Effects of Neo-Adjuvant Chemotherapy on CD8 + Serum Levels in Local Advanced Stage Breast Cancer Patients

Introduction.CD8 + CTLs are important components of tumor specific cellular adaptive immunity in breast cancer. Chemotherapy can increase the response of cytotoxic lymphocytes and provide anti-tumor immunity. Therefore, chemotherapy can cause cell death and trigger tumor antigens that are processed by APC and activate CD8 + cells to destroy tumor cells. Evaluation of the immune system before treatment can predict chemotherapy response. The purpose of this study was to assess the effect of neo-adjuvant chemotherapy on serum CD8 + levels in patients with locally advanced breast cancer. Methods.Non-comparative clinical trials were conducted at Dr. Mohammad Hoesin Hospital Palembang from October 2017 to June 2018.The sample of this study was 30 breast cancer patients who met the inclusion criteria. All samples underwent FAC neo-adjuvant chemotherapy and data were analyzed with SPSS version 21. Results.The mean age of advanced breast cancer patients was 45.97 ± 10,526 years with an age range of 30-66 years. Serum CD8 + levels after chemotherapy decreased significantly compared


Introduction
Breast cancer is the most common malignancy in women. In 2012 according to the International Agency for Research on Cancer (IARC) breast cancer was still a disease that ranked number one in the world as a new case, which was 43.3% and most often caused death, amounting to 12.9%. 1 In 2010, the incidence rate breast cancer in Indonesia 12 per 100,000 women, while in the USA about 92 per 100,000 women. 2 In Indonesia, more than 80% of cases are found to be at an advanced stage. 2 Locally advanced breast cancer (LABC) is still the largest part (50-60%) of cancer patients who come to polyclinics or hospitals in Indonesia. 3 According to Yudistira (2014) the distribution of the most clinical stadiums at Mohammad Hoesin Hospital Palembang is stage III or LABC, namely stage III A 26.53% and stage IIIB 48.98%. 4 Neo-adjuvant chemotherapy is a therapeutic choice in patients with locally advanced breast cancer. The use of this therapy is increasing in patients with inoperable breast cancer who are not candidates for breast conservation surgery or have been shown to have lymph node metastases.
Patients who show a complete pathology response to neo-adjuvant chemotherapy have a long-term disease-free survival. 5 This aims to reduce the tumor mass, so that surgery can be performed and continued adjuvant therapy. 6 Neo-adjuvant therapy is given in 3 cycles. 7.8 The first line used, namely anthracycline based chemotherapy. [9][10] The administration of neo-adjuvant chemotherapy reduces primary tumor mass, reduces lymphatic metastases in axilla and eradication of micrometastases, so that the operability of breast cancer changes. [11][12] The response of neo-adjuvant chemotherapy can be assessed by comparing mass sizes before and after chemotherapy. The clinical response criteria used are RECIST, which is complete, partial, stable and progressive. 13.
The immune system plays an important role in the development of cancer, especially breast cancer.
Response Adaptive and innate immunity plays an important role in tumor immune-surveillance and can limit the development and growth of neoplasms. Chemotherapy can trigger an immune response which contributes to the therapeutic response 14-16. In its development, tumor infiltrative lymphocytes (TILs) are important predictive factors for the chemotherapy response in breast cancer patients. 17 Cells from innate immune systems (neutrophils, monocytes, macrophages, and host APCs) and adaptive (B and T lymphocytes) work together to respond to various pathogens, including tumor antigens. Innate immunity cells are needed by B and T cells to identify immunogenic proteins, then allow adaptive immunity to form memory cells (lymphocytes that remain in the lymph nodes). Most antigens in breast cancer are self-proteins that can stimulate T cells and induce the regulator's immune response. T cell infiltration includes T Helper (CD4 +) and cytotoxic (CD8 +) where CD4 + T-helper facilitates antigen presentation through cytokine secretion and activation of presenting cell antigens (APC), while CD8 + cytotoxic T-cells play a role in tumor destruction. [17][18][19] In breast cancer, CD8 + cytotoxic T cell infiltration is closely related to the patient's long-term survival and good response to chemotherapy. Mao et al reported that CD8 + lymphocytes are the main cells that are effective in the immune response, which shows better disease-free survival in breast cancer patients. 20 Al Saleh et al in his study reported that high CD8 + expression could significantly predict pathologic complete responses after neo-adjuvant chemotherapy and illustrate an independent prognostic factor for Overall Survival (OS). 21 According to Seo et al, CD8 + cytotoxic T lymphocytes are important components of TILs that are associated with the chemotherapy response and can be used as predictors of response to anthracycline or anthracycline / taxane based on breast cancer. 22

Methods
This type of research is a clinical trial without comparison by looking at the level of CD8 + serum in patients with locally advanced breast cancer before and after neo-adjuvant chemotherapy.
The research was carried out in the oncology surgery clinic and inpatient installation of the Dr.

General Central Hospital Mohammad Hoesin Palembang. Research time starts from October 2017
to June 2018. The study population was local advanced-stage breast cancer patients who were going to get neo-adjuvant chemotherapy at the Central General Hospital Dr. Muhammad Hoesin Palembang. All local advanced breast cancer sufferers who meet the inclusion and exclusion criteria. The method of sampling is done by consecutive sampling, where all subjects who come and meet the sample selection criteria are included in the study until the required number of subjects is met. All patients provided informed consent before participating in the study sample. The parameter of the success of this study was a significant increase in CD8 + levels after neoadjuvant chemotherapy. Data in this study will be presented in tabular form and analyzed using paired T test to determine effectiveness with SPSS version 21.

Results
General characteristics of research subjects are shown in Table 5. Based on age, the average age of advanced breast cancer patients is 45.97 ± 10.526 years with an age range of 30-66 years, where there are 15 patients (50%) and ages (40%) < 40 years as many as 15 people (50%). The majority of patients with locally advanced breast cancer have given birth (96.7%) and only 1 (3.3%) has never been. Patients with locally advanced breast cancer with a history of family planning were found as many as 17 people (56.7%) and the rest (43.3%) did not have a family history of family planning. A total of 13 people (43.3%) patients with advanced breast cancer have a family history of the same disease.  Before conducting a statistical analysis, the Saphiro Wilk normality test is first performed.
Obtained variable probability value CD8 + before < 0.05 so that the statistical test used is the Wilcoxon test. From the statistical analysis it was found that there were differences in serum CD8 + levels before and after chemotherapy (p = 0,000) where the value of serum CD8 + after chemotherapy dropped significantly compared to before chemotherapy.
AUC value of the ROC curve CD8 + value can be seen below. AUC value is getting closer to 1, the better.  The receiver operating curve (ROC) curve was analyzed to find the cut-off point to obtain the sensitivity and specificity of serum CD8 + levels before chemotherapy. Determination of the cut-off value for serum CD8 + levels before chemotherapy, is done by making a curve between sensitivity, specificity and the value of serum CD8 + levels before chemotherapy for locally advanced breast cancer patients.  Based on table 6. The value of serum CD8 + before chemotherapy has a sensitivity of 42.86% and specificity of 43.48%.
The receiver operating curve (ROC) curve was analyzed to find the cut-off point to obtain the sensitivity and specificity of serum CD8 + levels after chemotherapy. Determination of the cut-off value for CD8 + serum levels after chemotherapy, is done by making a curve between sensitivity, specificity and the value of CD8 + serum levels after chemotherapy for locally advanced breast cancer patients.   Based on table 7. The value of serum CD8 + levels after chemotherapy has a sensitivity of 71.43% and specificity of 69.57%.

Discussion
The incidence of breast cancer increases with age, every ten years, the risk of cancer Immunohistochemistry is used not only for prognosis but also determines the therapy given to breast cancer sufferers.40 Chemotherapy is a cancer treatment with anti-neoplasm cytotoxic drugs.
Chemotherapy facilitates the antitumor immune response by reducing tumor burden and modifying the tumor microenvironment to enable a more effective immune response. Neo-adjuvant chemotherapy is chemotherapy that precedes primary therapy or primary therapy. Neo-adjuvant chemotherapy has been shown to reduce the size of the primary tumor and kill lymph node micrometastases. With neo-adjuvant chemotherapy, tumor respectability is expected to be better. 24. In this study we found patients with poor chemotherapy response of 23.3%.
In this study immunity was assessed as a serum CD8 + level wherein the serum CD8 + level values obtained after neo-adjuvant chemotherapy dropped significantly compared to before chemotherapy. From the statistical analysis it was found that there were differences in serum CD8 + levels before and after chemotherapy (p = 0,000 factor for pCR in breast cancer patients treated with antracycline / taxane based. This is different from this study where CD8 + which was examined through blood did not show an increase after chemotherapy. 22. In this study it was found that serum CD8 + levels before chemotherapy had a sensitivity of 42.86% and specificity of 43.48%. This means that the ability of serum CD8 + levels before therapy to detect a good therapeutic response is 42.86% while the ability of serum CD8 + levels before therapy to detect poor response to therapy is 43.48%. Therefore, serum CD8 + levels cannot be used as predictors for determining chemotherapy responses in breast cancer such as CD8 + in tumor tissue.

Conclusion
There was a significant decrease in serum CD8 + levels after chemotherapy compared to before chemotherapy. The value of serum CD8 + before chemotherapy has a sensitivity of 42.86% and specificity of 43.48% with a cut point of 660.7 cells/mm 3 The value of serum CD8 + after chemotherapy has a sensitivity of 71.43% and a specificity of 69.57% with a cut point of 280.2 cells/mm 3 .