Correlation of Peripheral Neutrophil-to-Lymphocyte Ratio (NLR) with Ki-67 and Other Clinicopathological Parameters of Breast Carcinoma

Abstract

Ki-67 is a protein expressed in the nucleus of cells during different phases of the cell cycle. Higher levels of Ki-67 expression in breast cancer are associated with high proliferation and poor prognosis. Neutrophil-to-lymphocyte ratio (NLR) is a peripheral marker of inflammation, an elevated pretreatment NLR is associated with an increased risk of relapse and worse survival among breast cancer patients. The present study was a retrospective and prospective study conducted on 140 patients under which the presurgical neutrophil–lymphocyte ratio was correlated with histological subtype, grade, stage, molecular subtype, and Ki-67 proliferation index in patients with breast carcinoma. There was a significant association between Ki67 expression groups and NLR ratio as well as between molecular subtypes groups and NLR ratio (p < 0.001). However, no significant association was found between Nottingham Bloom Richardson (NBR) grades and tumor size, nodal status, and metastasis (TNM) stages with NLR ratio (p > 0.001). NLR is a low-cost, minimally invasive, personal, and reliable marker of inflammation which has potential utility in predicting short- and long-term prognosis and risk stratification for anti-inflammatory or immune therapy for cancer patients in the future.

Introduction

Breast cancer is one of the leading malignancies in females worldwide, with approximately 2.3 million cases reported in 2020, resulting in about 685,000 deaths globally. In the past 5 years, 7.8 million women were diagnosed with breast cancer, making it the most prevalent cancer worldwide [1]. Among Indian females, breast cancer is the most common cancer, with an age-adjusted rate as high as 25.8 per 100,000 women and a mortality rate of 12.7 per 100,000 women [2]. Cancer of the breast is a heterogeneous disease, consisting of several separate entities with unique biological characteristics and behavior [3]. With the advent of microarrays, several genetic subgroups and extensive heterogeneity of cancer of the breast have been studied [4].

Systematic studies of gene expression patterns and their association with certain elements of phenotypic variation have led to a reclassification into molecular classification [5]. The classification of breast cancer is intricate and is often based on the intrinsic gene expression profiles. According to the IHC classification, breast cancer types include ER/PR + HER2 + with luminal B; ER/PR + , HER2/neu − with luminal A; ER/PR − , HER2 + ; and ER/PR − , HER2 − , commonly known as triple-negative or basal-like tumors. These classifications are essential as they influence the treatment approach and prognosis [2, 3]. Ki67 a protein expressed in the nucleus during various cell cycle phases except in the G0 quiescent state, is a widely used immunohistochemistry (IHC) proliferation antigen in breast cancer samples. High levels of Ki-67 expression in breast cancer are associated with increased proliferation and poor prognosis. Inflammation and host immune response also play a critical role in breast cancer prognosis by altering the tumor microenvironment and therapeutic efficacy. Components of the systemic inflammatory response, such as lymphocytes, neutrophils, and platelets, are important in carcinogenesis and tumor progression [6]. The neutrophil-to-lymphocyte ratio (NLR) is a peripheral marker of inflammation, increasingly studied since NLR can be derived from a complete blood count (CBC), it may provide a simple and inexpensive means for breast cancer prognostication. This study was done to investigate the association of pre-treatment NLR with various molecular subtypes of breast cancer and Ki 67 proliferation index and survival following breast cancer treatment [3], also as a potential predictor of chemotherapy response and long-term outcomes [7].

Materials and Methods

The present study was a retrospective and prospective study conducted in the Department of Pathology for a period of 18 months after obtaining approval from the Institutional Ethical Committee. A total of 140 patients were included in the study which included 36 retrospective and 104 prospective cases. The small sample size in our study is largely due to the specific inclusion criteria for breast carcinoma patients, time boundation of 18 months and feasibility. For retrospective cases, histopathological and immunohistochemistry (IHC) findings for breast carcinoma cases were retrieved from archive records and were re-evaluated. Also, their pre-surgical complete blood count reports were analyzed from the archived records to calculate absolute neutrophil and absolute lymphocyte count. The ratio of absolute neutrophil and absolute lymphocyte count was thus calculated to derive the NLR ratio from these reports and subsequently recorded on a predesigned Performa. For prospective cases, patients who underwent either core needle biopsy-wide local excision or modified radical mastectomy with newly diagnosed, early, or locally advanced breast cancer were included. The study process and its reasons were thoroughly explained to the patients in their native language, addressing any doubts. Detailed clinical examination and routine investigations were carried out in each case. Patients with ductal carcinoma in situ with or without microinvasion, and patients with a lack of information on pathologic or laboratory results. Known cases of any acute or chronic infection and post neoadjuvant chemotherapy (NACT) cases were excluded from the study. Two ml of peripheral blood sample in ethylenediaminetetraacetic acid (EDTA) vial was obtained at the time of clinical diagnosis, before treatment. The WBC count and differential count were determined by using a hematology analyzer (Beckman Coulter & Sysmex XN 1000). Absolute neutrophil count and absolute lymphocyte count were derived from total WBC count. The value of NLR ≥ 2.5 will be taken as high and < 2.5 as low [3]. Surgical specimen for histopathology was processed, stained, and microscopically examined as per standard protocols. After histopathological diagnosis, immunohistochemistry(IHC) staining for ER, PR, HER2, and Ki67 markers was done. IHC results were also recorded. Cases with ≥ 14% positive nuclei were classified as high Ki-67 expression and those with < 14% were classified as low Ki-67 expression [8].

Observations and Results

Of the total 140 cases, the maximum number of cases was seen in the age group of 41 to 60 years comprising 74 cases (52.9%). Minimum numbers of cases were seen in the age group more than 80 years comprising 2 cases (1.4%). One hundred thirty-nine cases (99.3%) were females while one was male. The majority of patients presented clinically with breast lumps with or without pain (46 and 48 cases respectively), male patient (0.7%). A maximum number of cases had a history of duration of clinical symptoms < 1 month comprising 80 cases (57.2%), and only 7 cases (5.0%) had a duration of more than 1 year. Lactation history was present in most of the cases (81 cases, 57.9%). Lactation history was absent in 58 cases (41.4%). A positive family history was noted in 11 cases (7.9%); however, 124 cases (88.6%) did not have any family history. Five cases (3.6%) were unaware of their positive or negative family history. Seventy-three cases (52.1%) had attained menopause whereas 66 cases (47.1%) did not attain menopause. Maximum number of cases had swelling size of 2 to 3 cm comprising 61 cases (43.6%). The tumors were most commonly present on the right side, with 72 cases (51.4%). Tumors on the left side were observed in 68 cases (48.6%). The majority of tumors were located in the upper outer quadrant comprising 40 cases (28.5%) followed by the lower outer quadrant, 37 cases (26.4%). A maximum number of patients (40 cases, 28.6%) had irregular tumor morphology followed by circumscribed tumor morphology comprising 36 cases (25.7%). Tumors with infiltrating and solid/cystic morphology were noted in 28 cases (20.0%) and 20 cases (14.3%) respectively. Only 16 patients (11.4%) had exophytic/fungating tumor morphology. The majority of cases comprising 125 patients (89.2%) of the study group did not have any nipple involvement. Of 140 patients, only 15 patients (10.7%) had nipple involvement. In the majority of patients comprising 116 cases (82.9%), lymph nodes were not palpable. Only in 24 cases (17.1%) lymph nodes were palpable. Of 140 cases, 99 cases (70.7%) were core biopsies, 23 cases (16.4%) were of modified radical mastectomy, and 18 cases (12.8%) were of wide local excision. Invasive ductal carcinoma was the most prevalent histopathological subtype observed, comprising 85 patients (60.7%), followed by lobular carcinoma observed in 32 patients (22.9%). Mucinous carcinoma was noted in 10 patients (7.1%), while metaplastic carcinoma and papillary carcinoma were observed in 8 patients (5.7%) and 5 patients (3.6%), respectively. Among the total study group, 32.1% of patients were found to have a total Nottingham Bloom Richardson (NBR) score of 8 whereas 1.4% of patients were found to have for score of 3, reflecting the diversity of breast cancer aggressiveness within the studied population. Grade 2 NBR was found to be the most prevalent grade group comprising 64 cases (45.7%) followed by Grade 3 NBR, observed in 58 cases (41.4%). Grade 1 NBR, representing well-differentiated tumors, was observed in only 18 cases (12.9%). Lymphovascular invasion was seen in 50 cases (35.7%). However, majority of cases showed an absence of lymphovascular invasion, 90 cases (64.3%). Tumor size, nodal status, and metastasis (TNM) staging was done in 41 cases (excluding core biopsies) and maximum cases were categorized under TNM stage II classification, comprising 25 cases (60.9%) followed by 12 cases (29.9%) with TNM stage III classification and 4 cases (9.7%) with TNM stage I classification. The majority of cases had a high neutrophil-to-lymphocyte ratio (NLR) of more than 2.5,86 cases (61.4%) whereas 54 cases (38.6%) had a low NLR of less than 2.5. The majority of cases showed a high Ki67 expression of more than 15%, comprising 130 cases (93.0%) and a low Ki67 (%) expression of less than 15% in 10 cases (7.0%).Molecular subtyping was done among 126 cases including HER2/neu equivocal cases which were further outsourced for FISH analysis and were further categorized into molecular subtypes. However, 14 cases with HER2/neu equivocal expression were excluded from the study as their further evaluation was not feasible due to cost limitations. The luminal B subtype was the most common subtype, observed in 64 cases (50.79%) followed by the triple-negative subtype in 31 cases (24.60%) and Her 2neu over-expression subtype in 23 cases (18.25%) respectively. Luminal A comprised the least common subtype, observed in 8 cases (6.35%). Further confirmation by FISH was not available at the place of study. When the association of NBR grades with NLR was studied, across all grades, 38.6% of the cases had low NLR status, while 61.4% had high NLR status, with a p-value of 0.885. This non-significant p-value of 0.885 indicated that there was no significant association between NBR grade and NLR status (Table 1). Further correlation between molecular subtypes and their NLR was done in which a significant association between Molecular subtype groups and NLR ratio was found (Table 2). Furthermore, there was no significant association between TNM stages and NLR (Table 3). There was a significant association between Ki-67 expression groups and NLR ratio (Table 4). 95% confidence interval was taken for all above correlations.

Table 1.

Correlation of cases based on Nottingham Bloom Richardson (NBR) grade with NLR

NBR grade	Total	NLR		p-value
		Low	High
Grade 1	18	6 (33.3%)	12 (66.7%)	0.885
Grade 2	64	25 (39.1%)	39 (60.9%)
Grade 3	58	23 (39.7%)	35 (60.3%)
Total	140	54 (38.6%)	86 (61.4%)

Table 2.

Correlation of cases based on molecular subtype distribution and NLR

Molecular subtype	NLR		Total	Chi-square value	p-value
	Low	High
Luminal A	4	4	8	9.26	0.026
	50.0%	50.0%	100.0%
Luminal B	20	44	64
	31.3%	68.8%	100.0%
HER2/neu	15	8	23
	65.2%	34.8%	100.0%
TNBC	10	21	31
	32.3%	67.7%	100.0%
Total	49	77	126
	38.9%	61.1%	100.0%

Table 3.

Correlation of cases based on TNM stage distribution and NLR

TNM stage	Total	NLR		p-value
		Low	High
Stage 1	3	1 (33.3%)	2 (66.7%)	0.550
Stage 2	25	13 (52%)	12 (48%)
Stage 3	12	5 (41.7%)	7 (58.3%)

Table 4.

Correlation of cases based on Ki-67 expression distribution and NLR

Ki-67 expression	NLR		Total	Chi-square value	p-value
	Low	High
≤ 15%	7 (70.0%)	3 (30.0%)	10 (100%)	4.48	0.034
> 15%	47 (36.2%)	83 (63.8%)	130 (100%)
Total	54 (38.6%)	86 (61.4%)	140 (100%)

Discussion

The presence of an elevated peripheral Neutrophil-to-lymphocyte (NLR) ratio, an indicator of systemic inflammation, has been recognized as a poor prognostic factor in various cancers. In a previous meta-analysis of 100 studies of patients conducted by Ethier et al. with unselected solid tumors, increased NLR was associated with decreased overall survival (OS) (hazard ratio (HR) 1.81; 95% confidence interval (CI) = 1.67–1.97; P < 0.001) [9]. In this study, most cases were in the 4th to 6th decade, this occurrence of breast cancer with advancing age could be due to the accumulation of a large number of cellular alterations and exposition to potential carcinogens resulting in an increase of carcinogenesis with time [2]. Incidence of breast cancer is higher in females because female breast parenchyma is sensitive to estrogen and hormone imbalance [2]. Present study highlighted a higher percentage of patients with lactation history, 57.9% whereas 41.4% of patients had no history of lactation. Concomitant with our study, Freudenheim et al. concluded that lactation has a weak protective effect against breast cancer. However, lactation may have a protective effect in premenopausal breast cancer [10]. The lower prevalence of positive family history in our study aligns with the broader trend. The majority of breast cancers are not directly linked to family history. About 90% of breast cancers result from gene changes acquired during an individual’s lifetime, rather than inherited mutations [11]. Our study observed that the most common location of the tumor was the upper outer quadrant (28.5%), followed closely by the lower outer quadrant (26.4%). This may be due to the fact that larger volume of breast tissue is present in upper and central quadrants [12]. High NLR indicates systemic inflammation which plays a role in angiogenesis and tumor progression. High NLR correlates with decreased lymphocytes leading to anticancer response with impaired immune suppression. Also, high neutrophil and lymphocyte counts reduce the effectiveness of chemotherapy and thus increase resistance to chemotherapy and poor response to treatment. Thus monitoring NLR during the course of breast cancer treatment can provide insights into disease progression and response to therapy [13]. In the present study, out of a total of 140 cases, Neutrophil- to- lymphocyte ratio was found to be more than 2.5% in 61.4% of cases and less than 2.5% in 38.6% of cases. Azab et al. in their study found that there were 62 patients with low NLR (< 2) and 61 patients with high NLR (≥ 2). Both low and high NLR groups had no statistically significant difference in most of their clinicopathological and treatment variables [14]. Arora et al. calculated NLR and found 64.4% of cases had high NLR (> 3) while 35.6 of the cases had low NLR (< 3) [15]. Ki67 can prove to be an important parameter for evaluating the disease aggressiveness, breast cancer proliferation and hence for taking therapeutic decisions concerning chemotherapy. Ki-67 is a proliferation marker which labels the nuclei of proliferating non-neoplastic and neoplastic cells [16]. In the present study, majority of cases showed a high Ki67 expression of more than 15%, comprising 130 cases (93.0%) and a low Ki67 (%) expression of less than 15% in 10 cases (7.0%). Similarly in a study by Arora et al. Ki67 IHC classification was done and found that 15.0% of the cases were of low grade, 28.8% of cases were of intermediate grade and 56.2% were of high grade [15]. Liang et al. in their study among a total of 2573 cases with breast cancer evaluated Ki67 immuno-expression and observed a high proliferation index in 1403 cases (Ki67 > 15%), a low proliferation index in 1170 cases (Ki67 < 15%) [17]. There was a significant association between Molecular Subtypes groups and NLR. “Gago-Dominguez et al. in their study showed a significant association of NLR with Molecular Subtypes of breast carcinoma with a p-value of < 0.001” [18]. Contrary to our study Arora et al. and Yersal et al. observed non-significant correlation of NLR with Molecular Subtypes, p > 0.5 and p = 0.51 respectively [15, 19]. In our study, we found no significant association between TNM stages and NLR, p = 0.550. Azab et al. in their study observed significant association between NLR and TNM stages with p < 0.0001 [14]. We found a significant association between Ki-67 expression groups and NLR, p = 0.034. Arora et al. in their study also found a significant correlation of NLR with Ki67, p < 0.001 [15]. With a cutoff of 14%, Ki67 was significantly correlated with NLR in the study of HER2 negative breast cancer patients done by Bae et al. [20].

Conclusion

Neutrophil–lymphocyte ratio (NLR) is a fast-emerging therapeutic and prognostic marker in breast carcinoma. Its significant correlation with Ki67 in this study supports this parameter as a useful marker to monitor disease progression and prognosis and possible response to treatment. Moreover, it is less time-consuming and overcomes the drawbacks associated with the estimation of Ki67. The NLR offers valuable insights into the immune-inflammatory response in patients with breast carcinoma. It is an affordable, straightforward parameter with high sensitivity but lower specificity. It responds rapidly to insults and reflects changes in clinical status. When combined with other valid infection or inflammation biomarkers, NLR can aid decision-making and management of acute or chronic diseases. Pre-surgical NLR ratio in patients of clinically suspected breast carcinoma will serve as a minimal invasive personalized marker for early diagnosis, guiding therapy, and prognosis and follow-up of patients.

Limitations

While the study contributes to the growing body of literature on inflammation-based markers in breast cancer, it has its own limitations including a smaller sample size due to time and cost limitations. Further studies are required to validate NLR's diagnostic and prognostic roles with a larger sample sizes and multi-center data as well as multivariate models to improve the study’s internal validity. Also, there is no universally agreed-upon NLR cutoff, and thresholds may vary depending on the population studied, disease subtype, or clinical context, further research exploring the optimal cutoff point for breast cancer, potentially through receiver operating characteristic (ROC) analysis or by evaluating larger, more diverse cohorts could be done.

Declarations

Conflict of Interest

The authors declare no competing interests.