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The Pretreatment Tumor Infiltrating T Lymphocytes (CD8+, CD4+, FOXP3+) and Systemic Neutrophil-Lymphocytes Ratio in Definitively Treated Cervical Cancer Patients: The Correlation to Clinicopathological Factors and Survival
ISSN: 1948-5956

Journal of Cancer Science & Therapy
Open Access

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The Pretreatment Tumor Infiltrating T Lymphocytes (CD8+, CD4+, FOXP3+) and Systemic Neutrophil-Lymphocytes Ratio in Definitively Treated Cervical Cancer Patients: The Correlation to Clinicopathological Factors and Survival

Hala Ahmed Zaghloul Isamil El Lathy1,2*, Ahlam Dohal2 and Miral Mashhour3
1Department of Clinical Oncology and Nuclear Medicine, Alexanderia University, Egypt
2Department of Radiation Oncology, King Fahad Specialist Hospital, Al-Dammam, Saudi Arabia
3Department of Pathology, King Fahad Specialist Hospital, Al-Dammam, Saudi Arabia
*Corresponding Author: Hala Ahmed Zaghloul Isamil El Lathy, Clinical Oncology and Nuclear Medicine Department, Faculty of Medicine, Alexandria University, Egypt & Radiation Oncology Department King Fahad Specialist Hospital Al Dammam KSA, Egypt, Tel: +00201223926059, Fax: +002034290746, Email: [email protected]

Received Date: Apr 12, 2018 / Accepted Date: May 08, 2018 / Published Date: May 10, 2018

Abstract

Purpose: To link pretreatment tumor infiltrating T-lymphocytes (TILs) (CD8+, CD4+, FOXP3+) and systemic neutrophil to lymphocyte ratio (NLR) to different clinical/pathological elements. Consequently, emphasizing their impact in predicting the outcome in definitively treated cervical cancer patients.

Methods: The most relevant clinical/pathological factors were used to establish a link with pre-treatment NLR and densities of TILs (CD8+, CD4+, FOXP3+) in cervical biopsies. The predictive significance of pre-treatment TILs and NLR, both for disease free survival (DFS) and overall survival (OS) were evaluated using Log rank alongside Cox regression analysis.

Results: Radical hysterectomies followed by adjuvant radiation with or without chemotherapy were offered to 28 patients, while the remaining twenty eligible patients received curative concurrent chemo-radiation. Augmented levels of CD8+, CD8+/CD4+, while reduced levels of FOXP33+ and NLR were linked to node negative, radical hysterectomies and early stages. Cox-regression demonstrated that augmented levels for NLR and nodal disease were individually correlated to dismal prognosis with HR 3.06 (95%confidence interval [CI], 3.45-9.24), 5.63 (95% CI, 2.61-9.32) for OS and (HR 8.21 (95% CI, 4.21-16.53) and 5.32 (95% CI, 2.37-10.24)for DFS, respectively. On the contrary, FOXP3+≥19 and CD8+/CD4+< 2 had a substantial link to reduced OS (HR 4.37(95% CI, 2.48-12.37), 2.31(95% CI, 2.34-9.32) and worsened DFS (HR 3.61 ( 95% CI , 1.38-9.32), 4.32(95%CI, 3.12-8.34).

Conclusion: The pretreatment NLR, CD8+, FOXP3+and C8+/CD4+showed a substantial link to various clinical/pathological prognostic characteristics for curatively treated cervical cancer patients. Furthermore, the prognostic prospective of the tested indicators could be emphasized.

Keywords: Tumour infiltrating lymphocytes; Neutrophil lymphocytes ratio; Cervical cancer patients; Haemoglobin; Antitumor immune capacity

Introduction

Cervical cancer claims the lives of around nine percent of all women plagued with cancer annually [1]. Platinum salts were established as the core regimen of curative chemo-radiation for locally advanced cervical cancer (stage IIB to IV) with an approximate 6% enhancement in 5-year overall survival [2]. The devised conventional clinical-pathological indicators of outcome in cervical cancer patients as lymph node, tumor size and pretreatment hemoglobin level, were proved to be incompetent due to unreliable specificity and sensitivity [3-6]. Accordingly, supplementary prognostic variables are considered indispensable to augment accuracy in clinical outcome predictions in definitively treated cervical cancer patients.

Of late cancer pathogenesis have developed greatly with substantial focus on the imperative power of the host immune system in tumor milieu. Emergent data showed that Neutrophil to lymphocyte ratio (NLR) possessed prognostic implication for patients with different types of cancers [713]. The (NLR) is an indicator for evaluating the systemic equilibrium between neutrophil-derived tumor induced inflammation and host lymphocyte-associated tumoricidal effect [14,15]. A higher level of NLR may highlight a possible trend towards augmented tumor induced inflammation alongside reduced capacity for tumor cell killing governed by host immunity. In addition, the immune response guarding against carcinogenesis, defined as cancer immunomodulation is showcased by tumor-infiltrating lymphocytes (TILs), permeating either the tumor associated epithelium or the surrounding stroma [16]. The Fork head box P3- positive (Foxp3+) regulatory T cells (Tregs), CD8+ T cells, and CD4+T cells are considered as essentials for immune tolerance and” surveillance [17]. “CD8+ T cells are cytotoxic inducing direct tumor cell killing [18]. The Substantial improvement in survival outcomes of many cancer patients had been directly associated with augmented levels of cytotoxic CD8+T cells [19-25]. Furthermore, CD4+ T cells can hold innumerable effector tasks, including promoting tumor-directed cytotoxic T cells involved in tumor cell lysis [18]. Conversely, regulatory T (Treg) cells reign a considerable oppressive impact on direct tumor cell killing modulated by host immune response. Moreover, Treg cells are further sub classified according to surface marker expression such as cluster of differentiation CD4+, the interleukin (IL)-2 receptor alpha chain CD25+, and the fork-head family transcription factor FOXP3+ [26,28]. The FOXP3 gene exerts dominant effect on CD4+ CD25+ T cells by reverting them toward a suppressive T regulatory modulation as confirmed in FoxP3+ Tregs cells retrieved from large cohort of cancer patients [29-32]. Consequently, FOXP3 is at present recognized as the most relevant indicator of Treg cells, and its identification provides a deeper comprehension of the control exerted by Treg cells on exacerbation of autoimmune diseases, alongside transplantation and immunity against carcinogenesis. Further to this, the amplified expression of suppressive Treg cells (CD4+CD25+FOXP3+), is significantly associated to worse clinical outcomes in certain cancers [33,34]. However, the precise role that each TILs exerts in optimizing host immune response in cervical cancer patients necessitates additional elaboration.”

As a result, our work reasoning revolved around correlating pretreatment of NLR and TILs (CD8+, CD4+ and FOXP3+) clinicopathological factors, so that their prognostic prospects could be highlighted in the context of definitively treated patients with cervical cancer.

Materials and Methods

A retrospective review of the patients getting treatment or getting referred to the Radiation Oncology Department at Clinical Oncology Department of the Alexandria University and King Fahad Specialist Hospital Al Dammam, during the time period between Jan 2013 and Dec 2016 was conducted once IRB approval have been acquired. The patients also consented to their participation. The medical records were examined to pick patients with non-metastatic cervical cancer and had undergone cervical biopsies before surgical procedure or radiation therapy. Age, grade, stage, histology, response to chemo, were included as prognostic variables. NLR, measured through the pretreatment peripheral blood cell count. We selectively included histologically proven, localized uterine cervix cancer FIGO stages IA1, IA2, IB1 and IIA1 who had radical hysterectomy and pelvic lymph nodes dissection. Additionally, adjuvant radiation was implemented based on risk factors (>1/3 stromal invasion, lympho-vascular invasion or tumor size >4 cm). While adjuvant chemo-radiation was offered to patients with positive pelvic nodes & surgical margins, alongside microscopic parametrial involvement. Whole pelvic irradiation 45-50.4 Gy in 1.8 Gy /25-28 fractions ± weekly cisplatin 40 mg/m2. We also included FIGO Stages IB2, IIA2, IIB, IIIA, IIIB and IVA patients who had definitive chemo-radiation 50.4 Gy in 1.8 Gy /28 fractions given to the tumor and pelvic lymph nodes given concurrently with weekly cisplatin 40 mg/m2 to be followed by Image guided. High Dose Rate Intra-cavitary Brachytherapy 7Gy x 4 fractions to deliver total dose of 85–90 Gy to high risk CTV. We excluded patients who demonstrated evidence of distant dissemination at initial inclusion or during treatment.

Immunohistochemistry

This staining was implemented on 4-5-mm paraffin sections. The main antibody consisted of mouse monoclonal antihuman anti-CD8 (DakoCytomation, Glostrup, Denmark; 1:100 dilution), anti-CD4 (Novocastra; 1:30), anti-FOXP3 (Abcam, Cambridge, UK; 1: 50). Tonsil tissue was used as a positive control for all the antibodies. T- lympocytes positive for CD4/CD8 exhibited cellular membranous immunostaining while FOXP3 positive T cells exhibited nuclear staining. The TILs were counted using ocular grid and a light microscope. Mean scoring of intraepithelial FOXP3+, CD8+ and CD4+ T cells in addition to positive cells within the immediate peri-tumoral stroma (within the same high-powered field; HPF) were manually counted twice for 3HPFs (x400) after which each marker’s positive cells mean was estimated.

Patient Follow-Up

All the follow ups took place at regular intervals till December 2016, or till the time of a participant’s death. This follow-up in essence took place every four months. The process included lab tests and imaging alongside physical exams. The period varied between a timespan of three to 48 months, while the median stood at 26 months. The OS was calculated from the diagnosis till the patient’s demise date. The patients that dropped out had their last follow-up dates were registered as the end point of follow up. DFS was recorded from surgery till relapse or the day of last visit.

Statistical consideration

The “link between the aforementioned TILs, NLR, and other clinic-pathological factors were analyzed with the Spearman test. The influences of the studied variables (TILs and NLR) on studied patients related clinical characteristics were evaluated by Mann-Whitney U test (between 2 groups) or Kruskal- Wallis test (≥3 groups). The receiver operator curves (ROC) were implemented to define cutoffs values of the tested variables. A 1.0 area under of the curve (AUC) meant the test was perfect, while 0.5 value” meant the test was non-informative. The variables for prognosis, as outlined through the univariate analysis at a value of P<0.1 were examined through the Cox model. The statistical significance was decided to be P<0.05. The OS and DFS was measured through the “Kaplan Meier method. Log-rank and Cox regression were conducted to link survival outcomes to different clinical and pathological factors. All these processes took place with the help of SPSS 16.0.”

Results

Forty-eight cervical cancer patients were eligible as per the defined inclusion criteria. Of this, around 41.7% or 20 patients had curative chemo-radiation, while another 28 or 58.3% had hysterectomy. This was then followed with adjuvant radiation for 12 or 25% of the sample, or chemo-radiation for 33.3% or 16 patients, as per the postoperative risks (Table 1). The age range for the patients registered between 35 and 72. In terms of histology, 79.2% or 38 patients in the study had squamous cell carcinoma, while 20.8% or 10 patients had uterine cervix adenocarcinoma.

Characteristics No. of Patients %
Age, years
Median 52 ---
Range  (35-72) --
<52 22 46%
≥ 52 26 54%
Zubrod performance scale
0 29 60.4%
1 15 31.3%
2 4 8.3%
Histopathological type
Squamous cell carcinoma 38 79.2%
Well differentiated- Grade 1 1 2.6 %
Moderately differentiated - Grade 2 9 23.7%
Poorly differentiated – Grade 3 28 73.7%
Adenocarcinoma 10 20.8%
Well differentiated- Grade 1 0  
Moderately differentiated- Grade 2 4 40%
Poorly differentiated- Grade 3 6 60%
Stage group
IB1 12 25%
IB2 11 23%
IIA1 5 10.4%
IIA2 4 8.3%
IIB 8 16.6%
IIIA 4 8.3%
IIIB 2 4.2%
IVA 2 4.2%
N stage
N0 25 52.1%
N1 23 47.9%
Definitive treatment
I-Radical hysterectomy 28 58.3%
II-Concurrent chemoradiation 20 41.7%
A-Radical hysterectomy and adjuvant radiation 12 25%
Tumor>4 cm 7 14.6%
>1/3 stromal invasion 3 6.3%
Lymphovascular invasion 2 4.1%
B-Radical hysterectomy and adjuvant chemoradiation 16 33.3%
Positive pelvic lymph nodes 3 6.3%
Parametrical involvement 7 14.5%
Positive margin 6 12.5%
C- Definitive concurrent chemoradiation 20 41.7%
IB2 5 10.4%
IIA2 4 8.3%
IIB 3 6.3%
IIIA 4 8.3%
IIIB 2 4.2%
IVA 2 4.2%
Inflammatory Response biomarkers (I-NLR)
Median 1.95 --
Mean ± SD 1.98 ± 6.7 --
Range  (0.6-24) --
ROC Cut off 2 --
<2 26 54.2%
≥2 22 45.8%
II-Tumor Infiltrating Lymphocytes (TILs)
1- CD8+TILs - Cells/HPF
Median 58 --
Mean ± SD 59 ± 31.3 --
Range 6.55-190.52 --
ROC Cutoff 64 --
<64 21 43.7%
≥64 27 56.3%
2-CD4+TILs - Cells/HPF
Median 30 --
Mean ±SD 31.7 ± 31.5 --
Range 8.7-220.5 --
ROC Cutoff 32 --
<32 23 47.9%
≥ 32 25 52.1%
3- CD4+ FOXP3+TILs - Cells/HPF
Median 18 --
Mean ± SD 17.3 ± 8.31 --
Range 0.98-32.4 --
ROC Cutoff 19 --
<19 28 58.3%
≥ 19 20 41.7 %
4-CD8+/CD4+Ratio
Median 1.9 --
ROC Cutoff 2 --
<2 19 39.6%
≥ 2 29 60.4%
Radiological /pathological response in Definitive concurrent chemoradiation patients 20 --
Complete response 12 60%
Partial response≥30% 4 20%
Stable disease 2 10%
Progressive disease 2 10%

Table 1: Patient characteristics at baseline (N=48).

NLR’s median value was 1.95 furthermore, the mean for CD8+, CD4+ and FOXP3+ CD4+ positive T lymphocytes were 58, 30 and 18 Cells/HPF. Furthermore, TILs ratio CD8+/ CD4+ was 1.9. Finally, the characteristics for all other baseline elements can be reviewed in (Table 1).”

ROC showed NLR baseline cutoff of 2, where the AUC was 0.873, for forecasting DFS with a specificity level of 82.9% and sensitivity level of 97.4%. Furthermore, CD8+, FOXP3+, CD8+/CD4+ and CD4+ cutoffs stood at 64,19, 2 and 32 with AUC that mounted up to 0.794, 0.854, 0.859 and 0.721 correspondingly. The estimated sensitivities for CD8+, FOXP3+, CD8+/CD4+ and CD4+were 93.3%, 94%, 92.3% and 85.1%, in that order. While their projected specificities were 84.3%, 84.5%, 81.5% and 75.3%, respectively in forecasting DFS (Figure 1 and 2). The sample was split into two based on the optimum cutoffs. It was observed that those with poorly differentiated histology, regional nodal disease, stage III-IV, and definitive chemo-radiation had an accentuated NLR value as opposed to others (Table 2). In addition, elevated expression for CD8+and CD8+/CD4+” were linked to early disease stages, node negative disease and radical hysterectomies. CD4+T lymphocytes had no link to any clinical-pathological factors (Table 2). Moreover, decreased expression of FOXP3+ T lymphocytes were associated with surgically treated patients who had limited tumor burden without nodal involvement (Table 2). More importantly, the baseline NLR was determined by regression analysis to be substantially linked with pretreatment FOXP3+ and CD8+/CD4+ ratio, as decreased values of NLR were linked with inferior expressions of FOXP3+ and augmented CD8+/CD4+ ratio (OR2.4, 95% CI 1.6-6.8, OR 2.1, 95%CI 1.1-7.8 respectively) (Table .3).”

Clinico- pathological parameters No. of Patients % Baseline NLR CD8+TILs - Cells/HPF CD4+TILs - Cells/HPF CD4+FOXP3+
TILs - Cells/HPF
CD8+ /CD4+
Median P value Median P value Median P value Median P value Median P value
Age, years
<52 22 46% 1.8 0.656 56 0.538 30 0.457 18 0.673 1.7 0.765
≥ 52 26 54% 1.96   54 -- 29 -- 21 -- 1.9 --
Histopathological type
Squamous cell carcinoma 38 -- 1.8 -- 51 -- 27 -- 19 -- 1.8 --
Well differentiated- Grade 1 1 2.6 % 2.3     0.0311* 48 -- 30     0. 121 15 -- 1.6     0.546
Moderately differentiated - Grade 2 9 23.7% 4.5 52 0.212 32 16 0. 136 1.63
Poorly differentiated – Grade 3 28 73.7% 3.6 56 -- 39 18 -- 1.44
Adenocarcinoma 10 -- 3.9 -- 47 -- 27 -- 17 -- 1.7 --
Well differentiated- Grade 1 0 -- --     0.0216* -- -- --     0. 316 -- -- 1.43       0.535
Moderately differentiated- Grade 2 4 40% 1.9 58 -- 42 15 -- 1.4
Poorly differentiated- Grade 3 6 60% 2.6 51 0.315 37 16 0. 126 1.37
N stage
N0 25 52.1% 1.8 0.0124* 78 0.0126* 34 0.324 18 0.0112* 2.8 0.0132*
N1 23 47.9% 2.9   35   20   29   1.3  
Stage group
IB1 12 25% 2.5     0.0145* 68     0. 0161* 27     0.141 14 0.011* 2.5     0.0167*
IB2 11 23% 3.7 64 28 18 2.4
IIA1 5 10.4% 3.9 65 26 17 2.5
IIA2 4 8.3% 3.8 50 29 16 1.7
IIB 8 16.6% 4.1 49 34 25 1.4
IIIA 4 8.3% 4.4 48 36 26 1.3
IIIB 2 4.2% 4.9 52 35 29 1.48
IVA 2 4.2% 5.1 48 33 28 1.45
Definitive treatment
I-Radical hysterectomy 28 58.3% 2.3   0.0132* 68   0.003* 34   0.111 17   0.002* 2.83   0.003*
II-Concurrent chemoradiation 20 41.7% 5.6 48 24 28 1.41

Table 2: Association between baseline inflammatory biomarkers (NLR, subtypes of TILs) and different clinico-pathological parameters.

Pretreatment TILs Pretreatment NLR
  Standardized
Coefficient
OR (95%CI) P-value
CD8+ -0.234 0.85 (0.41-3.2) 0.358
CD4+ -0.134 0.95 (0.44-2.9) 0.245
FOXP3+ +0.764* 2.4 (1.6-6.8) 0.001*
CD8/CD4+ -0.663* 2.1 (1.1-7.8) 0.024*

Table 3: Correlation between pretreatment NLR and TILs (CD8+, CD4+ and FOXP3+ CD4+ T lymphocytes).

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Figure 1: The receiver operator curve (ROC) of pretreatment NLR in cervical cancer patients.

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Figure 2: The receiver operator curve (ROC) of pretreatment tumor infiltrating lymphocytes (CD8+, CD4+, FOXP3+ CD4+ and CD8+ / CD4+ ratio) in cervical cancer patients.

Twenty patients who presented with stageIB2-IVA, were offered concurrent chemo-radiation. Pathological complete resolution was encountered in 12/20 (60%) of the patients. However, 20% or four of the 20 patients went through only a partial regression, while another four patients demonstrated stable progressive disease. Histopathological type (P=0.002), N Stage (P=0.001) and baseline T-stage (P=0.01) on top of the low cutoffs NLR (P=0.001) had a substantial link to path CR, which can be reviewed in (Table 3), alongside any pathological response. Furthermore, the higher cutoffs for CD8+/CD4+Ratio (0.002), while lower cutoffs of FOXP3+ (P=0.003) were thought to be substantially linked with CR path (Table 4) alongside any pathological response. Whereas CD4+ had no reportable link to any pathologic response (Table 4).”

Characteristics Complete pathologic response
Total CR =12Pts
% CR (12/20) =60%
P Value Partial pathologic response Total PR=4 Pts
% PR (4/20) =20%
P Value Stable /Progressive disease  Total=4Pts
%SD+PD (4/20) =20%
P Value
No %CR   No %PR   No %SD+PD  
Age, years
<52 7 35% 0.64 2 10% 0.89 2 10% 0.89
≥52 5 25% 2 10% 2 10%
Histopathological type
Squamous cell carcinoma 12 --    0.002* 2 --    0.036* 2 --    0.012*
Well differentiated- Grade 1 0 0% 0 -- 0 --
Moderately differentiated - Grade 2 9 45% 0 -- 0 --
Poorly differentiated – Grade 3 3 15% 2 10% 2 10%
Adenocarcinoma 0 0%   2 --   2 --  
Well differentiated- Grade 1 0 0%   0 0%   0.031* -- --   0.011*
Moderately differentiated- Grade 2 0 0% 2 10% -- --
Poorly differentiated- Grade 3 0 0% 0 0 2 10%
N stage
N1 (1-2) positive LNs 12 60%   0.001* 1 5%   0.01* 1 5%   0.01*
N1>2LNs 0 0% 3 15% 3 15%
                   
Stage group
IB2 5 25%   0.01*       0.013*     0.89
IIA2 4 20%        
IIB 3 15%        
IIIA     4 20%    
IIIB     0 0% 2  
IVA         2  
Inflammatory Response biomarkers
I-NLR
Median 5.6                
<4.3 11 55%   0.001* 3 15%   0.01* 0 --   0.013*
≥ 4.3 1 5% 1 5% 4 20%
II-Tumor infiltrating lymphocytes (TILs)
1- CD8+TILs - Cells/HPF
Median
ROC Cutoff
48
46
-- -- -- -- -- -- -- --
<46 2 10%   0.002* 1 5%   0.01* 1 5%   0.01*
≥ 46 10 50% 3 15% 3 15%
2-CD4+TILs - Cells/HPF
Median
ROC Cutoff
34
32
-- -- -- -- -- -- -- --
<32 7 35% 0.113 2 10% 0.215 2 10% 0.215
≥ 32 5 25% 2 10% 2 10%
3- CD4+ FOXP3+TILs - Cells/HPF
Median
ROC Cutoff
29
26
-- -- -- -- -- -- -- --
<26 10 50%   3 15%   4 20% --
≥ 26 2 10%   1 5%   0 0% --
4-CD8+/CD4+Ratio
Median
ROC Cutoff
1.41
1.8
-- -- -- -- -- -- -- --
<1.8 3 15%   0.002* 1 5%   0.01* 0 0%   0.013*
≥ 1.8 9 45% 3 15% 4 20%

Table 4: Association between pathological response and different clinicopathological parameters in patients treated with definitive concurrent chemoradiation (N=20).

The metrics for surgically treated 28 participants were illustrated in (Table 5). It was concluded from regression analysis that nodal disease was isolated as the unique prognosticator substantially connected to accentuated expressions of pretreatment NLR (P=0.02), FOXP3+ (P=0.001) and lower thresholds for CD8+ (P=0.01) ,CD8+/CD4+ratio (P=0.02), respectively CD4+ had no link to a single clinical/pathological factor”.

Characteristics No. of Patients %
Age, years    
Median
Range
52
(35-72)
--
<52 16 57%
≥52 12 43%
Histopathological type
Squamous cell carcinoma 22 78.6%
Well differentiated- Grade 1 1 3.5 %
Moderately differentiated - Grade 2 9 32.1%
Poorly differentiated – Grade 3 12 43%
Adenocarcinoma 6 21.4%
Well differentiated- Grade 1 0  
Moderately differentiated- Grade 2 4 14.3%
Poorly differentiated- Grade 3 2 7.1%
Stage group
IB1 12 42.8%
IB2 3 10.7%
IIA1 5 17.9%
IIA2 4 14.3%
IIB 4 14.3%
N stage    
N0 25 89.3%%
N1 3 10.7%
Adjuvant treatment
A-Radical hysterectomy and adjuvant radiation: 12 42.9%
1-Tumor > 4 cm 7 25%
2-> 1/3 stromal invasion 3 10.7%
3-Lymphovascular invasion 2 7.1%
B-Radical hysterectomy and adjuvant chemoradiation: 16 57.1%
  1. Positive pelvic lymph nodes
3 10.7%
  1. Parametrical involvement
7 25%
  1. Positive margin
6 21.4%
Inflammatory Response biomarkers
I-NLR
Median
Range
ROC Cutoff
2.3
(0.6-23)
2
 
<2 19 67.9%
≥ 2 9 32.1%
II-Tumor infiltrating lymphocytes (TILs)
1- CD8+TILs - Cells/HPF    
Median
Mean ±SD
Range
ROC Cutoff
68
58 ± 30.3
6.45-189.52
62
 
<62 21 75%
≥ 62 7 25%
2-CD4+TILs - Cells/HPF
Median
Mean ±SD
Range
ROC Cutoff
24
30.2 ± 32.5
8.9-215.5
22
 
<22 19 67.9%
≥ 22 9 32.1%
3- CD4+ FOXP3+TILs - Cells/HPF
Median
Mean ±SD
Range
ROC Cutoff
14
17.8 ± 9.31
0.97-31.6
16
--
<16 20 71.4 %
≥ 16 8 28.6%
4-CD8+/CD4+Ratio
Median
ROC Cutoff
2.83
2.6
--
<2.6 18 64.3%
≥ 2.6 10 35.7%

Table 5: Clinicopathological parameters of patients treated with radical hysterectomy +/-adjuvant radiation or concurrent chemoradiation (N=28).

The median follow-up stood at a period of around 26 months wherein 13 patients, who account for 27.1% of the sample, either developed distant dissemination or local relapse. From this group, around 20.8% or 10 patients succumbed secondary to cancer related complications. Further, the four-year DFS and OS stood at 73.7% and 81.2%. (Figure 3 and 4). With regards to survival analyses, the regional nodal spread, higher stages, poorly differentiated histology, gross residual disease following chemo-radiation and primary definitive chemo-radiation, were substantially linked to inferior DFS alongside worsened OS “(Table 6). Furthermore, patients that demonstrated decreased NLR <2 (Figure 5 and 6) and augmented TILs [CD8+≥ 64 (Figure 7 and 8), CD8+/CD4+≥ 2 (Fig 9 and 10) and reduced FOXP3+<19 (Figure 11 and 12) experienced substantially extended OS and DFS (Table 6). Conversely, CD4+ values had no effect on either of the two (Figure 13 and 14). More interestingly, the worst OS and DFS were robustly linked to accentuated expression of pretreatment NLR and nodal disease, with a hazard ratio “(HR 3.06 (95% confidence interval [CI], 3.45-9.24), 5.63 (95% CI, 2.61-9.32) for OS and hazard ratio (HR 8.21 (95% CI, 4.21-16.53) and 5.32 (95% CI, 2.37-10.24) for DFS, respectively. Furthermore, the augmented pretreatment thresholds of FOXP3+≥19 and lower CD8+/CD4+< 2 were seen as significantly related to reduced OS (HR 4.37 (95% CI, 2.48-12.37), 2.31(95%CI, 2.34-9.32) and worsened DFS (HR 3.61 (at 95% CI, 1.38- 9.32), 4.32(95%CI, 3.12-8.34), respectively.”

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Figure 3: The overall survival of cervical cancer patients in months.

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Figure 4: The disease-free survival of cervical cancer patients in months.

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Figure 5: The impact of NLR on overall survival of all cervical cancer patients in months.

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Figure 6: The impact of NLR on Disease Free survival of all cervical cancer patients in months.

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Figure 7: The impact of CD8+TILs on overall survival of all cervical cancer patients in months.

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Figure 8: The impact of CD8+TILs on Disease Free survival of all cervical cancer patients in months.

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Figure 9: The impact of FOXP3+ TILs on overall survival of all cervical cancer patients in months.

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Figure 10: The impact of FOXP3+ TILs on Disease Free survival of all cervical cancer patients in months.

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Figure 11: The impact of CD8+ /CD4+ TILs ratio on disease free survival of all cervical cancer patients in months.

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Figure 12: The impact of CD8+ /CD4+ TILs ratio on disease free survival of all cervical cancer patients in months.

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Figure 13: The impact of CD4+TILs on overall survival of all cervical cancer patients in months.

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Figure 14: The impact of CD4+TILs on disease free survival of all cervical cancer patients in months.

Discussion

A mounting volume of data has emphasized the link of TILs with an enhanced clinical result [35]. This study at hand contains a comprehensive evaluation of the performance of baseline intraepithelial “TILs i.e. CD8+, CD4+ and FOXP3+, in 48 cancer cervix patients and their prognosis as per FIGO stage IA-IVA, who were given treatment either with definitive concurrent chemo-radiation or through radical hysterectomies followed by adjuvant .chemo/radiation.

Our analysis illustrated that higher intensities of TILs ratios were linked to node negative disease “(P=0.0126, 0.003), radical hysterectomies (P=0.003, 0.003), and earlier stages of the disease (P=0.0161, 0.0167). Whereas CD4+T lymphocytes were linked to no clinical-pathological elements. Concordantly, Piersma stated that patients with nodal negative disease enjoyed the best clinical outcomes and they possessed as well a significantly greater densities of “CD8+ T cells(P<0.01), an augmented CD8+/CD4+ T-cell ratio(P=0.01), and increased CD8+/regulatory T-cell ratio” when compared to others with regional nodal involvements [25]. However, low pretreatment FOXP3+ T lymphocytes had a link to early stage disease(P=0.011) and node negative patients(P=0.0112). Wu had a similar result wherein they showed that regional nodal disease had substantially augmented densities for FOXP3+ T cells as opposed to those negative regional nodes (P= 0.045) [36]. Shah highlighted that FOXP3+was substantially augmented for higher clinical stages as opposed to lower stages (P=0.023) [37].”

It has also concluded from the current work, that amplified NLR was explicitly encountered in grade III cervical cancer (P=0.0311), lymph node involvement (P=0.0132) and advanced stage III-IV(P=0.0132) normally had an amplified NLR. Huang also showed in their meta-analysis that NLR had a substantial correlation with advanced FIGO stage (OR 2.12, 95% CI1 28–3.49) and regional nodal disease (OR 2.24, 95% CI 1.65–3.04) [38]. Furthermore, through regression scrutiny it was found that baseline NLR was substantially linked to pretreatment FOXP3+ and CD8+/CD4+ ratio, as reduced expressions of NLR were linked to the lower intensities of FOXP3+ and elevated ranks of CD8+/ CD4+ ratio (OR2.4, 95% CI 1.6-6.8, OR 2.1, 95%CI 1.1-7.8). It is pertinent to mention that this is the first study to outline the correlative link between baseline systemic bio variable. NLR and pretreatment. TILs (CD8+, CD4+ and FOXP3+) in tumor microenvironment. ROC” more so highlighted that baseline NLR and TILs could predict DFS in the group of patients suffering from cervical cancer. The 48 participants of the study were then segmented as per the definitive treatment given. Of this sample, 20 patients were ones who had locally advanced stages and were administered concurrent chemo-radiation. Several other clinical elements were seen to be linked with pathologic response as stage, grade, and lymph node contribution. “Furthermore, the accentuated cutoffs for pretreatment CD8+ (P=0.002) and CD8+/ CD4+Ratio (P=0.002) while a reducedcutoffs for FOXP3+ (P=0.003) were found to be considerably linked with path CR and any pathologic response.” “Whereas pretreatment CD4+ had no link to any kind of pathologic response. Draghiciu et al also outlined that decreased intensities CD8+ were observed in locally advanced cervical cancer that is dealt with using definitive concurrent chemo-radiation and achieved minimal response to treatment (OR=0.562; 95%CI=0.319-0.991; P=0.046) [39]. For the patients remaining, 28 in number, earlier stage were given radical hysterectomy after which adjuvant radiation and chemotherapy was implemented as per the postoperative pathological risk factor. For patients that had been through surgery, lymph nodal contribution was seen through a regression investigation, and was outlined as the single element most profoundly linked to accentuated intensities of pretreatment NLR (P=0.02), FOXP3+ (P=0.001) and reduced densities of pretreatment CD8+ (P=0.01), cutoffs of CD8+/ CD4+ratio (P=0.02), respectively.

The study results indicate that nodal involvement, advanced stage, adenocarcinoma, definitive concurrent chemo-radiation, poorly differentiated tumors, along with partial response to chemo-radiation can be considerably linked to reduced OS and DFS. Further, patients with reduced “NLR<2 and FOXP3+<19 while accentuated TILS. (CD8+≥64, CD8+/CD4+≥2) went through considerably extended DFS and OS. Cox regression analysis for survival demonstrated that augmented NLR and nodal involvement were individually linked to abysmal OS with a hazard ratio.” (HR 3.06 (95% confidence interval [CI], 3.45-9.24), 5.63 (95% CI, 2.61-9.32) and worsened DFS (HR 8.21 (95% CI, 4.21-16.53), 5.32 (95% CI, 2.37-10.24). Furthermore, the augmented FOXP3+≥19 and reduced CD8+/CD4+<2 were substantially linked to abysmal OS (HR 4.37 ( 95% CI, 2.48-12.37), 2.31(95%CI, 2.34-9.32) and much worse DFS (HR 3.61 (95% CI, 1.38- 9.32), 4.32(95%CI, 3.12-8.34) respectively. In the same vein, Huang demonstrated that high pretreatment NLR levels was substantially linked to dismal OS (HR:1.88, 95% CI 1.30–2.73) and shortened PFR (HR 1.65, 95% CI 1.18–2.29) [38]. Further to this, Draghiciu outlined that accentuated expression CD8+ was linked to augmented OS (HR=0.642; 95%CI=0.448-0.919; P=0.016) and DSS (HR=0.607; 95%CI=0.403-0.915; P=0.017) [39].”Jordanova also explained that augmented expressions of Treg (FoxP3+) “and decreased cutoffs of CD8+/regulatory T-cell ratio were linked to inferior survival (P=0.034 and0.02), respectively. In later Cox regression investigation, reduced CD8+/Treg ratio (P=0.047), decreased CD8+/Treg ratio (P=0.002) were seen as independent uncomplimentary predictive prognosticators in cervical carcinoma [40]. In the same vein, Piersma outlined a substantially more robust intratumoral densities of CD8+ and a greater CD8+/CD4+ T-cell ratio in localized cervical cancer without regional nodal involvement and were significantly correlated with superior prognoses [25].” Additionally, Shah stated that a substantially inferior survival rate was observed for patients with augmented expressions of CD4+FOXP3+ Tregs, as opposed to those with inferior expressions (35.3% versus 88.9%, P=0.001) [37].”

The study at hand has a potential input as it scrutinized on the role that TIL types (CD8+, FOXP3+, CD8 +/CD4+) and NLR exert as individual prognostic indicators. It is also confirmed the competencies of pretreatment TILs and NLR in forecasting prognoses of curatively treated patients who received either concurrent chemo-radiation or radical hysterectomies. However, the study confronted some restrictions because of its retrospective nature. The limited sample size alongside its heterogeneity, negatively impacted the generalizability of our results. Moreover, we did not delve into the details of how different TILs optimize and modulate each other’s activities.

Conclusion

The tested pretreatment TILs and NLR demonstrated a considerable link to various clinical-pathological prognostic variables in terms of definitively treated patients with cervical cancer. Furthermore, they could be perceived as independent prognostic forecasters of clinical outcomes.

Conflict Of Interest

The authors ascertain that they did not receive any financial support or funding from any institution or entity or any commercial operation. The authors further confirm that the study posed no conflicts of interest for them.

References

Citation: Isamil El-Lathy HAZ, Dohal A, Mashhour M (2018) The Pretreatment Tumor Infiltrating T Lymphocytes (CD8+, CD4+, FOXP3+) and Systemic Neutrophil-Lymphocytes Ratio in Definitively Treated Cervical Cancer Patients: The correlation to clinicopathological factors and Survival. J Cancer Sci Ther 10: 118-129. DOI: 10.4172/1948-5956.1000528

Copyright: © 2018 Isamil El-Lathy HAZ, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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