Accuracy of Neutrophil/Lymphocyte Ratio as Predictor of Neoadjuvant Chemotherapy Response in Locally Advanced Breast Cancer at

Introduction: Breast cancer is one of the most common neoplasm occurs in women. Neoadjuvant Chemotherapy (NACT) is widely used in Locally Advanced Breast Cancer (LABC) and Inflammatory Breast Cancer (IBC), to reduce tumor size (down-sizing), and further allowing breast-conserving surgery to be performed. Accurate markers are needed to prevent impractical chemotherapy that allows patients to receive definitive surgery in a timely manner. Previous study showed neutrophil/lymphocyte ratio (NLR) was superior in predicting long-term outcomes over PLR. This study aims to evaluate the relationship between the NLR value and neoadjuvant chemotherapy response in patients with locally advanced breast cancer at Dr. Mohammad Hoesin General Hospital Palembang. Method: This study is a prognostic test of the NLR accuracy to predict the response of neoadjuvant chemotherapy in patients with locally advanced breast cancer. The study included 113 patients as study subjects. Results: The obtained NLR cut off point value was ≤ 2.92 with an area under the ROC curve (AUC) of 92.2%. The diagnostic measures of the NLR prognostic test had sensitivity of 93%, specificity of 93%, Positive Predictive Value of 97%, Negative Predictive Value of 82%, and accuracy of 93%. There is a significant relationship between NLR and chemotherapy response in patients with locally advanced breast cancer (p = 0.000). Conclusion: There is a significant relationship between NLTR and chemotherapy response. NLR can be used as a predictor of neoadjuvant chemotherapy response with a good degree of accuracy.


Introduction
Breast cancer is the most common neoplasm in women and is the second leading cause of cancer death in women in industrialized countries. 1

Neoadjuvant Chemotherapy (NACT) is widely used in Locally
Advanced Breast Cancer (LABC) and Inflammatory Breast Cancer (IBC) to reduce the size of the tumor (down-sizing), and further allowing breast-conserving surgery to be performed. It has been suggested that the pathologic complete response (pCR) after NACT is thought to replace disease free survival (DFS) and overall survival (OS) markers, especially in patients with more aggressive subtypes, such as triple-negative or HER2-positive breast cancer. 1 Locoregional control (LRC), recurrence free survival (RFS), and overall survival (OS) rate were 99, 98, and 100% at the first year and 89, 69 and 77% at the fifth year, respectively.
Response to NACT was not correlated with LRC (p> 0.05) but was correlated with RFS and OS (p <0.05). 2 Apart from molecular subtypes, there are no other biomarkers that have been validated as predictive factor for pCR or prognostic factor for DFS and/or OS after NACT, including Ki67 and Residual Cancer Burden. Recently, the immune system has been shown to play an important role in breast cancer response to chemotherapy. Although breast cancer does not produce high neoantigen, breast cancer cells are often infiltrated by lymphocytes (tumor-infiltrating lymphocytes, TIL), which may vary widely across molecular subtypes. The presence of TIL in breast cancer is highly correlated with pCR after NACT was performed.
Different tumor infiltration cell subtypes, such as T-regulatory lymphocytes (Treg) and myeloid deriversuppressor cells (MDSC), lead to immune system suppression, and have been shown to reduce the success of NACT in patients with breast cancer. Consistently, breast cancer tends to respond to immune checkpoint inhibitors (ICI), although the success rate is lower compares to other neoplasms such as melanoma, kidney and lung cancers. 1 Some studies suggest that the use of markers, such as gene mutations, associated with response to neoadjuvant chemotherapy treatment is still difficult to assess, and no clinical biomarkers have been shown to generally predict cancer response after NACT to date. 1,3 Therefore, accurate markers are needed to prevent impractical chemotherapy that enables patients to receive definitive surgery in a timely manner.
The chronic systemic inflammatory response is continuously studied in terms of breast cancer progression and prognosis. Parameters using peripheral blood test have been investigated as factors that reflect the immune response. Circulating white blood cells, which result in changes in the proportion of neutrophils, lymphocytes, and monocytes, are associated with systemic inflammatory response. The relationship between cancer prognosis and absolute monocyte count (AMC), absolute lymphocyte count (ALC), lymphocyte-to-monocyte ratio (LMR), neutrophil-to-lymphocyte ratio (NLR), and platelet-tolymphocyte ratio (PLR) has been studied in various types of cancer. 4 In patients with breast cancer, Ni et al. reported that increased LMR was a beneficial prognostic factor after neoadjuvant chemotherapy (NACT). 5 In addition, Azab et al. reported that NLR was superior in predicting long-term outcomes over PLR. 6 In this study, we aimed to evaluate whether the neutrophil lymphocyte ratio (NLR) could predict the outcome of chemotherapy response in breast cancer patients with peripheral blood parameters. Additionally, we aimed to estimate the value of immune-related predictor markers in response to NACT.

Methods
This study is a prognostic test of the accuracy of the NLR to predict the response of neo-adjuvant chemotherapy in patients with locally advanced breast cancer.
The subjects of this study were patients with locally advanced breast cancer who received neoadjuvant chemotherapy at Dr. Muhammad Hoesin Palembang General Hospital who met the inclusion criteria.
The inclusion criteria were patients with locally advanced breast cancer who were diagnosed based on the criteria of the American Joint Committee of Cancer and received adjuvant chemotherapy with Karnofsky index ≥ 50. The exclusion criteria were patients who had undergone previous chemotherapy and were immunocompromised.
There were 113 subjects in this study. The independent variables were the immunohistochemistry subtypes and the neutrophil/lymphocyte ratio. The dependent variable was the adjuvant chemotherapy response. The sociodemographic characteristics were age, age at menarche, history of hormonal contraception usage, age at first pregnancy, family history of breast cancer.
The data are presented in distribution and narrative tables. The receiver operating characteristics (ROC) curve statistical test was performed using the MedCalc 2000 software to determine the NLR cut off point value in predicting chemotherapy response. Chi square test was used to evaluate the sociodemographic relationship to the NLR value, and to evaluate the relationship between the NLR value and the chemotherapy response.

Sociodemographic characteristics distribution
There were 113 patients who participated in the study. The average age was 47 years old, with the youngest was 27 years old and the oldest was 68 years old. There were 89 patients aged above 40

Relationship of sociodemographic characteristics and chemotherapy response in locally advanced stage breast cancer patients
The relationship between sociodemographic characteristics and chemotherapy response of patients with locally advanced breast cancer were analyzed using the chi square statistical test. It is found that there was no significant relationship between sociodemographic characteristics (age, age at menarche, age at first pregnancy, age at menopause, family history, contraception, parity and subtype) to chemotherapy response (p > 0.05).

Distribution of the Neutrophil/Lymphocyte Ratio (NLR)
The distribution of the NLR based on chemotherapy response showed the NLR value on good chemotherapy responses had a mean value of 1.90 ± 0.95, while the NLR value on poor chemotherapy responses had a mean value of 5.77 ± 4.26. The results of the study are presented in table 3.

Relationship of neutrophil/lymphocyte ratio (NLR) and chemotherapy response in locally advanced stage breast cancer patients
The analysis of the relationship between NLR and chemotherapy response in patients with locally advanced breast cancer found that 97.5% cancer patients who had NLR ≤ 2.92 had a good chemotherapy response, compared to 17.6% cancer patients who had NLR > 2.92. The results of statistical tests using the chi square test found that there was a significant relationship between NLR and chemotherapy response in patients with locally advanced breast cancer (p value = 0.000) with an alpha value of 0.05 (p <α).

Discussion
The chronic systemic inflammatory response is continuously studied in terms of breast cancer progression and prognosis. Parameters using peripheral blood test have been investigated as factors that reflect the immune response. Circulating white blood cells, which result in changes in the proportion of neutrophils, lymphocytes, and monocytes, are associated with systemic inflammatory response. The relationship between cancer prognosis and absolute monocyte count (AMC), absolute lymphocyte count (ALC), lymphocyte-to-monocyte ratio (LMR), neutrophil-to-lymphocyte ratio (NLR), and platelet-tolymphocyte ratio (PLR) has been studied in various types of cancer. 4 There is a known association between the systemic inflammatory response and changes in circulating

Advanced Stage Breast Cancer Patients
The relationship of sociodemographic characteristics and Neutrophil/Lymphocyte Ratio (NLR) in patients with locally advanced breast cancer were analyzed using the chi square statistical test. It is found that there was no significant relationship between sociodemographic characteristics (age, age at menarche, and better survival outcomes after NACT. 8 Neutrophil is a type of granulocyte leukocyte that is produced in 7-14 days, and remains in circulation for 6 hours. The main function of neutrophil is phagocytosis (killing and digesting microorganisms). Acute bacterial infections and trauma trigger neutrophil production. This increase in neutrophil number is referred as "shift to the left", which indicates an acute bacterial infection. 9,10 There is a known association between the systemic inflammatory response and changes in circulating white blood cells, particularly the presence of neutrophilia and relative lymphocytopenia. Routine blood test in cancer patients is an objective parameter that is easily measured and is able to show the degree of systemic inflammatory response. In 2017, a study by Lu et al. showed the components of the white blood cell count, particularly neutrophils and lymphocytes, have a role in predicting the life expectancy of cancer patients. 11 Immunohistochemically, lysis of tumor cells by chemotherapy agents can be associated with the release of tumor-related antigens. This can assist the immune response against cancer cells, which will be important in patients with immunity sensation to these tumor antigens before starting the chemotherapy regimen. This means, NLR can be a prognostic sign to predict the effectiveness of adjuvant chemotherapy after surgery or NACT. 12