CD8 and Foxp3 T Cells in Breast Cancer (Yasa INWT, et al.
Indones Biomed J.
: 287-94
DOI: 10.
18585/inabj.
RESEARCH ARTICLE
Expression of CD8 and Foxp3 T Lymphocyte as Predictor for Response to Neoadjuvant Chemotherapy in Stage i Breast Cancer I Nyoman Wawan Tirtha Yasa1,E.
I Wayan Sudarsa2.
I Wayan Niryana3.
Ni Putu Sriwidyani4.
I Made Jawi5.
I Wayan Putu Sutirta Yasa6.
Ida Bagus Tjakra Wibawa Manuaba2.
Ida Bagus Made Suryawisesa2 Doctoral Study Program.
Faculty of Medicine.
Universitas Udayana.
Jl.
Sudirman.
Denpasar 80232.
Indonesia Division Surgical Oncology.
Departement of Surgery.
Faculty of Medicine.
Universitas Udayana.
Jl.
Sudirman.
Denpasar 80232.
Indonesia Department of Neurosurgery.
Faculty of Medicine.
Universitas Udayana.
Jl.
Sudirman.
Denpasar 80232.
Indonesia Department ofAnatomical Pathology.
Faculty of Medicine.
Universitas Udayana.
Jl.
Sudirman.
Denpasar 80232.
Indonesia Department of Pharmacology.
Faculty of Medicine.
Universitas Udayana.
Jl.
Sudirman.
Denpasar 80232.
Indonesia Department of Clinical Pathology.
Faculty of Medicine.
Universitas Udayana.
Jl.
Sudirman.
Denpasar 80232.
Indonesia *Corresponding author.
Email: nyomanwawan@yahoo.
Received date: May 6, 2025.
Revised date: June 8, 2025.
Accepted date: June 10, 2025 Abstract ACKGROUND: Neoadjuvant chemotherapy (NAC) in breast cancer is usually utilized to eradicate micro-metastasis, induce apoptosis in tumor cells, and reduce the primary tumor size, enabling surgical intervention.
Recent studies have shown that tumor-infiltrating lymphocytes (TIL.
, especially cytotoxic CD8 T cells and immunosuppressive Foxp3 regulatory T cells, influence tumor response to treatment.
However, their role as predictive markers for NAC response remains unclear.
Therefore, this study was performed to investigate whether high expression of CD8 and low expression of Foxp3 T lymphocytes are associated with better response to NAC in stage i breast cancer patients.
METHODS: Total of 60 biopsy samples from stage i breast cancer patients were included, comprising 30 subjects in the response group ( ) and 30 subjects in the non-response group (O.
The expression levels of CD8 and Foxp3 T lymphocytes in tumor tissue were assessed semi-quantitatively by immunohistochemistry (IHC), using a scoring system based on the proportion and intensity of positively stained cells (BlackAos grading criteri.
RESULTS: Stage i breast cancer with high expression of CD8 T lymphocytes was significantly associated with a better response to NAC .
=0.
OR=6.
Meanwhile, low expression of Foxp3 T lymphocytes was not significantly associated with chemotherapy response .
=0.
OR=3.
A higher tumor grade was also associated with an improved response to The probability of achieving a positive response to NAC in subjects presenting with high CD8 expression, low Foxp3 expression, and high tumor grade was estimated at 96.
CONCLUSION: The combination of high expression of CD8 T lymphocyte, low expression of Foxp3 T lymphocyte and high tumor grade might be useful to predict good response to NAC in stage i breast cancer.
KEYWORDS: CD8 T lymphocyte.
Foxp3 T lymphocyte, neoadjuvant chemotherapy, breast cancer Indones Biomed J.
: 287-94
Introduction Breast carcinoma is the most commonly diagnosed cancer in women worldwide and ranks as the second leading cause of cancer-related deaths globally.
In Indonesia, breast carcinoma is the second most prevalent malignancy among women after cervical cancer.
In Bali, over 70% of breast cancer patients treated at Sanglah General Hospital present with advanced disease .
tage i and IV).
Copyright A 2025 The Prodia Education and Research Institute.
This work is licensed under a Creative Commons Attribution-NonCommercial 4.
0 International (CC-BY-NC) License.
The Indonesian Biomedical Journal.
Vol.
No.
June 2025, p.
Print ISSN: 2085-3297.
Online ISSN: 2355-9179 Patients with stage i breast cancer, categorized as locally advanced breast cancer (LABC), typically have large tumors and/or extensive regional lymph node involvement without distant metastasis.
These patients are commonly treated with combination neoadjuvant chemotherapy (NAC) to reduce tumor size and enable breast-conserving surgery while targeting micrometastases early.
But response rates to NAC remain variable and difficult to predict.
While some patients achieve substantial tumor shrinkage or pathological complete response, others show minimal or no response.
This variability underscores the need for predictive biomarkers for chemotherapy responsiveness.
The immune system is increasingly recognized as playing a key role in modulating tumor behavior and treatment outcomes in breast carcinoma.
Tumorinfiltrating lymphocytes (TIL.
, particularly cytotoxic CD8 T cells and regulatory Foxp3 T cells, have been associated with prognosis and therapeutic responses.
Ae.
CD8 T cells mediate anti-tumor immunity by directly killing tumor cells, and their presence has been correlated with improved .
Ae.
Conversely.
Foxp3 regulatory T cells (Treg.
suppress the immune response, promote immune evasion, and have been linked with poor prognosis when present in high numbers.
Ae.
Recent evidence suggests that chemotherapy can alter the immune microenvironment by modulating the balance of effector and suppressor T cells.
Anthracyclinebased chemotherapy, for instance, has been shown to reduce Foxp3 Treg levels and enhance cytotoxic T cell function in some breast cancer subtypes.
Despite these findings, the predictive role of CD8 and Foxp3 expression specifically in stage i breast cancer patients receiving NAC has not been fully elucidated.
Most previous studies have either evaluated overall TIL density or did not focus on this specific clinical stage and therapeutic setting.
Therefore, this study was performed to investigate the expression levels of CD8 and Foxp3 tumor-infiltrating lymphocytes as risk factors associated with the response to combination NAC in patients with stage i breast carcinoma.
The results might help identify potential immunological markers that can predict therapeutic outcomes and support personalized treatment strategies.
had received combination NAC Cyclophosphamide.
Adriamycin/Epirubicin and 5 Fluorouracil (CAF/CEF), with complete medical records and meeting the specified inclusion and exclusion criteria.
This study was conducted in the Surgical Oncology Division and Surgery Laboratory at the Faculty of Medicine.
Universitas Udayana/Sanglah Hospital, and the protocol of this study was approved by the Research Ethics Committee of the Faculty of Medicine.
Universitas Udayana (Approval No.
482/Skrt/XII/2.
Methods Study Design This was a nested case-control design study, involving samples of stage i breast carcinoma patients who Sample Recruitment The study population included all stage i breast carcinoma patients treated at Sanglah Hospital.
Denpasar, confirmed by histopathological examination and who had received 3 cycles of CAF/CEF NAC.
The inclusion criteria for this study consisted of patients diagnosed with stage i breast carcinoma who had undergone three cycles of combination NAC using either the CAF (Cyclophosphamide.
Adriamycin, and 5-Fluorouraci.
or CEF (Cyclophosphamide.
Epirubicin, and 5-Fluorouraci.
regimens, administered at three-week intervals.
The selection between CAF and CEF regimens was based on the oncologistAos clinical judgment, taking into account the subject's cardiac function, tolerance to anthracyclines, comorbidities, and drug availability at the time of treatment.
In cases where there was a delay in chemotherapy administration, the maximum allowable interval between cycles was four weeks.
Subjects were excluded from the study if they had previously received any form of treatment, including surgery, chemotherapy, radiotherapy, or hormonal therapy.
Additionally, those with incomplete medical records based on the evaluated variables or paraffin blocks that were unsuitable for IHC examination were also excluded.
There were final of 60 samples, selected through consecutive purposive sampling, comprising 30 subjects in the response group ( ) and 30 subjects in the non-response group (-).
SubjectAos Medical Record Collection The list of breast carcinoma subjects was obtained from the Cancer Patient Registration Data at the Surgical Oncology Division.
Faculty of Medicine.
Universitas Udayana/ Sanglah Hospital.
Denpasar.
Additional data were collected from subjectAos medical records.
Selected samples were those with histopathological diagnosis of infiltrating ductal carcinoma (IDC) and clinically categorized as stage i/LABC.
Collected data included demographic information, clinical data, pathology data, chemotherapy administration records, and chemotherapy response data.
All subjects received 3 cycles DOI: 10.
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of CAF/CEF combination NAC at 3-week intervals.
Tumor response was assessed every 2Ae3 weeks after chemotherapy by measuring tumor diameter.
Subjects were categorized into the case group .
ositive respons.
and the control group .
egative respons.
Paraffin blocks from biopsy specimens with good quality were collected from Department of Anatomical Pathology.
Faculty of Medicine.
Universitas Udayana/Sanglah Hospital for the immunohistochemical (IHC) examination of CD8 and Foxp3 .
Tumor grade was assessed by histopathological examination of biopsy or surgical tissue stained with hematoxylin and eosin (H&E).
The grading followed the modified Bloom-Richardson system, which evaluates three components: tubule formation, nuclear pleomorphism, and mitotic count.
Each component is scored from 1 to 3, and the total score classifies tumors into three grades.
Low grade (Grade .
includes well-differentiated tumors with a total score of 3 to 5, intermediate grade (Grade .
includes moderately differentiated tumors with scores of 6 to 7, and high grade (Grade .
consists of poorly differentiated tumors with scores of 8 to 9.
For the purpose of analysis in this study, tumors were grouped into two categories: low grade, combining Grade 1 and Grade 2, and high grade, which corresponds to Grade 3.
High-grade tumors generally exhibit more aggressive behavior, higher mitotic activity, and poorer differentiation compared to lowgrade tumors.
IHC Examination for CD8 and Foxp3 IHC staining for CD8 and Foxp3 was performed following the standard protocol at the Anatomical Pathology Laboratory of Sanglah Hospital.
Monoclonal antibodies used were CD8 (Clone RM9116-SO LV.
Thermo Vision/ LabVision.
Fremont.
CA) and Foxp3 (Flex RTU.
Can.
No.
GA0263-U.
DAKO.
Carpinteria.
CA.
USA), with the DAKO IHC-Paraffin Protocol.
After dewaxing and antigen retrieval using DAKO retrieval solution with microwave heating, slides were incubated overnight at room temperature with primary antibodies diluted 1:20 for CD8 and 1:100 for Foxp3 in 3% Normal Swine Serum.
Detection was completed using a biotinylated secondary antibody, streptavidin, and DAB chromogen for visualization, followed by MayerAos hematoxylin counterstaining.
Tonsil tissue served as the positive control.
CD8 expression was identified on the cell membrane and cytoplasm, while Foxp3 expression was localized in the nuclei.
Stained slides were examined under 400x magnification, and expression was quantified by averaging counts from ten high-power fields.
CD8 and Foxp3 T Cells in Breast Cancer (Yasa INWT, et al.
Indones Biomed J.
: 287-94
Data Analysis The association between independent variables (CD8 and Foxp3 ) and the dependent variable .
esponse to NAC) was tested using the Chi-square test.
If the assumptions for the Chi-square test were not satisfied.
FisherAos exact test was used as an alternative.
The association's strength was measured using the Odds Ratio (OR) along with a 95% Confidence Interval (CI).
A p-value of less than 0.
05 was considered statistically significant.
Potential confounding variables such as age, menopausal status, tumor grade.
Her2neu status, and TILs were controlled by multivariate analysis using logistic regression.
Results Subject Characteristics The histopathological grade examination revealed that there were 4 subjects .
7%) with grade I, 35 subjects .
with grade II, and 21 subjects .
0%) with grade i Meanwhile, based on the clinical staging, 11 subjects .
4%) were at stage iA, 44 subjects .
at stage iB, and 5 subjects .
3%) at stage iC (Table .
In terms of the TIL status, 46 subjects .
7%) were TIL-positive, while 14 subjects .
3%) were TIL-negative.
HER2/neu testing showed 52 .
7%) subjects were negative and 8 .
3%) subjects were positive.
Estrogen receptor (ER) and progesterone receptor (PR) testing was only performed on 9 subjects, resulting in 5 positives and 4 negatives (Table .
Lymphovascular invasion (LVI) and mitotic activity index (MAI) could not be fully evaluated due to incomplete histopathological reports.
The most common histopathological type was infiltrating ductal carcinoma (IDC) found in 58 subjects .
7%), with 2 subjects .
diagnosed with mucinous adenocarcinoma.
CD8 .
Foxp3 , and NAC Response IHC examination of biopsy tissue showed 16 subjects .
7%) had high CD8 expression and 44 subjects .
had low CD8 expression.
For Foxp3 , 46 subjects .
had low expression, while 14 subjects .
3%) had high After receiving three cycles of combination NAC (CAF/CEF), 30 subjects .
%) showed a positive response, and 30 subjects .
%) showed no response (Table .
Among the 30 subjects who responded to NAC, 28 subjects .
7%) experienced a partial response, and 2 subjects .
3%) achieved a clinical complete response.
No cases of pathological complete response were observed.
In the non- The Indonesian Biomedical Journal.
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Online ISSN: 2355-9179 Table 1.
Subject characteristics.
Variable Mean Age .
, meanASD Positive Response (%) Negative Response (%) p -value 33A10.
70A7.
Menstrual Status, n (%) Premenopause Menopause Clinical Stage, n (%) iA 6 .
%) 5 .
iC HER2/neu Status, n (%) 3 .
%) 2 .
Positive Negative TIL Status, n (%) 4 .
Positive 24 .
%) 22 .
%) Negative Tumor Grade, n (%) 6 .
%) 8 .
%) Low Grade High Grade 16 .
responsive group, 29 subjects .
3%) had stable disease, and 1 subject .
7%) experienced disease progression, with distant metastasis to the lungs.
Representative IHC results of CD8 and Foxp3 lymphocutes expression were shown in Figure 1 and Figure 2, respectively.
Association Between CD8 and NAC Response There was a significant association between high CD8 expression and a positive response to NAC .
=0.
Subjects with high CD8 expression were 6.
88 times more likely to respond to NAC compared to those with low CD8 expression (Table .
High CD8 expression acts as a predictive factor for better NAC response.
Association Between Foxp3 and NAC Response Although breast cancer patients with low Foxp3 expression 25 times more likely to respond to NAC compared to those with high Foxp3 , the association was not statistically significant .
=0.
(Table .
Low Foxp3 expression showed a trend toward better response but was not a significant predictor.
Association Between NAC Response and Other Variables Multivariate logistic regression analysis showed that high CD8 expression, low Foxp3 expression, and high tumor grade were significantly associated with a positive response to NAC .
<0.
(Table .
Other factors such as age, menstrual status.
TIL status, clinical stage, and HER2/ neu status were not significant predictive factors .
ata not Discussion In this study, most of the subjects with stage i breast carcinoma were 50 years old or younger, with many under The majority were also premenopausal.
This is different from developed countries, where breast cancer tends to occur in older women, with most patients being over 50 and many over 65.
Early-onset breast cancer is often linked to internal factors, especially when diagnosis is delayed and the cancer is already in an advanced stage, leading to a poorer prognosis.
Risk factors for early-onset breast cancer include family history, early menarche, radiation exposure, and oral contraceptive use.
Breast cancer in young women is usually more aggressive, estrogen receptornegative, poorly differentiated, and shows high p53 and Ki67 expression.
All subjects in this study were married, and many had completed high school or higher education.
However.
Table 2.
IHC results for CD8 .
Foxp3 , and NAC Variable High CD8 Low CD8 High Foxp3 Low Foxp3 Positive NAC Response Negative NAC Response Frequency Proportion (%) DOI: 10.
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Figure 1.
CD8A T lymphocytes detected on the cell membrane of infiltrating immune cells in tumor tissue.
Positive CD8A expression appears as brown membrane staining, indicating localization on the cell surface.
Immunohistochemical staining was performed using the CD8 RM9116-SO LV monoclonal antibody.
White bar = 10 AAm.
this did not ensure early medical consultation or treatment Most subjects first tried alternative treatments, which delayed proper diagnosis and treatment.
Lack of health insurance also contributed to these delays.
Health education and awareness about breast cancer need to be improved to promote earlier detection.
High-grade tumors are known to have active cell growth and good blood supply, even if there is tissue These findings support previous studies.
Tumor grade is an important prognostic factor.
Higher-grade tumors grow more aggressively and are more likely to recur than lower-grade tumors.
Some types of breast cancer, such as tubular, mucinous, and medullary carcinoma, are linked to better outcomes.
Most the subjects in this study were Her2/neu-negative, and Her2/neu overexpression was rare and not statistically Some of those with overexpression responded better to CAF than Cyclophosphamide.
Methotrexate, and Figure 2.
Foxp3A regulatory T cells identified by nuclear localization in tumor-infiltrating lymphocytes.
Positive Foxp3A expression is indicated by brown staining confined to the cell Immunohistochemical staining was performed using the DAKO monoclonal antibody for Foxp3.
White bar = 10 AAm.
CD8 and Foxp3 T Cells in Breast Cancer (Yasa INWT, et al.
Indones Biomed J.
: 287-94
Fluorouracil (CMF) chemotherapy, showing improved .
However.
Her2/neu overexpression is also connected with more aggressive disease, especially in patients with lymph node involvement.
Several factors such as age, menstrual status, tumor type, and tumor grade are believed to influence the response to NAC (NAC).
However, most studies, including this one, found no significant relationship between these factors and treatment response.
Ae.
These are considered nonregimen-dependent factors.
Although most patients showed positive TILs, they did not significantly affect the NAC response.
TILs include various immune cells such as CD4 .
CD8 , and Foxp3 .
The ratio between CD8 and Foxp3 might be more meaningful.
A higher number of Foxp3 cells can suppress immune responses and help tumors avoid detection by the immune .
The role of TILs in cancer prognosis has been studied in ovarian, colorectal, and lung cancer.
The immune system is believed to support the effectiveness of NAC, especially when using agents like doxorubicin and .
After NAC, an increase in CD8 cells has been seen in patients with complete clinical response.
NAC
may stimulate the immune system by reducing Foxp3 cells and enhancing CD8 activity.
In this study.
CD8 positivity was common.
A portion of patients had high CD8 expression, which was associated with a good response to NAC.
CD8 cells attack tumor cells, and NAC can enhance their effect.
This agrees with other research showing better outcomes when CD8 levels are high.
Chemotherapy and radiotherapy have been shown to lower Foxp3 levels, while CD8 levels remain unchanged.
These treatments also increase immune-stimulating cytokines like interferon (IFN)-, interleukin (IL)-4, and IL-2, which are likely triggered by cyclophosphamide.
High CD8 levels were also associated with cancer spread to lymph nodes.
Some patients with high CD8 later had recurrence or metastasis, suggesting that immune response is related to tumor behavior.
CD8 could be useful for predicting prognosis and lymph node involvement.
Foxp3 was found in several patients, but high levels were less common.
Low Foxp3 expression may suggest weaker immune suppression, although it was not statistically significant.
Still, patients with lower Foxp3 were more likely to respond to NAC (OR=3.
Foxp3 can reduce CD8 activity.
Therefore, patients with lower Foxp3 may have better immune responses against tumors.
NAC reduces Foxp3 and boosts CD8 function, which may help explain the better outcomes in patients with low Foxp3 .
The Indonesian Biomedical Journal.
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June 2025, p.
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Online ISSN: 2355-9179 Table 3.
Association of CD8 expression with NAC Table 4.
Association of Foxp3 expression with NAC CD8 Status Positive Response Negative Response Total Foxp3 Status Positive Response High CD8 13 .
Low Foxp3 26 .
High Foxp3 4 .
Total Low CD8 Total Chi-square (XA) = 8.
p-value = 0.
Odds Ratio (OR) = 6.
95% CI = 1.
707Ae27.
Negative Response Chi-square (XA) = 3.
p-value = 0.
Odds Ratio (OR) = 3.
95% CI = 0.
888Ae11.
Patients with low Foxp3 before and after NAC had better recurrence-free survival than those with high levels.
This means Foxp3 could be used to predict treatment response.
In patients with a complete pathological response.
Foxp3 decreased and CD8 increased after NAC.
Similar findings were seen in ovarian cancer, where NAC reduced Foxp3 and increased CD8 cells due to immune reactions triggered by dying tumor cells.
In this study, logistic regression analysis results showed that patients with a combination of high CD8 , low Foxp3 , and high-grade tumors had a significantly better response to CAF or CEF chemotherapy.
This combination of markers can help predict which patients are likely to benefit from NAC.
In this study, the tumor response was measured using calipers, which only provide two-dimensional data.
Ideally.
MRI should be used for more accurate measurement.
The Foxp3 IHC test was done for the first time and required several antibody dilutions to get clear results.
Further research is recommended to validate the predictive value of CD8 and Foxp3 expression for response to NAC in a larger, multi-center cohort using more advanced and quantitative immunological assays.
Future studies should consider incorporating flow cytometry or real-time PCR for more precise measurement of immune markers.
Additionally, longitudinal studies examining changes in CD8 and Foxp3 expression before and after NAC may help clarify their dynamic roles in treatment response.
Advanced imaging modalities such as MRI should be utilized for more accurate assessment of tumor response.
Given the observed immune involvement, it would also be beneficial to explore the potential synergistic effects of combining NAC with immunotherapeutic agents, particularly in patients with high CD8 and low Foxp3 profiles.
Conclusion The results of this study showed that high CD8 T cell infiltration and high histopathological grade were significantly associated with a favorable response to NAC in stage i breast carcinoma.
Although low Foxp3 expression showed a trend toward better response, the association was not statistically significant.
The combination of high CD8 , low Foxp3 , and high-grade tumors may serve as a useful predictive marker for treatment response.
These findings suggest potential biomarkers for optimizing neoadjuvant therapy strategies.
Acknowledgments The authors would like to thank the Faculty of Medicine.
Udayana University, and Sanglah General Hospital.
Denpasar, for their support and facilities.
We are also grateful to the staff of the Oncology Surgery Division and the Department of Anatomical Pathology for their assistance, and to all patients who participated in this study.
Table 5.
Logistic regression analysis for factors associated with NAC response.
Variable Wald Sig.
Exp(B) 95% CI for Exp(B) 527Ae83.
Low Foxp3 202Ae44.
High Grade 267Ae17.
Constant High CD8 Total DOI: 10.
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Authors Contribution INWTY and IWS contributed to the conceptualization and planning of the research.
IWN and NPS conducted data acquisition and sample collection.
IMJ and IWPSY performed data calculations and statistical analysis.
INWTY drafted the manuscript.
IBTWM contributed to data interpretation and critical revision.
technical assistance and critical input.
All authors read and approved the final manuscript.
References