The survey in the role of LAG3+ tumor-infiltrating lymphocytes in colorectal cancer prognosis; Relationship with sidedness

doi:10.21203/rs.3.rs-1746250/v1

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Research Article

The survey in the role of LAG3+ tumor-infiltrating lymphocytes in colorectal cancer prognosis; Relationship with sidedness

https://doi.org/10.21203/rs.3.rs-1746250/v1

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published 10 Feb, 2023

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Background: The interplay between tumor-infiltrating lymphocytes (TILs) and tumor cells is a major determining factor in cancer progression. CD45RO seems to be a reliable indicator for predicting prognosis and disease outcome, along with CD3 and CD8 markers. LAG-3 is another important marker that overexpresses on TILs in a variety of cancers and is associated with disease prognosis; however, its prognostic impact is controversial. Hence, in the present study, we aimed to investigate the presence of CD45RO+, LAG3+, CD3+, and CD8+ lymphocytes in CRC tumor tissues and their association with clinicopathological parameters of the disease as well as patients' survival, according to primary tumor locations.

Methods: Expression of CD45RO, LAG3, CD3, and CD8 was Immunohistochemically analyzed in tissue sections from 136 patients with CRC. The percentage of TILs expressing these markers were separately determined in the invasive margin (IM) and center of the tumor (CT). Their associations with clinical factors and survival were analyzed in the entire cohort, and in the subgroups of patients with right and left/rectum tumors.

Results: Based on our observation, CD45RO+ and CD3+ lymphocytes were the most frequent infiltrated cells in both CT and IM regions of colon tumor tissue. On the other hand, LAG3+ lymphocytes were the least frequent subset in both areas. In univariate analysis, high score of CD45RO in IM was associated with better overall survival in the entire cohort. High score of CD8 and CD45RO in IM were also associated with improved overall survival in right-sided tumors. However, CD45RO expression correlated with advanced TNM stages (III/IV), in the entire cohort and in right-sided tumors. In the case of LAG3, although the association between the expression of this marker and survival was not found, LAG3+ TILs in CT were associated with higher T-stages in the entire cohort.

Conclusions: Our findings suggest that the clinicopathological and prognostic significance of immune system-related markers such as CD45RO and LAG3 depends on the primary tumor sides. Our results collectively demonstrated that infiltration of CD45RO+ lymphocytes in IM could be an independent prognostic factor in a site-dependent manner.

Colorectal cancer

immunohistochemistry

prognostic biomarker

tumor microenvironment

Lymphocyte activation gene 3

Colorectal cancer (CRC) is the second most common cause of cancer death in the world ¹.

CRC can be identified by the location of the primary tumor in the large intestine and accordingly includes tumors of the right, left colon, and rectum ². Based on primary tumor location, CRC shows differences in chromosomal and molecular characteristics, microbiome, incidence, pathogenesis, and outcome ³. It has been found that tumors on the left and right sides of CRC have different immune landscapes. For example, the level of immune infiltration is higher in right-sided CRC than in left-sided tumors, thus, the prognosis may be different among different primary tumor locations ⁴.

CRC prognosis is largely based on histopathological grading, according to the American Joint Committee on Cancer (AJCC) TNM system ⁵. However, significant differences in clinical outcomes are observed among patients within the same TNM stage, showing the weaknesses of this classification system ⁶. One of the failure reasons for this system seems to be the lack of attention to the impact of the tumor microenvironment (TME), especially the immune system elements, on tumor progression ⁷. TME is generally defined as the environment surrounding the tumor, including the extracellular matrix, blood vessels, endothelial cells, tumor cells, and immune cells ⁸. Therefore, the identification of immune-related biomarkers in TME might help to predict the prognosis and clinical outcome of the disease.

Tumor-infiltrating lymphocytes (TILs) are the main immune cells of TME and their presence is an indication of host immune reaction to tumor cells and prevention of tumor invasion ⁹. TILs compose a variety of subpopulations of T cells ¹⁰, including CD4 + and CD8 + T lymphocytes, FOXP3 + regulatory T cells, and CD45RO + memory T cells ¹¹. There are many reports that suggest that CD3+, CD8 + and CD45RO + TILs are correlated with favorable clinical outcomes in several cancers ^12–14 due to their critical roles in tumor immunity ¹⁵.

CD45RO + memory T lymphocytes, as one of the important components of TILs are responsible for more reinforced responses to secondary exposures to antigens ¹⁶. In particular, CD45RO + memory T cells have been demonstrated to play specific roles in several human cancers ¹⁷. Although studies have shown that CD45RO + T cell infiltration had a positive prognostic effect on many types of tumors such as gastric, cervical and hepatocellular carcinoma, but the accurate clinical significance of these T cells was not clear ¹².

Immune checkpoints (ICPs) are membrane proteins that regulate immune cells by either activating or inhibiting their immune functions and have the potential of acting as prognostic biomarkers ¹⁸. Inhibitory immune checkpoints (iICPs) like T-cell immunoglobulin mucin-domain containing-3 (TIM-3), programmed cell death protein-1 (PD-1), and Lymphocyte activation gene 3 (LAG3), are all present on T-cells and bind to ligands that are commonly produced by tumor cells (18). LAG3 or CD223 is expressed on T cells, especially on activated CD8 + and CD4 + T cells, B cells and DCs ¹⁹. Interaction of LAG3 with its ligands i.e. MHC class II molecules, galectin-3, liver sinusoidal endothelial cell lectin (LSECtin), and fibrinogen-like protein 1 (FGL1)²⁰, mediates various signaling in TILs function impairment, including inhibition of Th1 cell proliferation and reduced production of interleukin-2 (IL-2), interferon-γ, and tumor necrosis factor (TNF) in T cells, resulting in tumor escape ²¹. LAG3 expression is frequently associated with exhausted T cells, and it is generally considered an exhaustion marker for CD4 + and CD8 + T cells in response to repetitive antigen stimulation in cancer ^22–25. Overexpression of LAG3 is found in many types of human tumors such as CRC, breast cancer, liver carcinoma, follicular lymphoma, and squamous cell carcinoma of the head and neck, which is significantly associated with the development of an invasive tumor and clinicopathological outcomes ^26–28. Moreover, LAG3 can be an interesting target for immunotherapy and there are a number of ongoing early-phase clinical trials testing anti-LAG3 therapeutic antibodies in different types of cancer ²⁹. Although there are limited studies that showed the association between tumor-infiltrating LAG3 T-cells and CRC prognosis, their results were controversial. In addition, the majority of studies aimed at investigating the prognostic impact of total population of TILs, and fewer studies have focused on the prognostic significance of specific subpopulations. Accordingly, we aimed to evaluate the presence of CD45RO + and LAG3 + TILs in the CT and IM and their impacts on outcomes in CRC with special focus on tumor location (right and left colon tumors).

2.1 Patients and specimens

136 patients with CRC who had complete data were enrolled in the study. The study population was retrospectively selected from patients with CRC who underwent surgical resection of their primary tumor between April 2013 to December 2016 in the Al-Zahra Hospital (Isfahan University of Medical Sciences, Isfahan, Iran). Exclusion criteria were receiving preoperative chemotherapy, having history of other cancers or autoimmune diseases, and insufficient and inappropriate tissue. Information of survival status (dead/alive), date and type of recurrence (if present), metastatic organ, and for patients who died, date and cause of death were collected from their records and telephone contact. Pathological data including TNM-stage, histological grade, lymph node involvement and lymphovascular or perineural invasion were collected from patient files. An expert pathologist reviewed the H&E-stained tumor sections to confirm the patient's pathological data and select suitable blocks, preferably having both CT and IM. Formalin-fixed and paraffin-embedded (FFPE) tissue blocks were then selected and prepared for immunohistochemical staining.

2.2 Immunohistochemistry

Tissue sections with 3- µ-m thickness were prepared and fixed on slides coated with poly-L-lysine. Sections were dewaxed at 44°C overnight, immersed in xylene for deparaffinization, and rehydrated in a graded series of ethanol )100% and 96%). Antigen retrieval was then performed by boiling the tissue sections in a pressure cooker for 20 min in Tris-EDTA buffer (pH: 9.0). Endogenous peroxidase activity was blocked by incubation in 10% hydrogen peroxide (Master diagnostica, Spain) for 10 min, and subsequently, the sections were blocked with 10% goat serum (Cyto Matin Gene, Iran) at room temperature for 30 min. Primary anti-human antibodies against LAG3 (1:1000, ab209236, clone: EPR20261; Abcam, USA), CD45RO (1:320, 304202, Clone UCHL1, Biolegend, USA), CD3 (1/2.5, MAD-000621QD, clone EP41, Master diagnostica), CD8 (1:150, 372902, clone C8/144B, Biolegend), respectively were added and followed by overnight incubation at 4°C. The slides were then washed and incubated with a horseradish peroxidase-conjugated secondary antibody for 40 min. The chromogenic reaction was visualized by incubation with diaminobenzidine (DAB) (Master diagnostica) for 3–5 min. Sections were counterstained with hematoxylin, dehydrated by increasing alcohol solutions, cleared in xylene, and mounted with a permanent mounting medium. Human tonsil tissue in which lymphocytes were abundant was used as a positive control to titrate the proper concentration of primary antibodies.

2.3 Evaluation of immunochemistry staining

Two expert pathologists blindly reviewed the sections. The slides related to each patient were first examined with a magnification of 100× and normal area, invasive margin (IM), and tumor center (TC) were identified and an area representing the average expression of the marker was selected. The selected area was then examined in high-power field (× 400) and the expression of CD45RO, LAG3, CD3, and CD8 were determined. The percentage indicated the ratio of the positive cells to total tumor stromal cells. In addition to the percentages, the intensity of LAG3 expression in each region was also reported as negative, low, moderate, and high. LAG3 expression was also scored by multiplying the number of LAG-3 + TILs by the intensity using R software (version 4.0.3). Accordingly, patients were classified into high score (LAG + ^hi) and low score (LAG3 + ^Lo) groups. The IHC score for CD3, CD8, and CD45RO was determined based on their median expression and patients were grouped into high score and low score.

2.4 Statistical analysis

Statistical analyses were performed using SPSS software version 24 (IBM SPSS, USA). The normality of CD45RO and LAG3 expression in different groups was checked. The significance of differences in CD45RO and LAG3 expression between several clinicopathological variables was assessed by t-test, non-parametric Mann-Whitney test, Chi-square test or Fisher’s exact test as appropriate. The Chi-square test was performed in the entire cohort and based on primary tumor location (right-sided and left-sided colon tumors). Survival times were defined as overall survival (OS), the time from surgery to cancer-related death and disease-free survival (DFS), between the surgery and any type of relapse or last follow-up. For univariate analyses, Cox regression method was used to determine predictors of DFS and OS in patients with CRC, while only variables in the univariate analysis that had a P-value < 0.1 were included in the multivariate Cox’s regression model to investigate the independent predictive factors. P-values < 0.05 were considered statistically significant.

3.1. Patients characteristics

Final study population consisted of 136 patients with CRC with the mean age (± SD) of 62.35 (± 14.10) years (19 to 92 years) at the time of diagnosis. Of them, 83 (61.0%) patients were male, and 53 (39.0%) patients were female. According to the pathology reports, 109 (80.1%) patients had colonic tumors and 27 (19.9%) had rectal tumors. Among colon tumors, 56 (41.2%) were located on the right side and 49 (36%) were located on the left side. Based on pathologic stages, most patients were in stage II (33.8%) and stage III (29.4%). Distant metastasis was seen in 12 patients (8.8%) (M1) and 46 patients (33.8%) had lymph node involvement at the time of surgery. Regarding tumor histological grade, most cases (76/136, 55.9%) had well-differentiated tumors. The mean ± SD follow-up period was 54.08 ± 27.6 months. During follow-up period, 57(41.9%) patients died and 79(58.1%) patients were alive up to October 2021. During this time, 30 patients (22.1%) experienced recurrence. The summarized clinical and pathological information of the patients are listed in detail in Table 1.

Table 1

**Clinicopathological characteristics of patients with colorectal cancer**
Parameters	No. of Cases (%)	Mean ± SD	Parameters	No. of Cases (%)
Total	136		TNM stage
Sex			I/II	84 (61.8)
Male	83 (61.0)		III/IV	52 (38.2)
Female	53 (39.0)		Lymphovascular invasion (LVI)
Age		62.35 ± 14.102	Absent	79 (58.1)
< 63	65 (47.8)		Present	57 (41.9)
≥ 63	71 (52.2)		Perineural invasion
Tumor side			Absent	111 (81.6)
Right	56 (41.2)		Present	25 (18.4)
Left	76(55.9)		Metastasis
Unknown	4 (2.9)		Absent	89 (65.4)
Tumor size		5.56 ± 2.47	Present	34 (25.0)
< 5	56 (41.2)		Unknown	13 (9.6)
≥ 5	78 (57.4)		Recurrence
Unknown	2 (1.5)		Absent	97(71.3)
Differentiation grade			Present	29(21.3)
Low grade	76 (55.9)		Unknown	10(7.4)
Moderate to high grade	60 (44.1)		Tumor budding
T stage			Low	89 (65.4)
T1/T2	47 (34.6)		High	47 (34.6)
T3/T4	89 (65.4)		Tertiary lymphoid structure (TLS)
Lymph node involvement			Absent	104 (76.5)
Absent	90 (66.2)		Present	32 (23.5)
Present	46 (33.8)		Survival
M stage			alive	79 (58.1)
M0	124 (91.2)		dead	57 (41.9)
M1	12 (8.8)

3.2. Distribution of CD45RO+, LAG3+, CD3+, and CD8 + TILs in patients with CRC

Tissue samples were immunohistochemically stained for CD45RO, LAG3, CD3 and CD8 (Figure. 1). The mean frequencies of CD45RO+, LAG3+, CD3+, and CD8 + TILs were assessed in both CT and IM as well as normal-like adjacent tissue. The mean frequency of all markers in both CT and IM was significantly higher than in normal-like tissue. The mean frequency of CD45RO + lymphocytes in the IM area was significantly higher than CT (P < 0.001). The mean frequency of LAG3 + lymphocytes in CT was significantly higher than normal tissue. In addition, comparison of the frequency of CD3 + and CD8 + lymphocytes showed that the mean frequency of these cells in both CT and IM areas was higher than normal tissue (P < 0.001) (Table 2).

Table 2

Mean frequency of CD45RO+, LAG3+, CD3+, and CD8 + lymphocytes in patients with colorectal cancer
		n	Mean ± SD	P
Pair 1	CD45RO.CT	136	45.7721 ± 13.99575	< 0.001*
Pair 1	CD45RO.IM	136	51.8750 ± 12.54529	< 0.001*
Pair 2	CD45RO.CT	47	46.8085 ± 13.32803	< 0.001*
Pair 2	CD45RO.Normal	47	36.4894 ± 12.41996	< 0.001*
Pair 3	CD45RO.IM	47	52.2340 ± 11.64738	< 0.001*
Pair 3	CD45RO.Normal	47	36.4894 ± 12.41996	< 0.001*
Pair 4	LAG3.CT	66	5.4632 ± 11.13680	0.571
Pair 4	LAG3.IM	66	5.4485 ± 9.13019	0.571
Pair 5	LAG3.CT	16	6.4237 ± 12.10187	0.026*
Pair 5	LAG3.Normal	16	1.1695 ± 2.19837	0.026*
Pair 6	LAG3.IM	15	6.1356 ± 10.79025	0.058*
Pair 6	LAG3.Normal	15	1.1695 ± 2.19837	0.058*
Pair 7	CD3.CT	118	39.8729 ± 12.23806	< 0.001*
Pair 7	CD3.IM	118	46.1017 ± 12.10946	< 0.001*
Pair 8	CD3.CT	51	40.8824 ± 12.59785	< 0.001*
Pair 8	CD3.Normal	51	27.3529 ± 10.74025	< 0.001*
Pair 9	CD3.IM	51	46.8627 ± 11.17859	< 0.001*
Pair 9	CD3.Normal	51	27.3529 ± 10.74025	< 0.001*
Pair 10	CD8.CT	119	19.1597 ± 9.44025	< 0.001*
Pair 10	CD8.IM	119	24.0756 ± 9.52175	< 0.001*
Pair 11	CD8.CT	49	19.0816 ± 9.27999	0.002*
Pair 11	CD8.Normal	49	15.0000 ± 7.56913	0.002*
Pair 12	CD8.IM	47	25.2128 ± 9.02638	< 0.001*
Pair 12	CD8.Normal	47	14.7872 ± 7.65829	< 0.001*
*: statistically significant / CT: Central of tumor, IM: Invasive margin

3.3. Mean frequency of CD45RO + and LAG3 + lymphocytes in patients with different clinicopathological features

The mean frequency of CD45RO+, LAG3+, CD3+, and CD8 + lymphocytes was evaluated in patients with different clinicopathological features. No significant relationship was observed between the mean frequency of CD3 + and CD8 + cells and clinicopathological features. As shown in Table 4, the mean frequency of CD45RO in CT was higher in patients with lymph node metastasis (P = 0.016) and patients with advanced TNM stages (III/IV) (P = 0.037). In addition, the mean frequency of CD45RO in IM in females was higher than males (P = 0.041). The mean frequency of LAG3 in CT was higher in patients with lower T stages (T1/T2) (P = 0.025) and metastasis (P = 0.055). The mean frequency of LAG3 in IM was higher in patients with larger tumor size (P = 0.024).

3.4. Associations of CD45RO + and LAG3 + TILs with clinicopathological features

We next scored CD45RO, LAG3, CD3 and CD8 markers (as described in section 2.3) and patients were classified into high score and low score. We then compared the score of immune markers in patients with different clinicopathological features. No significant relationship was observed between the score of CD3 and CD8 markers in CT and IM and clinicopathological features.

Associations between the score of CD45RO and LAG3 in CT and IM and clinicopathological characteristics in detail are demonstrated in supplementary Table 5–6. Our results demonstrated that the score of CD45RO in CT was lower in patients without lymph node involvement (P = 0.013) and patients with earlier TNM stages (I/II) (P = 0.031). No significant association was observed between the score of CD45RO and other clinicopathological factors including age, sex, grade, tumor location, tumor budding.

In the case of LAG3, Low score of LAG3 in CT was significantly associated with no metastasis (P = 0.023) and no recurrence (P = 0.015). The score of LAG3 in CT showed a decreased trend for an association with earlier stages (I/II) (P = 0.076).The score of LAG3 in IM was observed to be higher in patients with higher T stages (T3/T4) (P = 0.027). In addition, high score of LAG3 in IM was associated with the absence of TLS in CRC tissues (P = 0.014). In addition, there was an association between high score of LAG3 in IM and larger tumor size (P = 0.052).

3.5. Associations of CD45RO + and LAG3 + TILs with clinicopathological features based on primary tumor location

We then classified patients based on their primary tumor location into two groups: left/ rectum and right, and repeated the analyses in patients with high and low scores of LAG3 and CD45RO. Our results indicated that in right-sided colon tumors, low score of CD45RO in CT was associated with earlier TNM stages (I/II) (P = 0.017) and the absence of lymph node involvement (P = 0.015).

In left-sided colon tumors, Low score of LAG3 in CT was also significantly associated with no metastasis (P = 0.039), no recurrence (P = 0.027) and low tumor budding (P = 0.05). The score of LAG3 in IM was lower in patients with higher T-stage (T3/T4) (P = 0.015) in these tumors. Associations between the score of CD45RO and LAG3 in CT and IM and clinicopathological characteristics based on primary tumor location in detail are demonstrated in additional Table 1–2.

3.6. Prognostic value of CD45RO+, LAG3+, CD3+, and CD8 + TILs

Cox regression analysis was performed to evaluate the effects of CD45RO+, LAG3+, CD3+, and CD8 + TILs in CT and IM on OS and DFS in patients with CRC. In univariate Cox regression model, high expression of CD45RO in IM was obtained to be associated with improved OS (hazard ratio [HR] = 0.582, 95% CI = 0.339–0.997, P = 0.049). Whereas no association between LAG3 expression and OS was observed neither in CT nor in IM (Fig. 2). The univariate Cox proportional hazards model for DFS revealed no significant relationship between the expression of any of investigated markers and the risk of post-operative disease relapse (data not shown). Our analysis also showed that early TNM-stages (HR = 3.529, 95% CI = 2.074–6.006, P < 0.001) was associated with improved OS. Moreover, low tumor budding showed a trend for an association with better OS (P = 0.065).

All variables in the univariate analysis with a p-value < 0.1 were then entered into a multivariate Cox regression model. No association was found between the expression of CD45RO, LAG3, CD3, and CD8 and OS along with the clinicopathological variables (Table 3).

Table 3

Univariate Cox regression analysis of overall survival (OS) in patients with colorectal cancer
Parameters	Univariable analysis HR (95% CI)	P	Parameters	Univariable analysis HR (95% CI)	P
Sex			TLS
Male	1	0.174	Absent	1	0.415
Female	0.679 (0.388–1.187)	0.174	Present	0.760 (0.394–1.469)	0.415
Age			CD45RO.CT
< 63	1	0.799	Low	1	0.137
≥ 63	1.070 (0.635–1.802)	0.799	High	1.511 (0.877–2.603)	0.137
Tumor side			CD45RO.IM
Right	1	0.119	Low	1	0.049*
Left	1.569 (0.891–2.762)	0.119	High	0.653 (0.380–1.123)	0.049*
Tumor size			LAG3.CT
< 5	1	0.549	Low	1	0.226
≥ 5	1.178 (0.689–2.015)	0.549	High	0.656 (0.331–1.299)	0.226
Differentiation grade			LAG3.IM
Low grade	1	0.205	Low	1	0.495
Moderate to high grade	1.399 (0.832–2.353)	0.205	High	1.206 (0.704–2.066)	0.495
T stage			CD3.CT
T1/T2	1	0.060	Low	1	0.211
T3/T4	1.760 (0.976–3.176)	0.060	High	1.394 (0.828–2.344)	0.211
Lymph node involvement			CD3.IM
Absent	1	< 0.001*	Low	1	0.640
Present	2.713 (1.611–4.568)	< 0.001*	High	1.133 (0.672–1.908)	0.640
M stage			CD8.CT
M0	1	< 0.001*	Low	1	0.533
M1	7.443 (3.741–14.808)	< 0.001*	High	1.180 (0.702–1.983)	0.533
TNM stage			CD8.IM
I/II	1	< 0.001*	Low	1	0.482
III/IV	3.529 (2.074–6.006)	< 0.001*	High	0.821 (0.473–1.424)	0.482
Lymphovascular invasion (LVI)			CD45RO.IM/CT
Absent	1	0.663	Low to low	1	0.047
Present	1.123 (0.666–1.896)	0.663	CT high / IM low	1.699(0.773–3.734)	0.187
Perineural invasion			CT low / IM high	0.444(0.209–0.941)	0.034
Absent	1	0.140	High to high	0.956(0.486–1.880)	0.896
Present	1.576 (0.862–2.881)	0.140	LAG3.IM/CT
Tumor budding			Low to low	1	0.290
Low	1	0.065	CT high / IM low	1.362(0.182–10.175)	0.763
High	1.643 (0.970–2.783)	0.065	CT low / IM high	1.509(0.846–2.691)	0.164
			High to high	0.778(0.354–1.710)	0.532
CT: Center of tumor, IM: Invasive margin, TLS: Tertiary lymphoid structure
*: statistically significant

3.7. Associations of CD45RO+, LAG3+, CD3+, and CD8 + TILs with OS based on primary tumor location

We next analyzed the association between the expression of immune markers and OS based on the tumor location using a univariate Cox regression model. It was observed that there was a significant association between high score of CD45RO in IM and improved OS in right-sided tumors (HR = 0.199, 95% CI = 0.057–0.689, P = 0.011). In addition, high expression of CD8 in IM was associated with better OS (HR = 0.288, 95% CI = 0.083–0.995, P = 0.049), while no correlation was found between CD3 + and LAG3 + TILs and OS (Additional Table 3 and Fig. 3).

Table 4

Mean frequency of CD45RO + and LAG3 + lymphocytes in patients with different clinicopathological features
Parameters	CD45RO.CT		CD45RO.IM		LAG3.CT		LAG3.IM
Parameters	Mean ± SD	P-value	Mean ± SD	P-value	Mean ± SD	P-value	Mean ± SD	P-value
Sex
Male	45.00 ± 14.86	0.903 ª	50.12 ± 12.90	0.041*	4.95 ± 10.06	0.696	5.27 ± 8.46	0.907
Female	46.98 ± 12.57	0.903 ª	54.62 ± 11.55	0.041*	6.26 ± 12.70	0.696	5.71 ± 10.17	0.907
Age
< 63	46.00 ± 13.11	0.857	52.00 ± 11.95	0.912	5.35 ± 11.22	0.531	4.47 ± 8.07	0.407
≥ 63	45.56 ± 14.84		51.76 ± 13.15		5.56 ± 11.13		6.33 ± 9.97
Tumor side
Right	45.00 ± 13.75	0.448	49.55 ± 12.90	0.263 ª	5.32 ± 11.38	0.807	6.96 ± 12.28	0.322
Left	46.84 ± 13.75		53.42 ± 12.28		5.64 ± 11.20		4.39 ± 5.99
Tumor size
< 5	44.82 ± 13.31	0.555	53.30 ± 13.11	0.240	6.21 ± 12.91	0.809	3.44 ± 6.95	0.024*
≥ 5	46.28 ± 14.60		50.70 ± 12.16		5.06 ± 9.85		6.96 ± 10.29
Differentiation grade
Low grade	44.08 ± 12.16	0.113	52.17 ± 10.40	0.768	4.42 ± 9.84	0.369 ª	5.38 ± 9.41	0.841
Moderate to high grade	47.91 ± 15.87		51.50 ± 14.91		6.78 ± 12.54		5.53 ± 8.83
T stage
T1/T2	44.46 ± 11.52	0.432	51.91 ± 9.97	0.979	6.42 ± 11.66	0.025* ª	5.23 ± 6.58	0.603
T3/T4	46.46 ± 15.15		51.85 ± 13.76		4.95 ± 10.88		5.56 ± 10.26
Lymph node involvement
Absent	43.72 ± 13.50	0.016*	52.44 ± 12.92	0.461	6.15 ± 11.92	0.422	6.35 ± 10.63	0.335
Present	49.78 ± 14.22		50.76 ± 11.83		4.11 ± 9.40		3.67 ± 4.66
M stage
M0	45.52 ± 14.07	0.859 ª	51.97 ± 12.78	0.556 ª	5.45 ± 11.16	0.911 ª	5.68 ± 9.44	0.278
M1	48.33 ± 13.54		50.83 ± 10.18		5.58 ± 11.36		3.00 ± 4.43
TNM stage
I/II	43.80 ± 13.85	0.037*	52.55 ± 13.27	0.421	6.38 ± 12.25	0.388	6.65 ± 10.92	0.200
III/IV	48.94 ± 13.77		50.77 ± 11.30		3.98 ± 8.95		3.5000 ± 4.50
Lymphovascular invasion
Absent	44.68 ± 12.20	0.287	51.58 ± 12.70	0.750	5.21 ± 11.01	0.301	5.99 ± 9.30	0.709
Present	47.28 ± 16.15		52.28 ± 12.43		5.80 ± 11.39		4.70 ± 8.91
Perineural invasion
Absent	44.59 ± 13.64	0.212 ª	51.75 ± 12.73	0.818	5.63 ± 11.62	0.952	5.89 ± 9.84	0.667
Present	51.00 ± 14.65		52.40 ± 11.91		4.72 ± 8.80		3.48 ± 4.46
Metastasis
Absent	45.22 ± 13.35	0.595	51.85 ± 11.99	0.663	6.33 ± 12.30	0.055 ª	6.28 ± 10.35	0.064
Present	46.76 ± 16.69		50.73 ± 14.41		3.03 ± 7.22		2.97 ± 4.21
Recurrence
Absent	45.10 ± 13.55	0.592	51.65 ± 12.18	0.928	6.49 ± 12.36	0.040*	6.38 ± 10.06	0.131
Present	46.72 ± 16.49		51.89 ± 15.20		1.86 ± 2.86		2.96 ± 4.01
Survival
Alive	44.87 ± 13.89	0.380	53.54 ± 12.61	0.068	6.28 ± 12.68	0.366	6.16 ± 10.85	0.158
Dead	47.01 ± 14.16		49.56 ± 12.18		4.33 ± 8.52		4.45 ± 5.92
ª P-values were obtained from non-parametric Mann-Whitney test.
CT: Central of tumor, IM: Invasive margin
*: statistically significant

In recent years, the prognostic significance of TILs has been well-documented. Infiltrating TILs, composing of different lymphocyte subtypes, into the TME represent the immune response status between host immune cells and tumors ³⁰. Previous studies have shown that the infiltration of TILs correlates with tumor development, clinicopathological features of patients, clinical outcome, and survival in several cancers, including CRC ³¹. Among them, Kasurinen et al. investigated the prognostic value of different combinations of CD3 + and CD8 + immune cells in patients with CRC and demonstrated that the infiltration of a large number of CD3 + and CD8 + inflammatory cells in tumor tissue correlated with an improved survival, in many types of cancers ^32–34. In the present study, regarding the importance of memory (CD45RO+) and cytotoxic (CD8+) lymphocytes in eradication of tumor cells, we attempted to delineate the presence of CD3+, CD8+, CD45RO + TILs and the expression of LAG3 as a novel inhibitory marker in both IM and CT of CRC tissues and then assessed their correlation with clinical outcomes with focus on tumor location. Our results showed that CD45RO + lymphocytes followed by CD3 + lymphocytes were the most frequent subsets in both areas of CRC tumor tissues, while LAG3 + cells showed the lowest frequency.

CD45RO is generally thought to be a marker of memory T cells which represent effector activity and rapid reaction in TME and can also affect the outcome of the disease ¹⁷. Our survival analysis revealed that high score of CD45RO in IM was associated with better OS. Similar results were reported in several other cancers such as renal cell carcinoma, gastric cancer, breast cancer and ovarian carcinoma ^35–38. In these studies, CD45RO + TILs were also positively associated with DFS and OS. The protective role of CD45RO + T-cell in anti-tumor responses and their association with better prognosis in most cancers might be explained by providing a pool of effector cells or direct killing effect of these cells in TME that repress the proliferation and migration of tumor cells. Additionally, it has been showed that CD45RO + T cells could decrease the production of high-mobility group box 1 (HMGB1) in CRC, and since HMGB1 accelerates cell growth, invasion, and angiogenesis in cancer tissues, CD45RO + lymphocytes might subsequently suppress the proliferation and migration of colorectal tumor cells ³⁹.

In the next step, to investigate the role of primary tumor localization in CRC outcomes, we classified the study population into two groups of right-sided and left-sided tumors. To best of our knowledge, none of the previous studies have examined our target markers expressions in CRC and their effects on prognosis based on primary tumor location. Our survival analysis showed that CD45RO score in IM in right-sided tumors was directly associated with better survival. Moreover, high score of CD8 in IM was associated with better OS in these patients. These findings further supported previous evidence suggesting that proximal and distal CRC may represent different immunological entities as they also showed differences in epidemiological, pathological, and clinical properties ⁴⁰.

Apart from survival, we examined the association between the score ofCD45RO in CT and IM, with clinicopathological properties in CRC, as well. We observed that low score of CD45RO in CT was correlated with no lymph node metastasis and earlier TNM stages (I/II) in both entire cohort and right-sided tumors. There was also a trend for high score of CD45RO and lesser tumor size. A study by Hu et al. on patients with lung adenocarcinoma was demonstrated that CD45RO + TILs are negatively correlated with tumor size and lymph node involvement ³⁰. However, controversially, they observed that high density of CD45RO + lymphocytes was correlated with earlier TNM stages. Although the reasons for this discrepancy remain unknown, a larger scale study is needed to reach a more definitive conclusion.

Thus, in this study, we examined the association of LAG3 score in both IM and CT of CRC with survival. Beside effector functions, some subpopulations of T cells express LAG3 and exert inhibitory functions to regulate immune responses. Overall, although there was a trend for high score of LAG3 in CT and improved OS we observed no statistically significant association between LAG3 expression and OS. Consistently, most studies including a meta-analysis on NSCLC ,renal, ovarian, gastric and a few other cancers have shown that expression of LAG3 is associated with better survival ⁴¹. In a study on patients with stage I–IIIB NSCLC, Hald et al. also showed that LAG3 expression on TILs was correlated with improved survival ⁴². The presence of LAG3 + TILs was also associated with longer Disease-specific survival in breast cancer patients, as well ²⁹. Despite these results, some studies reported that LAG3 expression is associated with poor survival. Chen et al. in a study on CRC demonstrated that patients with high percentage of LAG3 + cells in their tumor tissues had shorter survival compared with those with a low percentage of LAG3 + cells ⁴³. Considering that LAG3 is an inhibitory immune checkpoint that elicits suppressive immune responses and facilitates tumor escape, such a correlation between the expression of this marker and improved survival may be paradoxical. One hypothesis could be that the low level of LAG3 expression may lead to an exhausted phenotype, but up-regulation of LAG3 may initiate negative feedback of inhibitory signals that creates an active immune environment in the tumor and improve prognosis ⁴⁴. However, most previous studies mainly focused on the role of LAG3 in T cell immunity, but a recent study suggested that LAG3 expression correlated with NK-mediated cytotoxicity pathways, activation of B-cells, B cell-mediated immunity, and B cell receptor signaling pathways ⁴⁵. Another new aspect of the LAG3 marker is the expression of LAG3 on effector memory T cells with an activated phenotype. In this regard, Slevin et al. showed that LAG3 expression is elevated in the inflamed colonic mucosa of active ulcerative colitis and stimulated colonic LAG3 + T cells were able to produce IFNγ and IL-17A ⁴⁶. This suggests that, at the active inflammation site, LAG3 + cells may have effector rather than suppressor phenotypes. Given this view and the importance of LAG3 expression on memory T cells, we also examine the effect of the simultaneous expression of LAG3 and CD45RO on TILs on survival, although the results did not show any statistically significant relationship (data not shown).

Regarding clinical features, our study results showed a relationship between high score of LAG3 in IM in and higher T-stage and larger tumor size. However, many studies have not found a significant correlation between expression and any of the clinical features in NSCLC, CRC and Hodgkin lymphoma ^{42, 44, 47}, a similar result was reported by Que et al. which showed that LAG3 + T lymphocytes in tumor tissue were associated with larger tumor size in soft tissue sarcoma ⁴⁸. We also found that high score of LAG3 in IM was significantly correlated with the absence of TLS formation. Similarly, a previous study on CRC also demonstrated that LAG3 + T lymphocytes in tumor tissue were associated with higher invasion depth and metastasis ⁴³. Whilst, in CT of left-sided tumors, low score of LAG3 was associated with no metastasis and no recurrence. Controversial to survival analysis, our results may be suggest that the presence of LAG3 on lymphocytes infiltrated into tumor tissue may trigger suppressive immune responses and facilitates tumor cell proliferation, resulting in disease progression and tumor recurrence or metastasis. This issue should be a topic that warrants further investigation in a larger population.

In conclusion, our results indicated that infiltration of CD45RO + cells in IM is an independent prognostic factor in right-sided colon cancer, but not in left-sided tumors. In addition, higher numbers of LAG3 TILs in IM correlate with higher T-stage and low score of LAG3 in CT correlated with the absence of metastasis and recurrence in left-sided colon tumors. These findings probably suggest that the interaction between TILs and tumor cells in TME varies depending on the tumor side. The results of this study need validation but may be clinically relevant, as they indicate that tumor location might be an important factor to take into consideration in therapeutic decisions according to the patients' immune status and give physicians a good idea of the prognosis and clinical outcomes of the disease

Ethics approval

This project confirmed by Isfahan University of medical sciences ethics committee (IR.MUI.MED.REC.1398.610).

Consent for publication

Not applicable

Availability of data and materials

The data used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors have no competing interests that might be perceived to influence the results and/or discussion reported in this paper.

Funding

The present study was part of an MSc thesis written by Shirin Tavana, financially supported by grants from the Isfahan University of Medical Sciences, Isfahan, Iran [Grant No.398899] and technically supported by the Department of Immunology, School of Medicine, Isfahan University of Medical Sciences.

Authors' contributions

S. H and Z.M carried out the experiments. S.T wrote the main manuscript text and prepared figures. M.H.S carried out pathology sections analysis and data collection. Z.M data collection and statistical analysis. Z.H helped in statistical analysis. Z.S.F helped shape the research, analysis and manuscript edition. M.R conceived the original idea and supervised the project and manuscript final edition. All authors provided critical feedback and helped shape the research, analysis and manuscript.

Arnold M, Sierra MS, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global patterns and trends in colorectal cancer incidence and mortality. Gut. 2017;66(4):683–91.
Bufill JA. Colorectal cancer: evidence for distinct genetic categories based on proximal or distal tumor location. Annals of internal medicine. 1990;113(10):779–88.
Mirón Fernández I, Mera Velasco S, Turiño Luque JD, González Poveda I, Ruiz López M, Santoyo Santoyo J. Right and Left Colorectal Cancer: Differences in Post-Surgical-Care Outcomes and Survival in Elderly Patients. Cancers (Basel). 2021;13(11).
Baek SK. Laterality: Immunological Differences Between Right-Sided and Left-Sided Colon Cancer. Annals of coloproctology. 2019;35(6):291–3.
Sobin LH, Gospodarowicz MK, Wittekind C. TNM classification of malignant tumours: John Wiley & Sons; 2011.
Nagtegaal ID, Quirke P, Schmoll HJ. Has the new TNM classification for colorectal cancer improved care? Nature reviews Clinical oncology. 2011;9(2):119–23.
Pagès F, Galon J, Dieu-Nosjean MC, Tartour E, Sautès-Fridman C, Fridman WH. Immune infiltration in human tumors: a prognostic factor that should not be ignored. Oncogene. 2010;29(8):1093–102.
Jahanban-Esfahlan R, Seidi K, Banimohamad-Shotorbani B, Jahanban-Esfahlan A, Yousefi B. Combination of nanotechnology with vascular targeting agents for effective cancer therapy. Journal of cellular physiology. 2018;233(4):2982–92.
Yu P, Fu YX. Tumor-infiltrating T lymphocytes: friends or foes? Laboratory investigation; a journal of technical methods and pathology. 2006;86(3):231–45.
Nosho K, Baba Y, Tanaka N, Shima K, Hayashi M, Meyerhardt JA, et al. Tumour-infiltrating T-cell subsets, molecular changes in colorectal cancer, and prognosis: cohort study and literature review. The Journal of pathology. 2010;222(4):350–66.
Chew A, Salama P, Robbshaw A, Klopcic B, Zeps N, Platell C, et al. SPARC, FOXP3, CD8 and CD45 correlation with disease recurrence and long-term disease-free survival in colorectal cancer. PloS one. 2011;6(7):e22047.
Lee HE, Chae SW, Lee YJ, Kim MA, Lee HS, Lee BL, et al. Prognostic implications of type and density of tumour-infiltrating lymphocytes in gastric cancer. British journal of cancer. 2008;99(10):1704–11.
Angell HK, Bruni D, Barrett JC, Herbst R, Galon J. The Immunoscore: Colon Cancer and Beyond. Clinical cancer research: an official journal of the American Association for Cancer Research. 2020;26(2):332–9.
Galon J, Lanzi A. Immunoscore and its introduction in clinical practice. The quarterly journal of nuclear medicine and molecular imaging: official publication of the Italian Association of Nuclear Medicine (AIMN) [and] the International Association of Radiopharmacology (IAR), [and] Section of the So. 2020;64(2):152 – 61.
Naito Y, Saito K, Shiiba K, Ohuchi A, Saigenji K, Nagura H, et al. CD8 + T cells infiltrated within cancer cell nests as a prognostic factor in human colorectal cancer. Cancer research. 1998;58(16):3491–4.
Hu G, Wang S. Tumor-infiltrating CD45RO(+) Memory T Lymphocytes Predict Favorable Clinical Outcome in Solid Tumors. Scientific reports. 2017;7(1):10376.
Wakatsuki K, Sho M, Yamato I, Takayama T, Matsumoto S, Tanaka T, et al. Clinical impact of tumor-infiltrating CD45RO⁺ memory T cells on human gastric cancer. Oncology reports. 2013;29(5):1756–62.
Blackley EF, Loi S. Targeting immune pathways in breast cancer: review of the prognostic utility of TILs in early stage triple negative breast cancer (TNBC). Breast (Edinburgh, Scotland). 2019;48 Suppl 1:S44-s8.
Andrews LP, Marciscano AE, Drake CG, Vignali DA. LAG 3 (CD 223) as a cancer immunotherapy target. Immunological reviews. 2017;276(1):80–96.
Wang J, Sanmamed MF, Datar I, Su TT, Ji L, Sun J, et al. Fibrinogen-like protein 1 is a major immune inhibitory ligand of LAG-3. Cell. 2019;176(1–2):334–47. e12.
Huang CT, Workman CJ, Flies D, Pan X, Marson AL, Zhou G, et al. Role of LAG-3 in regulatory T cells. Immunity. 2004;21(4):503–13.
Chihara N, Madi A, Kondo T, Zhang H, Acharya N, Singer M, et al. Induction and transcriptional regulation of the co-inhibitory gene module in T cells. Nature. 2018;558(7710):454–9.
Grosso JF, Goldberg MV, Getnet D, Bruno TC, Yen HR, Pyle KJ, et al. Functionally distinct LAG-3 and PD-1 subsets on activated and chronically stimulated CD8 T cells. Journal of immunology (Baltimore, Md: 1950). 2009;182(11):6659-69.
Huang RY, Eppolito C, Lele S, Shrikant P, Matsuzaki J, Odunsi K. LAG3 and PD1 co-inhibitory molecules collaborate to limit CD8 + T cell signaling and dampen antitumor immunity in a murine ovarian cancer model. Oncotarget. 2015;6(29):27359–77.
Williams JB, Horton BL, Zheng Y, Duan Y, Powell JD, Gajewski TF. The EGR2 targets LAG-3 and 4-1BB describe and regulate dysfunctional antigen-specific CD8 + T cells in the tumor microenvironment. The Journal of experimental medicine. 2017;214(2):381–400.
Hemon P, Jean-Louis F, Ramgolam K, Brignone C, Viguier M, Bachelez H, et al. MHC class II engagement by its ligand LAG-3 (CD223) contributes to melanoma resistance to apoptosis. Journal of immunology (Baltimore, Md: 1950). 2011;186(9):5173-83.
Gandhi MK, Lambley E, Duraiswamy J, Dua U, Smith C, Elliott S, et al. Expression of LAG-3 by tumor-infiltrating lymphocytes is coincident with the suppression of latent membrane antigen-specific CD8 + T-cell function in Hodgkin lymphoma patients. Blood. 2006;108(7):2280–9.
Meng Q, Liu Z, Rangelova E, Poiret T, Ambati A, Rane L, et al. Expansion of Tumor-reactive T Cells From Patients With Pancreatic Cancer. Journal of immunotherapy (Hagerstown, Md: 1997). 2016;39(2):81 – 9.
Burugu S, Gao D, Leung S, Chia SK, Nielsen TO. LAG-3 + tumor infiltrating lymphocytes in breast cancer: clinical correlates and association with PD-1/PD-L1 + tumors. Annals of oncology: official journal of the European Society for Medical Oncology. 2017;28(12):2977–84.
Hu Z, Gu X, Zhong R, Zhong H. Tumor-infiltrating CD45RO(+) memory cells correlate with favorable prognosis in patients with lung adenocarcinoma. Journal of thoracic disease. 2018;10(4):2089–99.
Fridman WH, Pagès F, Sautès-Fridman C, Galon J. The immune contexture in human tumours: impact on clinical outcome. Nature reviews Cancer. 2012;12(4):298–306.
Kasurinen J, Hagström J, Kaprio T, Beilmann-Lehtonen I, Haglund C, Böckelman C. Tumor-associated CD3- and CD8-positive immune cells in colorectal cancer: The additional prognostic value of CD8+-to-CD3 + ratio remains debatable. Tumour biology: the journal of the International Society for Oncodevelopmental Biology and Medicine. 2022;44(1):37–52.
Rathore AS, Kumar S, Konwar R, Makker A, Negi MP, Goel MM. CD3+, CD4+ & CD8 + tumour infiltrating lymphocytes (TILs) are predictors of favourable survival outcome in infiltrating ductal carcinoma of breast. The Indian journal of medical research. 2014;140(3):361–9.
Tian C, Lu S, Fan Q, Zhang W, Jiao S, Zhao X, et al. Prognostic significance of tumor-infiltrating CD8⁺or CD3⁺ T lymphocytes and interleukin-2 expression in radically resected non-small cell lung cancer. Chinese medical journal. 2015;128(1):105–10.
Hotta K, Sho M, Fujimoto K, Shimada K, Yamato I, Anai S, et al. Prognostic significance of CD45RO + memory T cells in renal cell carcinoma. British journal of cancer. 2011;105(8):1191–6.
Osinsky S, Kovelskaya A, Bubnovskaya L, Osinsky D, Merentsev S. CD8 and CD45RO T lymphocytes in bone marrow of gastric cancer patients: correlation with disseminated tumor cells and disease outcome. Experimental oncology. 2015;37(1):48–52.
Kryczek I, Gryboś M, Dlubek D, Klimczak A, Rabczyński J, Lange A. Accumulation of CD45RO(+) cells in peritoneal carcinomatous fluid favours survival of ovarian carcinoma patients. Cancer Immunol Immunother. 2002;51(9):513–9.
Ahmadvand S, Faghih Z, Montazer M, Safaei A, Mokhtari M, Jafari P, et al. Importance of CD45RO + tumor-infiltrating lymphocytes in post-operative survival of breast cancer patients. Cellular oncology (Dordrecht). 2019;42(3):343–56.
Peng RQ, Wu XJ, Ding Y, Li CY, Yu XJ, Zhang X, et al. Co-expression of nuclear and cytoplasmic HMGB1 is inversely associated with infiltration of CD45RO + T cells and prognosis in patients with stage IIIB colon cancer. BMC cancer. 2010;10:496.
Lee GH, Malietzis G, Askari A, Bernardo D, Al-Hassi HO, Clark SK. Is right-sided colon cancer different to left-sided colorectal cancer? - a systematic review. European journal of surgical oncology: the journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology. 2015;41(3):300–8.
Saleh RR, Peinado P, Fuentes-Antrás J, Pérez-Segura P, Pandiella A, Amir E, et al. Prognostic Value of Lymphocyte-Activation Gene 3 (LAG3) in Cancer: A Meta-Analysis. Front Oncol. 2019;9:1040.
Hald SM, Rakaee M, Martinez I, Richardsen E, Al-Saad S, Paulsen EE, et al. LAG-3 in Non-Small-cell Lung Cancer: Expression in Primary Tumors and Metastatic Lymph Nodes Is Associated With Improved Survival. Clinical lung cancer. 2018;19(3):249 – 59.e2.
Chen J, Chen Z. The effect of immune microenvironment on the progression and prognosis of colorectal cancer. Medical oncology (Northwood, London, England). 2014;31(8):82.
Moerdler S, Ewart M, Friedman DL, Kelly K, Pei Q, Peng M, et al. LAG-3 is expressed on a majority of tumor infiltrating lymphocytes in pediatric Hodgkin lymphoma. Leukemia & lymphoma. 2021;62(3):606–13.
Liu Q, Qi Y, Zhai J, Kong X, Wang X, Wang Z, et al. Molecular and Clinical Characterization of LAG3 in Breast Cancer Through 2994 Samples. Front Immunol. 2021;12:599207.
Slevin SM, Garner LC, Lahiff C, Tan M, Wang LM, Ferry H, et al. Lymphocyte Activation Gene (LAG)-3 Is Associated With Mucosal Inflammation and Disease Activity in Ulcerative Colitis. Journal of Crohn's & colitis. 2020;14(10):1446–61.
Rhyner Agocs G, Assarzadegan N, Kirsch R, Dawson H, Galván JA, Lugli A, et al. LAG-3 Expression Predicts Outcome in Stage II Colon Cancer. Journal of Personalized Medicine. 2021;11(8):749.
Que Y, Fang Z, Guan Y, Xiao W, Xu B, Zhao J, et al. LAG-3 expression on tumor-infiltrating T cells in soft tissue sarcoma correlates with poor survival. Cancer biology & medicine. 2019;16(2):331–40.

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The survey in the role of LAG3+ tumor-infiltrating lymphocytes in colorectal cancer prognosis; Relationship with sidedness

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Patients and specimens

2.2 Immunohistochemistry

2.3 Evaluation of immunochemistry staining

2.4 Statistical analysis

3. Results

3.1. Patients characteristics

3.2. Distribution of CD45RO+, LAG3+, CD3+, and CD8 + TILs in patients with CRC

3.3. Mean frequency of CD45RO + and LAG3 + lymphocytes in patients with different clinicopathological features

3.4. Associations of CD45RO + and LAG3 + TILs with clinicopathological features

3.5. Associations of CD45RO + and LAG3 + TILs with clinicopathological features based on primary tumor location

3.6. Prognostic value of CD45RO+, LAG3+, CD3+, and CD8 + TILs

3.7. Associations of CD45RO+, LAG3+, CD3+, and CD8 + TILs with OS based on primary tumor location

4. Discussion

Declarations

References

Additional Declarations

Supplementary Files

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