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Abstract

BACKGROUND:

The presence of human cytomegalovirus (HCMV) in breast cancer has been reported, suggesting a potential association between HCMV infection and breast carcinogenesis.

OBJECTIVE:

To evaluate the association between HCMV infection and immune activation and inflammatory markers in breast cancer.

METHODS:

HCMV DNA was detected from all patients using real-time PCR, Anti HCMV IgM and IgG antibodies were measured. IL-17 and IL-22 concentrations were detected by ELISA. Assessment of NLR and PLR was done, and cell proliferation was assessed using MTT assay.

RESULTS:

The results revealed a significantly increased prevalence of anti-HCMV IgG and HCMV DNA in patients compared to both benign and control groups where positive HCMV prevalence was significantly associated with vascular invasion, proliferation rate, high neutrophil-to-lymphocyte ratio (NLR), and elevated IL-17 serum level. Furthermore, we demonstrated that increased serum IL-17 in patients was markedly associated with tumor stage, vascular invasion, and high NLR.

CONCLUSION:

It can be concluded that HCMV infection may have vital roles in breast cancer pathogenesis. Moreover, altered peripheral blood cells and cytokines may result in disordered immune response in breast cancer patients.

Keywords

HCMV breast cancer NLR PLR IL-17 IL-22

1. Introduction

Breast cancer is the most common cancer among women worldwide, where incidence and mortality are still increasing in both developing and developed countries. It accounts for 30% of all new cancer diagnoses among females [1]. Epidemiological studies from Arab countries show an alarmingly rising burden with associated incidence and mortality [2]. In Egypt, it is also the most common cancer among women, accounting for approximately 37.7% of cancers with a peak incidence in the age group of 40–59 years and a mortality rate of 29.1% [3].

It has been estimated that over 15% of all cancers worldwide are attributable to infectious agents especially viruses [4]. Human cytomegalovirus (HCMV) is an opportunistic DNA virus that belongs to the herpesviridae family. It is carried by up to 90% of the adult population worldwide causing a lifelong latent infection that may reactivate during an inflammatory process including cancer [5]. In Egypt, primary HCMV infection occurs in 0.15–2.0% of all pregnancies and may be transmitted to the fetus in up to 40% of cases [6].

The frequent presence of HCMV in breast cancer has been reported, suggesting a potential association between HCMV infection and breast carcinogenesis [7]. However, the precise role of HCMV in human cancers has not been clarified and some of the previous results have been inconsistent due to alternative behavior of HCMV in tumor cells and high level of genetic variance of HCMV [8,9].

A close link between HCMV infections and inflammation has been established [10] and it was shown that HCMV significantly influences the immune status in chronic inflammatory conditions [11]. The neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) are attractive biomarkers of systemic inflammation and immune status with a prognostic value in patients with breast cancer [12]. In addition, it has been shown that HCMV is associated with myelosuppression [13] and that the NLR/PLR combined might provide information for the identification of HCMV infection [14].

It has been reported that high level circulating pro-inflammatory cytokines are the hallmark of latent HCMV reactivation [15]. The interleukin-17 (IL-17) family of cytokines (such as IL-17 and IL-22) has been greatly involved in chronic inflammatory diseases acquiring an interest in cancer immunity [16]. IL-17 is a pleiotropic cytokine participating in cancer progression and was suggested to play varied and crucial roles in viral infections [17]. However, its role in breast cancer, as well as its relationship with HCMV infection, needs further exploration.

The current study aimed to evaluate the clinicopathological significance of HCMV as well as its relationship with different inflammatory immune markers in primary and recurrent subgroups of Egyptian breast cancer patients.

2. Material and methods

2.1. Subjects

The current study included 160 patients with different breast lesions [benign breast lesions (n = 44), primary breast cancer that was classified into early breast cancer “stage I and II” (n = 37) and advanced breast cancer “stage III and IV” (n = 45), and recurrent breast cancer (n = 34)], as well as 40 age, matched females as a control group who were healthy females attended to the breast clinic in the Surgery Department for checkup or just mastalgia and proved normal by triple assessment (clinical examination, radiological assessment +/- pathological assessment). Patients were recruited from the Surgery Department outpatient clinic or ward and Cancer Management and Research Department, Medical Research Institute, Alexandria University during the period from January 2019 to May 2020. All patients were examined and reported for age, tumor type, stage, grade, lymph node involvement, vascular invasion and tumor metastasis as well as estrogen receptor/progesterone receptor and human epidermal growth factor receptor 2 (HER2) status. Exclusion criteria included subjects who had used any form of vitamin D in the last two years, patients with a history of any other cancer or any immune-mediated disease and those with equivocal histologic results.

2.2. Methods

Venous blood samples (5 ml) were obtained from all subjects in plain vacutainers for serum analyses.

Tissue samples were collected from the lesions after being excised and sent for histopathological examination in benign lesions, early and recurrent breast cancer whereas core tissue biopsy was taken from the lesion before being sent to neoadjuvant chemotherapy in advanced breast cancer. These samples were obtained for quantification of HCMV DNA using real-time PCR technique and 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay.

The collection and use of tissue and blood samples for the current study were approved by the medical ethical committee of Alexandria University. Written consents were obtained from all subjects participating in this study. The current study follows the principles outlined in the Declaration of Helsinki for use of human tissue or subjects.

2.2.1. Analysis of blood sample

Peripheral blood samples taken from the study population were collected in plain vacutainers and left for 20 min at 37 °C to clot followed by 1 h at 4 °C to retract blood clot. Blood samples were then centrifuged (at 1800 rpm for 10 min) where sera were collected and stored in aliquots at −80 °C for HCMV antibodies, IL-17 and IL-22 assessment.

2.2.1.1. Detection of anti-HCMV IgM and IgG antibodies:

Anti HCMV IgM and IgG antibodies were measured in all subjects under study by using AccuDiag^{^TM} HCMV IgM and IgG ELISA kits (Cat. No. 1202-2 and 1201-11, respectively) according to the manufacturer’s instructions.

2.2.1.2. Detection of IL-17and IL-22 cytokine levels:

Serum cytokines concentrations were detected strictly in accordance with the instructions of the commercially available ELISA kits (IL-17 and IL-22 ELISA kits purchased from Cusabio; Cat. No. CSB-E12819h and Abcam; Cat. No. ab216170, respectively).

2.2.1.3. Assessment of NLR and PLR:

At the time of histopathological examination, NLR was calculated by dividing the absolute neutrophil count by the absolute lymphocyte count. The PLR was calculated by dividing the absolute platelet count by the absolute lymphocyte count. Based on previous studies, an NLR value of 3.0 was used as the cut-off value to differentiate between high-NLR (≥3.0) and low-NLR (<3.0) [12] while a PLR value of 150.0 was used as the cut-off value to discriminate between high-PLR (≥150.0) and low-PLR (<150.0) [18].

2.2.2. Preparation of tissue samples

Breast cancer tissue samples removed from all patients undergoing surgery and Tru cut biopsy as well as tumor-free breast tissue obtained from a benign group that served as controls were transferred in sterile phosphate buffer saline. Part of the tissue samples (≤25 mg) were cut into small pieces and placed in a 1.5 ml microcentrifuge tube to be used for DNA extraction and the rest of the tissue samples were transferred to RPMI-1640 containing 1% Penicillin/Streptomycin/Amphotracine B antibacterial and antimycotic (Lonza, BioWhittaker, Vervier) for using in MTT assay.

2.2.3. DNA extraction

DNA was isolated from all tissue samples using QIAamp DNA Mini Kit (cat. nos. 51304), according to the manufacturers’ instructions.

2.2.4. Detection and quantification of HCMV-DNA by real-time PCR

HCMV-DNA in tissue samples was quantified by the StepOne real-time PCR system using (GeneProof CMV-PCR Kit), according to the manufacturers’ instruction for the specific amplification of conservative DNA sequence of a single-copy gene encoding the 4 IE antigen in all genotypes of HCMV.

The amplification program started with an initial holding stage at 37 °C for 2 min and 10 min at 95 °C for Taq activation followed by 45 cycles of 95 °C denaturation for 5 s, 60 °C annealing for 40 s and 72 °C extension for 20 s.

2.2.5. Assessment of cell proliferation rate by MTT assay

Cell proliferation was assessed on the tumor cells taken from patients using TACS^® MTT assay (Bio-techne; Cat. No. 4890-025-K). The cells were adjusted to a concentration of 2 × 10⁶ cells/ml/well before they were cultured at 37 °C and 5% CO₂ for 48 h. Thereafter, 10 μL of the tetrazolium salt (MTT reagent) was added to each well where the culture plate was maintained for 3 h at 37 °C. The absorbance was then measured at 570 nm while the cells were suspended in 100 μL of MTT detergent.

2.2.6. Statistical analysis

Values were expressed as mean ± SD and were analyzed using SPSS statistical software version 20.0. (Armonk, NY: IBM Corp). Chi-square test (Fisher or Monte Carlo) was used to compare between groups for categorical variables, Mann Whitney test was used to compare between two groups for not normally distributed quantitative variables. Significance of the obtained results was judged at the 5% level. Cut-off values for different biomarkers were included in this study.

3. Results

3.1. Demographic and pathological data of the studied breast cancer patients

During the designed duration of the study, 160 patients with different breast lesions were enrolled in the study. They were classified as follows [benign breast lesions (n = 44), early breast cancer “stage I and II” (n = 37), advanced breast cancer “stage III and IV” (n = 45) and recurrent breast cancer (n = 34)]. The demographic and pathological characteristics of the studied patients are summarized in Table 1.

Table 1
Demographic and pathological data of the studied breast cancer patients

Early-stage patients Advanced stage Recurrent Benign Control

(n = 37) (n = 45) (n = 34) (n = 44) (n = 40)

Age (years)

Min.–Max. 30–60 30–60 38–72 32–68 35–64

Mean ± SD 49.43 ± 7.7 49.7 ± 7.8 50 ± 9 47.36 ± 8.43 48.97 ± 7.1

Pathology

Invasive ductal carcinoma 36(97.3%) 37(82.2%) 34(100%)

Invasive lobular carcinoma 0 3(6.7%) 0

Mixed 1(2.7%) 5(11.1%) 0

Lymph node involvement

N0 31(83.8%) 16(35.6%) 25(73.5%)

N1 6(16.2%) 22(48.8%) 7(20.6%)

N2 — 4(8.9%) 2(5.9%)

N3 — 3(6.7%) —

Vascular invasion

No 16(43.2%) 11(24.4%)

Yes 21(56.8%) 34(75.6%) 34(100%)

Stage

I 14(37.8%) — 8(23.5%)

II 23(62.2%) — 13(38.2%)

III — 30(66.7%) 13(38.2%)

IV — 15(33.3%) —

Grade

I 2(5.4%) 2(4.4%) 1(2.9%)

II 27(73%) 31(68.9%) 27(79.4%)

III 8(21.6%) 12(26.7%) 6(17.6%)

Estrogen receptors

Positive/Negative 22/15 25/20 21/13

Progesterone receptors

Positive/Negative 22/15 23/22 19/15

HER2

Positive/Negative 20/17 20/25 15/19

	Early-stage patients	Advanced stage	Recurrent	Benign	Control
Age (years)
Min.–Max.	30–60	30–60	38–72	32–68	35–64
Mean ± SD	49.43 ± 7.7	49.7 ± 7.8	50 ± 9	47.36 ± 8.43	48.97 ± 7.1
Pathology
Invasive ductal carcinoma	36(97.3%)	37(82.2%)	34(100%)
Invasive lobular carcinoma	0	3(6.7%)	0
Mixed	1(2.7%)	5(11.1%)	0
Lymph node involvement
N0	31(83.8%)	16(35.6%)	25(73.5%)
N1	6(16.2%)	22(48.8%)	7(20.6%)
N2	—	4(8.9%)	2(5.9%)
N3	—	3(6.7%)	—
Vascular invasion
No	16(43.2%)	11(24.4%)
Yes	21(56.8%)	34(75.6%)	34(100%)
Stage
I	14(37.8%)	—	8(23.5%)
II	23(62.2%)	—	13(38.2%)
III	—	30(66.7%)	13(38.2%)
IV	—	15(33.3%)	—
Grade
I	2(5.4%)	2(4.4%)	1(2.9%)
II	27(73%)	31(68.9%)	27(79.4%)
III	8(21.6%)	12(26.7%)	6(17.6%)
Estrogen receptors
Positive/Negative	22/15	25/20	21/13
Progesterone receptors
Positive/Negative	22/15	23/22	19/15
HER2
Positive/Negative	20/17	20/25	15/19

3.2. Prevalence of HCMV in different studied groups

As shown in Table 2, the results revealed that all individuals enrolled in this study were seronegative for anti-HCMV IgM. However, there was a statistically significant difference in the seroprevalence of anti-HCMV IgG between different groups (p < 0.001). All patients (100%) in the recurrent group, 88.9% of advanced-stage patients, and 86.5% of early-stage patients were positive for anti-HCMV IgG compared to 81.8% and 60% in the benign and healthy control groups, respectively.

Table 2
Comparison between different studied groups regarding HCMV prevalence

Breast cancer patient stage (n = 82)

Early Advanced Recurrent Benign Control p

(stage I + II) (stage III + IV) (n = 34) (n = 44) (n = 40)

Anti-HCMV IgM

Negative 37 (100%) 45 (100%) 34 (100%) 44 (100%) 40 (100%)

Positive 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) —

Mean ± SD. 0.04 ± 0.01 0.05 ± 0.02 0.05 ± 0.02 0.05 ± 0.02 0.04 ± 0.01

p ₀ 0.131 0.062 0.087 0.121

Anti-HCMV IgG

Negative 5 (13.5%) 5 (11.1%) 0 (0%) 8 (18.2%) 16 (40%)

Positive 32 (86.5%) 40 (88.9%) 34 (100%) 36 (81.8%) 24 (60%) <0.001^∗

Mean ± SD. 1.6 ± 0.4 1.7 ± 0.6 1.9 ± 0.2 1.7 ± 0.5 1.1 ± 0.5

p ₀ <0.001^∗ <0.001^∗ <0.001^∗ <0.001^∗

Sig. bet. grps. p₁ = 0.026^∗, p₂ = 0.276, p₃ = 0.245, p₄ = 0.207, p₅ = 0.240, p₆ = 0.933

HCMV DNA in tissue

Negative 21 (56.8%) 18 (40%) 4 (11.8%) 38 (86.4%) — <0.001^∗

Positive 16 (43.2%) 27 (60%) 30 (88.2%) 6 (13.6%) —

	Breast cancer patient stage (n = 82)
Anti-HCMV IgM
Negative	37 (100%)	45 (100%)	34 (100%)	44 (100%)	40 (100%)
Positive	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	—
Mean ± SD.	0.04 ± 0.01	0.05 ± 0.02	0.05 ± 0.02	0.05 ± 0.02	0.04 ± 0.01
p ₀	0.131	0.062	0.087	0.121
Anti-HCMV IgG
Negative	5 (13.5%)	5 (11.1%)	0 (0%)	8 (18.2%)	16 (40%)
Positive	32 (86.5%)	40 (88.9%)	34 (100%)	36 (81.8%)	24 (60%)	<0.001^∗
Mean ± SD.	1.6 ± 0.4	1.7 ± 0.6	1.9 ± 0.2	1.7 ± 0.5	1.1 ± 0.5
p ₀	<0.001^∗	<0.001^∗	<0.001^∗	<0.001^∗
Sig. bet. grps.	p₁ = 0.026^∗, p₂ = 0.276, p₃ = 0.245, p₄ = 0.207, p₅ = 0.240, p₆ = 0.933
HCMV DNA in tissue
Negative	21 (56.8%)	18 (40%)	4 (11.8%)	38 (86.4%)	—	<0.001^∗
Positive	16 (43.2%)	27 (60%)	30 (88.2%)	6 (13.6%)	—

The data were assessed using chi square test (χ²), kruskal wallis test (H), pairwise comparison bet. Each 2 groups was done using post hoc Test.p: p value for comparing between the studied groups; p₀: p value for comparing between control and each studied group; p₁: comparing between recurrent and early; p₂: comparing between recurrent and advanced; p₃: comparing between recurrent and benign; p₄: comparing between early and advanced; p₅: comparing between benign and early; p₆: comparing between benign and advanced.^∗: Statistically significant at p ≤ 0.05.

Additionally, there was a statistically significant difference in the prevalence of HCMV DNA in tissue between different groups (p < 0.001). Our results showed that positive tissue prevalence of HCMV DNA was higher in recurrent (88.2%) followed by advanced stage and early-stage groups (representing 60% and 43.2%, respectively) in comparison to the benign group (13.6%).

3.3. Association of HCMV prevalence with pathological characteristics in primary and recurrent breast cancer patients

To explore the relationship between HCMV and disease severity, we analyzed the association between anti-HCMV IgG and DNA with different pathological parameters in breast cancer patients as shown in Table 3. There was a statistically significant association between seropositivity of anti-HCMV IgG and tissue prevalence of HCMV DNA with vascular invasion (p < 0.001; p = 0.036, respectively). The results showed that serum and tissue positivity of HCMV were higher in patients with vascular invasion. There was also a statistically significant association between seropositivity of anti-HCMV IgG as well as tissue prevalence of HCMV DNA and proliferation rate (p < 0.001).

Table 3
Relation between prevalence of HCMV prevalence in primary and recurrent breast cancer patients according to pathological characteristics and proliferation rate

Positive Anti – HCMV IgG Positive HCMV DNA in Tissue

Pathological characteristics Early Advanced Recurrent Early Advanced Recurrent

(stages I + II) (stages III + IV) (n = 34) (stages I + II) stages III + IV (n = 30)

(n = 32) (n = 40) (n = 16) (n = 27)

Tumor grades

I 2 (6.3%) 1 (2.5%) 1 (2.9%) 2 (12.5%) 1 (3.7%) 1 (3.3%)

II 22 (68.8%) 29 (72.5%) 27 (79.4%) 12 (75%) 19 (70.4%) 23 (76.7%)

III 8 (25%) 10 (25%) 6 (17.6%) 2 (12.5%) 7 (25.9%) 6 (20%)

𝜒² (^MCp) 1.571 (0.814) 2.815 (0.589)

Type of tumor

Invasive ductal carcinoma 31 (96.9%) 33 (82.5%) 34 (100%) 15 (93.8%) 23 (85.2%) 30 (100%)

Mixed type 1 (3.1%) 4 (10%) 0 (0%) 1 (6.3%) 3 (11.1%) 0 (0%)

Invasive lobular carcinoma 0 (0%) 3 (7.5%) 0 (0%) 0 (0%) 1 (3.7%) 0 (0%)

𝜒² (^MCp) 7.399^∗ (0.036^∗) 5.227 (0.145)

Vascular invasion

No 13 (40.6%) 10 (25%) 0 (0%) 3 (18.8%) 2 (7.4%) 0 (0%)

Yes 19 (59.4%) 30 (75%) 34 (100%) 13 (81.3%) 25 (92.6%) 30 (100%)

𝜒² (p) 16.425^∗ (<0.001^∗) 5.321^∗ (^MCp = 0.036^∗)

Estrogen receptor (ER)

Negative 12 (37.5%) 19 (47.5%) 13 (38.2%) 6 (37.5%) 13 (48.1%) 11 (36.7%)

Positive 20 (62.5%) 21 (52.5%) 21 (61.8%) 10 (62.5%) 14 (51.9%) 19 (63.3%)

𝜒² (p) 0.953 (0.621) 0.883 (0.643)

Progesterone receptor (PR)

Negative 12 (37.5%) 21 (52.5%) 15 (44.1%) 6 (37.5%) 14 (51.9%) 13 (43.3%)

Positive 20 (62.5%) 19 (47.5%) 19 (55.9%) 10 (62.5%) 13 (48.1%) 17 (56.7%)

χ² (p) 1.642 (0.440) 0.907 (^MCp = 0.635)

HER2

Negative 16 (50%) 21 (52.5%) 19 (55.9%) 6 (37.5%) 13 (48.1%) 16 (53.3%)

Positive 16 (50%) 19 (47.5%) 15 (44.1%) 10 (62.5%) 14 (51.9%) 14 (46.7%)

χ² (p) 0.232 (^MCp = 0.891) 1.049 (0.592)

Proliferation rate

Mean ± SD. 0.9 ± 0.4 1.7 ± 0.2 0.9 ± 0.4 0.9 ± 0.4 1.7 ± 0.1 1 ± 0.4

H (p) 57.825^∗ (<0.001^∗) 46.386^∗ (<0.001^∗)

	Positive Anti – HCMV IgG	Positive HCMV DNA in Tissue
Tumor grades
I	2 (6.3%)	1 (2.5%)	1 (2.9%)	2 (12.5%)	1 (3.7%)	1 (3.3%)
II	22 (68.8%)	29 (72.5%)	27 (79.4%)	12 (75%)	19 (70.4%)	23 (76.7%)
III	8 (25%)	10 (25%)	6 (17.6%)	2 (12.5%)	7 (25.9%)	6 (20%)
𝜒² (^MCp)	1.571 (0.814)	2.815 (0.589)
Type of tumor
Invasive ductal carcinoma	31 (96.9%)	33 (82.5%)	34 (100%)	15 (93.8%)	23 (85.2%)	30 (100%)
Mixed type	1 (3.1%)	4 (10%)	0 (0%)	1 (6.3%)	3 (11.1%)	0 (0%)
Invasive lobular carcinoma	0 (0%)	3 (7.5%)	0 (0%)	0 (0%)	1 (3.7%)	0 (0%)
𝜒² (^MCp)	7.399^∗ (0.036^∗)	5.227 (0.145)
Vascular invasion
No	13 (40.6%)	10 (25%)	0 (0%)	3 (18.8%)	2 (7.4%)	0 (0%)
Yes	19 (59.4%)	30 (75%)	34 (100%)	13 (81.3%)	25 (92.6%)	30 (100%)
𝜒² (p)	16.425^∗ (<0.001^∗)	5.321^∗ (^MCp = 0.036^∗)
Estrogen receptor (ER)
Negative	12 (37.5%)	19 (47.5%)	13 (38.2%)	6 (37.5%)	13 (48.1%)	11 (36.7%)
Positive	20 (62.5%)	21 (52.5%)	21 (61.8%)	10 (62.5%)	14 (51.9%)	19 (63.3%)
𝜒² (p)	0.953 (0.621)	0.883 (0.643)
Progesterone receptor (PR)
Negative	12 (37.5%)	21 (52.5%)	15 (44.1%)	6 (37.5%)	14 (51.9%)	13 (43.3%)
Positive	20 (62.5%)	19 (47.5%)	19 (55.9%)	10 (62.5%)	13 (48.1%)	17 (56.7%)
χ² (p)	1.642 (0.440)	0.907 (^MCp = 0.635)
HER2
Negative	16 (50%)	21 (52.5%)	19 (55.9%)	6 (37.5%)	13 (48.1%)	16 (53.3%)
Positive	16 (50%)	19 (47.5%)	15 (44.1%)	10 (62.5%)	14 (51.9%)	14 (46.7%)
χ² (p)	0.232 (^MCp = 0.891)	1.049 (0.592)
Proliferation rate
Mean ± SD.	0.9 ± 0.4	1.7 ± 0.2	0.9 ± 0.4	0.9 ± 0.4	1.7 ± 0.1	1 ± 0.4
H (p)	57.825^∗ (<0.001^∗)	46.386^∗ (<0.001^∗)

The data were assessed using chi square test (𝜒²), monte carlo (MC) and kruskal wallis test (H).p: p value for comparing between the studied groups.∗: Statistically significant at p ≤ 0.05.

3.4. Altered immune activation and inflammatory markers in primary and recurrent breast cancer patients

We assessed NLR and PLR in every sample and the results revealed a significant increase in the mean of both NLR and PLR in patients compared to a healthy control group where the advanced stage group displayed higher NLR relative to recurrent, early-stage, and benign groups. Regarding serum IL-17 and IL-22 levels, a marked increase was observed in breast cancer patients compared to both benign and healthy control groups where serum IL-17 was significantly increased in the recurrent group in comparison to early and advanced stage groups. On the other hand, no significant difference was shown between primary and recurrent breast cancer patients as regards IL-22 serum level (Table 4).

Table 4
Comparison between different groups regarding immune activation and inflammatory markers

Breast cancer patient stage (n = 82)

Early Advanced Recurrent Benign Control p

(stage I + II) (stage III + IV) (n = 34) (n = 44) (n = 40)

NLR

Mean ± SD. 3.2 ± 0.7 4 ± 0.9 3.4 ± 0.6 3.4 ± 0.8 2.1 ± 0.4 <0.001*

p ₀ <0.001^∗ <0.001^∗ <0.001^∗ <0.001^∗

Sig. bet. grps. p₁ = 0.202, p₂ = 0.001^∗ , p₃ = 0.1000, p₄ < 0.001^∗, p₅ = 0.239, p₆ = 0.001^∗

PLR

Mean ± SD. 126.7 ± 21.7 167.1 ± 33.9 157.7 ± 35.4 124 ± 13.9 105.7 ± 15.2 <0.001^∗

p ₀ <0.001^∗ <0.001^∗ <0.001^∗ <0.001^∗

Sig. bet. grps. p₁ < 0.001^∗, p₂ = 0.117, p₃ < 0.001^∗, p₄ < 0.001^∗, p₅ = 0.252, p₆ < 0.001^∗

IL-17

Mean ± SD. 116.9 ± 36.1 136.3 ± 35.1 163.8 ± 41.1 29.5 ± 8.8 28.5 ± 8.9 <0.001^∗

p ₀ <0.001^∗ <0.001^∗ <0.001^∗ 0.1000

Sig. bet. grps. p₁ < 0.001^∗, p₂ < 0.001^∗, p₃ < 0.001^∗, p₄ = 0.024^∗, p₅ < 0.001^∗, p₆ < 0.001^∗

IL-22

Mean ± SD. 156.5 ± 46.9 162.1 ± 51.2 156.9 ± 51.9 44.1 ± 13.8 43.1 ± 14.4 <0.001^∗

p ₀ <0.001^∗ <0.001^∗ <0.001^∗ 0.1000

Sig. bet. grps. p₁ = 0.1000, p₂ = 0.977, p₃ < 0.001^∗, p₄ = 0.968, p₅ < 0.001^∗, p₆ < 0.001^∗

	Breast cancer patient stage (n = 82)
NLR
Mean ± SD.	3.2 ± 0.7	4 ± 0.9	3.4 ± 0.6	3.4 ± 0.8	2.1 ± 0.4	<0.001*
p ₀	<0.001^∗	<0.001^∗	<0.001^∗	<0.001^∗
	Sig. bet. grps.	p₁ = 0.202, p₂ = 0.001^∗ , p₃ = 0.1000, p₄ < 0.001^∗, p₅ = 0.239, p₆ = 0.001^∗
PLR
Mean ± SD.	126.7 ± 21.7	167.1 ± 33.9	157.7 ± 35.4	124 ± 13.9	105.7 ± 15.2	<0.001^∗
p ₀	<0.001^∗	<0.001^∗	<0.001^∗	<0.001^∗
Sig. bet. grps.	p₁ < 0.001^∗, p₂ = 0.117, p₃ < 0.001^∗, p₄ < 0.001^∗, p₅ = 0.252, p₆ < 0.001^∗
IL-17
Mean ± SD.	116.9 ± 36.1	136.3 ± 35.1	163.8 ± 41.1	29.5 ± 8.8	28.5 ± 8.9	<0.001^∗
p ₀	<0.001^∗	<0.001^∗	<0.001^∗	0.1000
Sig. bet. grps.	p₁ < 0.001^∗, p₂ < 0.001^∗, p₃ < 0.001^∗, p₄ = 0.024^∗, p₅ < 0.001^∗, p₆ < 0.001^∗
IL-22
Mean ± SD.	156.5 ± 46.9	162.1 ± 51.2	156.9 ± 51.9	44.1 ± 13.8	43.1 ± 14.4	<0.001^∗
p ₀	<0.001^∗	<0.001^∗	<0.001^∗	0.1000
Sig. bet. grps.	p₁ = 0.1000, p₂ = 0.977, p₃ < 0.001^∗, p₄ = 0.968, p₅ < 0.001^∗, p₆ < 0.001^∗

The data were assessed using anova test (F), kruskal wallis test (H), pairwise comparison bet. Each 2 groups was done using post hoc Test.p: p value for comparing between the studied groups; p₀: p value for comparing between control and each studied group; p₁: comparing between recurrent and early; p₂: comparing between recurrent and advanced; p₃: comparing between recurrent and benign; p₄: comparing between early and advanced; p₅: comparing between benign and early; p₆: comparing between benign and advanced.^∗: Statistically significant at p ≤ 0.05.

3.5. Association of NLR and PLR with pathological characteristics in primary and recurrent breast cancer patients

As shown in Table 5, 54 patients (46.6%) had low NLR and 62 patients (53.4%) had high NLR whereas 73 patients (62.9%) had low PLR and 43 patients (37.1%) had high PLR. We analyzed the association between NLR and PLR with different pathological parameters and proliferation rate to elucidate the clinical relevance of these inflammatory markers in breast cancer. The results revealed that low NLR and PLR were significantly associated with early-stage (stage II) and lower proliferation rate while high NLR and PLR showed an opposite pattern (NLR: p = 0.016; p = 0.004, respectively) (PLR: p < 0.001; p = 0.001, respectively). However, no significant association was observed between both NLR and PLR and other pathological parameters.

Table 5
Relation between NLR and PLR with pathological parameters and proliferation rate in primary (n = 82) and recurrent breast cancer patients (n = 34) (total n = 116)

NLR PLR

Low-NLR < 3.0 High-NLR (≥3.0) p Low PLR < 150 High PLR ≥ 150 p

(n = 54) (n = 62) (n = 73) (n = 43)

Age

≤45 20 (37%) 18 (29%) 0.360 26 (35.6%) 12 (27.9%) 0.421

>45 34 (63%) 44 (71%) 47 (64.4%) 31 (72.1%)

Tumor stages

I 14 (25.9%) 8 (12.9%) 19 (26%) 3 (7%)

II 21 (38.9%) 15 (24.2%) 0.016^∗ 30 (41.1%) 6 (13.9%) <0.001^∗

III 16 (29.6%) 27 (43.5%) 17 (23.3%) 26 (60.5%)

IV 3 (5.6%) 12 (19.4%) 7 (9.6%) 8 (18.6%)

Tumor grade

I 3 (5.6%) 2 (3.2%) 4 (5.5%) 1 (2.3%) 0.729

II 36 (66.7%) 49 (79%) 0.323 53 (72.6%) 32 (74.4%)

III 15 (27.8%) 11 (17.7%) 16 (21.9%) 10 (23.3%)

Vascular invasion

No 19 (35.2%) 8 (12.9%) 0.005^∗ 18 (24.7%) 9 (20.9%) 0.646

Yes 35 (64.8%) 54 (87.1%) 55 (75.3%) 34 (79.1%)

Estrogen receptor (ER)

Negative 18 (33.3%) 30 (48.4%) 0.101 32 (43.8%) 16 (37.2%) 0.484

Positive 36 (66.7%) 32 (51.6%) 41 (56.2%) 27 (62.8%)

Progesterone receptor (PR)

Negative 19 (35.2%) 33 (53.2%) 0.051 32 (43.8%) 20 (46.5%) 0.780

Positive 35 (64.8%) 29 (46.8%) 41 (56.2%) 23 (53.5%)

HER2

Negative 29 (53.7%) 32 (51.6%) 0.822 39 (53.4%) 22 (51.2%) 0.814

Positive 25 (46.3%) 30 (48.4%) 34 (46.6%) 21 (48.8%)

Proliferation rate

Mean ± SD. 1.1 ± 0.4 1.3 ± 0.5 0.004^∗ 1.1 ± 0.5 1.4 ± 0.5 0.001^∗

	NLR		PLR
Age
≤45	20 (37%)	18 (29%)	0.360	26 (35.6%)	12 (27.9%)	0.421
>45	34 (63%)	44 (71%)		47 (64.4%)	31 (72.1%)
Tumor stages
I	14 (25.9%)	8 (12.9%)		19 (26%)	3 (7%)
II	21 (38.9%)	15 (24.2%)	0.016^∗	30 (41.1%)	6 (13.9%)	<0.001^∗
III	16 (29.6%)	27 (43.5%)		17 (23.3%)	26 (60.5%)
IV	3 (5.6%)	12 (19.4%)		7 (9.6%)	8 (18.6%)
Tumor grade
I	3 (5.6%)	2 (3.2%)		4 (5.5%)	1 (2.3%)	0.729
II	36 (66.7%)	49 (79%)	0.323	53 (72.6%)	32 (74.4%)
III	15 (27.8%)	11 (17.7%)		16 (21.9%)	10 (23.3%)
Vascular invasion
No	19 (35.2%)	8 (12.9%)	0.005^∗	18 (24.7%)	9 (20.9%)	0.646
Yes	35 (64.8%)	54 (87.1%)		55 (75.3%)	34 (79.1%)
Estrogen receptor (ER)
Negative	18 (33.3%)	30 (48.4%)	0.101	32 (43.8%)	16 (37.2%)	0.484
Positive	36 (66.7%)	32 (51.6%)		41 (56.2%)	27 (62.8%)
Progesterone receptor (PR)
Negative	19 (35.2%)	33 (53.2%)	0.051	32 (43.8%)	20 (46.5%)	0.780
Positive	35 (64.8%)	29 (46.8%)		41 (56.2%)	23 (53.5%)
HER2
Negative	29 (53.7%)	32 (51.6%)	0.822	39 (53.4%)	22 (51.2%)	0.814
Positive	25 (46.3%)	30 (48.4%)		34 (46.6%)	21 (48.8%)
Proliferation rate
Mean ± SD.	1.1 ± 0.4	1.3 ± 0.5	0.004^∗	1.1 ± 0.5	1.4 ± 0.5	0.001^∗

The data were assessed using chi square test (𝜒²), fisher Exact (FE), monte carlo (MC), and mann whitney (U) test. p: p value for comparing between the studied groups.^∗: Statistically significant at p ≤ 0.05.

3.6. Association of serum IL-17 and IL-22 with pathological characteristics, NLR and PLR in primary and recurrent breast cancer patients

We further assessed the association between serum cytokines (IL-17 and IL-22) and different pathological parameters in both primary and recurrent breast cancer patients. The results showed that serum IL-17 was significantly associated with tumor stage (p = 0.036) and vascular invasion (p < 0.001) being higher in patients with stages (stages II, IV) and those with vascular invasion. Regarding IL-22 serum level, a significant association was observed between high IL-22 levels and vascular invasion (p = 0.007). Regarding association NLR and PLR, it was apparent from the current results that high serum IL-17 level was significantly associated with high NLR (p = 0.001). However, no significant association was observed between serum IL-22 with either NLR or PLR (Table 6).

Table 6
Relation between serum IL-17 and IL-22 with pathological parameters, NLR and PLR in primary (n = 82) and recurrent breast cancer patients (n = 34) (total n = 116)

Pathological parameters Serum IL-17 Serum IL-22

Mean ± SD. P Mean ± SD. P

Tumor stages

I 120.2 ± 30.7 133.9 ± 36.8

II 132.9 ± 42.5 0.036^∗ 160.4 ± 48.2 0.053

III 146.9 ± 44.3 164.9 ± 53.9

IV 152.2 ± 34 174.1 ± 49

Tumor grade

I 133.3 ± 32.4 165.3 ± 39.2

II 142.3 ± 42.8 0.220 159.2 ± 51.4 0.867

III 125.9 ± 37.4 155.1 ± 48.8

Vascular invasion

No 102.7 ± 23 <0.001^∗ 136.4 ± 44.5 0.007^∗

Yes 148.9 ± 39.6 165.6 ± 49.4

Estrogen receptor (ER)

Negative 138.3 ± 36.1 0.978 161.6 ± 47.4 0.613

Positive 138.1 ± 44.9 156.8 ± 51.5

Progesterone receptor (PR)

Negative 143.1 ± 40.4 0.248 161.1 ± 49.3 0.662

Positive 134.2 ± 41.9 157 ± 50.3

HER2

Negative 138.1 ± 42.3 0.971 161.9 ± 52 0.487

Positive 138.3 ± 40.5 155.4 ± 47.3

Other immune activation and inflammatory markers

NLR

Low-NLR (<3.0) 124.7 ± 38.6 0.001^∗ 152.7 ± 50.8 0.220

High-NLR (≥3.0) 149.9 ± 40.2 164.1 ± 48.5

PLR

Low-PLR (<150) 131.7 ± 40.5 0.061 153.2 ± 47.4 0.181

High-PLR (≥150) 146.2 ± 41.3 165.7 ± 52

Pathological parameters	Serum IL-17		Serum IL-22
Tumor stages
I	120.2 ± 30.7		133.9 ± 36.8
II	132.9 ± 42.5	0.036^∗	160.4 ± 48.2	0.053
III	146.9 ± 44.3		164.9 ± 53.9
IV	152.2 ± 34		174.1 ± 49
Tumor grade
I	133.3 ± 32.4		165.3 ± 39.2
II	142.3 ± 42.8	0.220	159.2 ± 51.4	0.867
III	125.9 ± 37.4		155.1 ± 48.8
Vascular invasion
No	102.7 ± 23	<0.001^∗	136.4 ± 44.5	0.007^∗
Yes	148.9 ± 39.6		165.6 ± 49.4
Estrogen receptor (ER)
Negative	138.3 ± 36.1	0.978	161.6 ± 47.4	0.613
Positive	138.1 ± 44.9		156.8 ± 51.5
Progesterone receptor (PR)
Negative	143.1 ± 40.4	0.248	161.1 ± 49.3	0.662
Positive	134.2 ± 41.9		157 ± 50.3
HER2
Negative	138.1 ± 42.3	0.971	161.9 ± 52	0.487
Positive	138.3 ± 40.5		155.4 ± 47.3
Other immune activation and inflammatory markers
NLR
Low-NLR (<3.0)	124.7 ± 38.6	0.001^∗	152.7 ± 50.8	0.220
High-NLR (≥3.0)	149.9 ± 40.2		164.1 ± 48.5
PLR
Low-PLR (<150)	131.7 ± 40.5	0.061	153.2 ± 47.4	0.181
High-PLR (≥150)	146.2 ± 41.3		165.7 ± 52

^∗: Statistically significant at p ≤ 0.05.

3.7. Association of HCMV prevalence with different immune activation and inflammatory markers in primary and recurrent breast cancer patients

Concerning the relationship between HCMV prevalence and the studied immune activation and inflammatory markers, our results revealed a significant association between positive anti-HCMV IgG and serum IL-17 (p < 0.001) as well as a significant relation was observed between positive tissue prevalence of HCMV with both high NLR (p = 0.007) and serum IL-17 (p < 0.001). On the other hand, no significant association was observed between HCMV prevalence with either PLR or serum IL-22 (Table 7).

Table 7
Relation between HCMV prevalence and different immune activation and inflammatory markers in primary breast cancer patients and recurrent groups

Immune activation and inflammatory markers Positive anti-HCMV IgG (n = 106) Positive HCMV DNA in tissue (n = 73)

NLR

Low-NLR (<3.0) 49 (90.7%) 27 (50%)

High-NLR (≥3.0) 57 (91.9%) 46 (74.2%)

p 1.000 0.007^∗

PLR

Low-PLR (<150) 67 (91.8%) 45 (61.6%)

High-PLR (≥150) 39 (90.7%) 28 (65.1%)

p 0.841 0.708

Serum IL-17

Mean ± SD. 141.1 ± 41.7 156.2 ± 39.1

p < 0.001^∗ <0.001^∗

Serum IL-22

Mean ± SD. 160.2 ± 49.8 163.9 ± 50.7

p 0.321 0.150

Immune activation and inflammatory markers	Positive anti-HCMV IgG (n = 106)	Positive HCMV DNA in tissue (n = 73)
NLR
Low-NLR (<3.0)	49 (90.7%)	27 (50%)
High-NLR (≥3.0)	57 (91.9%)	46 (74.2%)
p	1.000	0.007^∗
PLR
Low-PLR (<150)	67 (91.8%)	45 (61.6%)
High-PLR (≥150)	39 (90.7%)	28 (65.1%)
p	0.841	0.708
Serum IL-17
Mean ± SD.	141.1 ± 41.7	156.2 ± 39.1
p	< 0.001^∗	<0.001^∗
Serum IL-22
Mean ± SD.	160.2 ± 49.8	163.9 ± 50.7
p	0.321	0.150

The data were assessed using chi-square test (χ²) and student (t) test. ^∗: Statistically significant at p ≤ 0.05.

4. Discussion

It has been suggested that viruses may be an additional high-risk factor closely associated with human breast cancer [19]. Although some viruses have been detected in breast cancer specimens, findings regarding the HCMV’s role in breast cancer are still controversial.

In the present study, we investigated a possible involvement of HCMV in the pathogenesis of breast cancer. Our results showed that the seropositivity of anti-HCMV IgG and tissue prevalence of HCMV DNA were higher in recurrent and primary breast cancer patients’ groups compared to benign and control groups. This may be related to the initiator or promoter characters of the virus where it can act directly on the cancer cells or evade immune response and get a shelter in immunologically poor tumor environment [20]. Therefore, HCMV and cancerous cells are considered two players for one goal which is to escape from immune response. In concordance, Cox et al. [21] reported a statistically significant correlation between high HCMV IgG levels and breast cancer development. However, Mohammedizaden et al. [22] failed to find any relationship between HCMV and breast cancer development that might be attributed to the limitation of molecular methods and an absence of virus after carcinogenesis.

Regarding the association between HCMV prevalence and clinicopathological parameters in breast cancer, the present study revealed that high anti-HCMV IgG and positive tissue prevalence of HCMV were significantly associated with vascular invasion and proliferation rate. In line with our results, it was reported that US28 HCMV gene induced vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX2) and Stat3 activation through IL-6 upregulation [23] as well as cmvIL-10 was a potent enhancer of invasion and expression of genes strongly linked to the metastatic spread of breast cancer [24]. Moreover, HCMV-encoded proteins may enable the cells to exceed the G1-phase to facilitate rapid cell division [25].

It has been reported that inflammation plays a crucial role in the defence against several pathogens, including viruses, as well as it could mediate the initiation and promotion of tumours, angiogenesis, and metastasis [26].

Peripheral blood-derived inflammation-based markers such as NLR and PLR have been recently evaluated as prognostic markers in solid tumors [27]. It has been reported that patients with an increased NLR/PLR were associated with advanced stages of malignancy and unfavorable tumor characteristics [28]. In agreement, our results revealed that high NLR and PLR were significantly associated with advanced stage and high proliferation rate in primary and recurrent breast cancer patients. Neutrophils were observed to be associated with tumor proliferation and distant metastasis by the release of several inflammatory mediators, such as matrix metalloproteinase-9, neutrophil elastase, and IL-8 [29]. In addition, NLR and PLR are thought to be representatives of the ongoing inflammatory process in the tumor microenvironment where neutrophils and platelets are indicated to be incriminated in tumor activities in vivo [30]. These results can suggest that imbalances of the peripheral immune cells may provide insight into underlying tumor progression in breast cancer.

In addition, our results revealed a marked increase in serum IL-17 and IL-22 levels in patients compared to both benign and control groups where high serum IL-17 level was significantly associated with advanced stage, vascular invasion and high NLR. On the other hand, high IL-22 levels showed a significant association with vascular invasion only. In line, it has been reported that Th17 cells and their cytokines (IL-17 and IL-22) could contribute to cancer [31]. IL-17 was observed to enhance tumour aggressiveness via induction of angiogenic factors, such as chemokine CXCL8, MMP-2, MMP-9, and VEGF [32] as well as it was found that IL-17A affected different steps of metastasis such as migration of tumorigenic neutrophils and tumour cells to distant metastatic sites with production of IL-6 and CCL20 [33].

More importantly, the present study showed that high NLR and serum IL-17 levels were significantly associated with positive tissue prevalence of HCMV DNA. In line, it has been shown that HCMV can be reactivated as a part of inflammatory response and that HCMV infected cells promote inflammatory and angiogenic secretome through secretion of cytokines that induce haemangiogenesis, lymphangiogenesis, cell proliferation as well as immune evasion/immunosuppression [34]. IL-17 was also shown to play an important role in protecting permissive cells from virus-induced apoptosis via induction of anti-apoptotic proteins (i.e., Bcl-xL and Bcl-2), as well as through the desensitization of CTL killing of these cells [35].

It can be concluded from these findings that the presence of HCMV infection may have a vital role in breast cancer development. Therefore, HCMV immunization or elimination may represent a potential therapeutic target in breast cancer. In addition, NLR could have a possible connection with HCMV infection and that imbalanced peripheral blood cells and cytokines may result in disordered immune response.

Footnotes

Acknowledgements

We would like to thank all the patients included in this study; without them it could not have been done. We would also like to show our gratitude to the Pathology Department, Medical Research Institute, Alexandria University.

Ethical statement

Written consents were obtained from all subjects participating in the study. The collection and use of blood and tissue samples for this study were approved previously by the ethical committee of the medical research institute, Alexandria University. The study fulfills the principles delineated in the Declaration of Helsinki for use of human tissue or subjects.

Conflicts of interest

The authors declare that there are no conflicts of interest.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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	Breast cancer patient stage (n = 82)
	Early	Advanced	Recurrent	Benign	Control	p
	(stage I + II)	(stage III + IV)	(n = 34)	(n = 44)	(n = 40)
Anti-HCMV IgM
Negative	37 (100%)	45 (100%)	34 (100%)	44 (100%)	40 (100%)
Positive	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	—
Mean ± SD.	0.04 ± 0.01	0.05 ± 0.02	0.05 ± 0.02	0.05 ± 0.02	0.04 ± 0.01
p ₀	0.131	0.062	0.087	0.121
Anti-HCMV IgG
Negative	5 (13.5%)	5 (11.1%)	0 (0%)	8 (18.2%)	16 (40%)
Positive	32 (86.5%)	40 (88.9%)	34 (100%)	36 (81.8%)	24 (60%)	<0.001^∗
Mean ± SD.	1.6 ± 0.4	1.7 ± 0.6	1.9 ± 0.2	1.7 ± 0.5	1.1 ± 0.5
p ₀	<0.001^∗	<0.001^∗	<0.001^∗	<0.001^∗
Sig. bet. grps.	p₁ = 0.026^∗, p₂ = 0.276, p₃ = 0.245, p₄ = 0.207, p₅ = 0.240, p₆ = 0.933
HCMV DNA in tissue
Negative	21 (56.8%)	18 (40%)	4 (11.8%)	38 (86.4%)	—	<0.001^∗
Positive	16 (43.2%)	27 (60%)	30 (88.2%)	6 (13.6%)	—

	Positive Anti – HCMV IgG			Positive HCMV DNA in Tissue
Pathological characteristics	Early	Advanced	Recurrent	Early	Advanced	Recurrent
	(stages I + II)	(stages III + IV)	(n = 34)	(stages I + II)	stages III + IV	(n = 30)
	(n = 32)	(n = 40)		(n = 16)	(n = 27)
Tumor grades
I	2 (6.3%)	1 (2.5%)	1 (2.9%)	2 (12.5%)	1 (3.7%)	1 (3.3%)
II	22 (68.8%)	29 (72.5%)	27 (79.4%)	12 (75%)	19 (70.4%)	23 (76.7%)
III	8 (25%)	10 (25%)	6 (17.6%)	2 (12.5%)	7 (25.9%)	6 (20%)
𝜒² (^MCp)	1.571 (0.814)			2.815 (0.589)
Type of tumor
Invasive ductal carcinoma	31 (96.9%)	33 (82.5%)	34 (100%)	15 (93.8%)	23 (85.2%)	30 (100%)
Mixed type	1 (3.1%)	4 (10%)	0 (0%)	1 (6.3%)	3 (11.1%)	0 (0%)
Invasive lobular carcinoma	0 (0%)	3 (7.5%)	0 (0%)	0 (0%)	1 (3.7%)	0 (0%)
𝜒² (^MCp)	7.399^∗ (0.036^∗)			5.227 (0.145)
Vascular invasion
No	13 (40.6%)	10 (25%)	0 (0%)	3 (18.8%)	2 (7.4%)	0 (0%)
Yes	19 (59.4%)	30 (75%)	34 (100%)	13 (81.3%)	25 (92.6%)	30 (100%)
𝜒² (p)	16.425^∗ (<0.001^∗)			5.321^∗ (^MCp = 0.036^∗)
Estrogen receptor (ER)
Negative	12 (37.5%)	19 (47.5%)	13 (38.2%)	6 (37.5%)	13 (48.1%)	11 (36.7%)
Positive	20 (62.5%)	21 (52.5%)	21 (61.8%)	10 (62.5%)	14 (51.9%)	19 (63.3%)
𝜒² (p)	0.953 (0.621)			0.883 (0.643)
Progesterone receptor (PR)
Negative	12 (37.5%)	21 (52.5%)	15 (44.1%)	6 (37.5%)	14 (51.9%)	13 (43.3%)
Positive	20 (62.5%)	19 (47.5%)	19 (55.9%)	10 (62.5%)	13 (48.1%)	17 (56.7%)
χ² (p)	1.642 (0.440)			0.907 (^MCp = 0.635)
HER2
Negative	16 (50%)	21 (52.5%)	19 (55.9%)	6 (37.5%)	13 (48.1%)	16 (53.3%)
Positive	16 (50%)	19 (47.5%)	15 (44.1%)	10 (62.5%)	14 (51.9%)	14 (46.7%)
χ² (p)	0.232 (^MCp = 0.891)			1.049 (0.592)
Proliferation rate
Mean ± SD.	0.9 ± 0.4	1.7 ± 0.2	0.9 ± 0.4	0.9 ± 0.4	1.7 ± 0.1	1 ± 0.4
H (p)	57.825^∗ (<0.001^∗)			46.386^∗ (<0.001^∗)

	NLR			PLR
	Low-NLR < 3.0	High-NLR (≥3.0)	p	Low PLR < 150	High PLR ≥ 150	p
	(n = 54)	(n = 62)		(n = 73)	(n = 43)
Age
≤45	20 (37%)	18 (29%)	0.360	26 (35.6%)	12 (27.9%)	0.421
>45	34 (63%)	44 (71%)		47 (64.4%)	31 (72.1%)
Tumor stages
I	14 (25.9%)	8 (12.9%)		19 (26%)	3 (7%)
II	21 (38.9%)	15 (24.2%)	0.016^∗	30 (41.1%)	6 (13.9%)	<0.001^∗
III	16 (29.6%)	27 (43.5%)		17 (23.3%)	26 (60.5%)
IV	3 (5.6%)	12 (19.4%)		7 (9.6%)	8 (18.6%)
Tumor grade
I	3 (5.6%)	2 (3.2%)		4 (5.5%)	1 (2.3%)	0.729
II	36 (66.7%)	49 (79%)	0.323	53 (72.6%)	32 (74.4%)
III	15 (27.8%)	11 (17.7%)		16 (21.9%)	10 (23.3%)
Vascular invasion
No	19 (35.2%)	8 (12.9%)	0.005^∗	18 (24.7%)	9 (20.9%)	0.646
Yes	35 (64.8%)	54 (87.1%)		55 (75.3%)	34 (79.1%)
Estrogen receptor (ER)
Negative	18 (33.3%)	30 (48.4%)	0.101	32 (43.8%)	16 (37.2%)	0.484
Positive	36 (66.7%)	32 (51.6%)		41 (56.2%)	27 (62.8%)
Progesterone receptor (PR)
Negative	19 (35.2%)	33 (53.2%)	0.051	32 (43.8%)	20 (46.5%)	0.780
Positive	35 (64.8%)	29 (46.8%)		41 (56.2%)	23 (53.5%)
HER2
Negative	29 (53.7%)	32 (51.6%)	0.822	39 (53.4%)	22 (51.2%)	0.814
Positive	25 (46.3%)	30 (48.4%)		34 (46.6%)	21 (48.8%)
Proliferation rate
Mean ± SD.	1.1 ± 0.4	1.3 ± 0.5	0.004^∗	1.1 ± 0.5	1.4 ± 0.5	0.001^∗

The association of human cytomegalovirus with biomarkers of inflammation and immune activation in breast cancer

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Material and methods

2.1. Subjects

2.2. Methods

2.2.1. Analysis of blood sample

2.2.1.1. Detection of anti-HCMV IgM and IgG antibodies:

2.2.1.2. Detection of IL-17and IL-22 cytokine levels:

2.2.1.3. Assessment of NLR and PLR:

2.2.2. Preparation of tissue samples

2.2.3. DNA extraction

2.2.4. Detection and quantification of HCMV-DNA by real-time PCR

2.2.5. Assessment of cell proliferation rate by MTT assay

2.2.6. Statistical analysis

3. Results

3.1. Demographic and pathological data of the studied breast cancer patients

Footnotes

Acknowledgements

Ethical statement

Conflicts of interest

Funding

References