in our cohort which is consistent with the previous literature. Almost half of our patients had intermediate global TIL counts while high global TIL counts were seen in only 7 patients. Poor PR was significantly more common in patients with lower global TIL counts similar to previous studies.[13]

On multivariate analysis, low CD3, low CD4, low CD20, and low CD56 were independently associated with increased odds of poor PR. The highest OR for poor PR was observed with low CD4 (adjusted OR: 228.46) even after adjusting for tumor stage and lymph-nodal status. Although low CD8 was associated with significantly decreased odds of poor PR on univariate analysis (OR: 0.26), it was not found to be an independent predictor of PR on multivariate logistic regression. Overall, our observations are consistent with the findings of a study on 121 patients of breast carcinoma treated with NACT which reported that good PR was predicted by baseline high CD4, high CD20, and low CD8. Evidence also suggests that increased lymphocytic infiltration with CD4 + Th1 and Tfh cells is associated with improved survival and increased complete PR.[25] Increased CD8 + TIL and decreased CD4/CD8 percentage has been found to be significantly associated with stage progression.[26] Decreased tumoral and circulating CD4 counts have also been associated with worse outcomes in early and metastatic breast cancer.[27],[28]

A novel finding of our study was that low CD56 is independently associated with poor PR (adjusted OR: 24.35). While data on the expression of NK-cell ligands suggest a key role of NK cells in tumor biology and response to treatment, there is little direct evidence of the association between stromal NK-cell infiltration and clinically relevant outcomes.[26] A study on gene expression in the tumor stroma of 53 patients of breast cancer identified increased expression of NK-cell markers in clusters with good outcome (relapse-free survival and recurrence rate) and decreased expression of CXCL14 (chemokine involved in NK-cell chemotaxis) in clusters with poor outcomes.[29] Our study provides direct evidence of the association between low infiltration by NK-cells in the tumoral stroma and poor PR to NACT in a well-defined, homogeneous cohort of TNBC patients. This finding may also have important implications for NK-cell-based therapies and further, larger, exploratory studies are needed to validate the same.

The data on the change in TIL profile induced by NACT are limited and contradictory.[30],[31] In a study on 56 patients with breast cancer, Ladoire et al. reported no change in CD3 or CD8 following NACT.[31] In contradistinction, García-Martínez et al. demonstrated significant NACT-induced change in CD3, CD4 and CD20 but not CD8.[8] In our study, NACT did not induce any change in global TIL counts or TIL subpopulations as compared to baseline. These discrepancies may be related to the NACT regimen used as it is plausible that different chemotherapeutic agents and their scheduling may induce distinct immune changes. Almost 90% of the patients in the series by García-Martínez et al. received sequential anthracycline and taxane compared to 18 (22.5%) in our study and 20% patients in the study by Ladoire et al., respectively. Moreover, it should be noted that these previous studies included a heterogeneous cohort of different breast carcinoma subtypes with very few patients of TNBC. In fact, one of the biggest strengths of our study is that we looked at only TNBC patients. Given the different biological behavior of the various breast carcinoma subtypes, it is plausible that they may have distinct TIL subpopulation and varying degrees of immune-escape with different responses to NACT. Thus, the findings in one particular subtype should not be necessarily extrapolated to another subtype.

We acknowledge the limitations of our study. We used a semi-quantitative approach for the evaluation of global TIL and TIL subpopulations. Computer-based assessment of TIL may help in reducing interobserver bias.[6],[8],[32] In our study, all observations were carried out by two independent pathologists and there was a good interobserver agreement between them. Moreover, while the computer-based approach has numerous advantages, it should be mentioned that such approaches are not standardized or validated. Another limitation of our study is that the degree and type of immune cells infiltrating the tumor stroma is incompletely representative of the functional immune response in the tumor microenvironment. Comprehensive analysis of functional subpopulations and gene-expression studies would have added further granularity in this aspect.


 > Conclusions Top


TIL subpopulation CD3, CD4, CD8, CD20, and CD56 are independently associated with poor PR. Further studies are needed to validate our findings and explore its' implications in immunotherapy combined with NACT.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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