Retrospective Study Open Access
Copyright ©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastrointest Oncol. Jun 15, 2024; 16(6): 2487-2503
Published online Jun 15, 2024. doi: 10.4251/wjgo.v16.i6.2487
Association between Helicobacter pylori infection, mismatch repair, HER2 and tumor-infiltrating lymphocytes in gastric cancer
Carlos A Castaneda, Faculty of Health Sciences, Universidad Cientifica del Sur, Lima 15038, Peru
Carlos A Castaneda, GECO PERU, Grupo de Estudios Clinicos Oncologicos del Peru, Lima 15038, Peru
Miluska Castillo, Luis A Bernabe, Joselyn Sanchez, Katherine Tello, Raul Alatrista, Department of Research, Instituto Nacional de Enfermedades Neoplasicas, Lima 15038, Peru
Joselyn Sanchez, Faculty of Human Medicine, Universidad Ricardo Palma, Lima 15039, Peru
Matteo Fassan, Department of Medicine, Surgical Pathology & Cytopathology Unit, University of Padua, Padua 35121, Italy
Ignacio Ivan Wistuba, Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, United States
Ivan Chavez Passiuri, Eloy Ruiz, Department of Abdominal Surgery, Instituto Nacional de Enfermedades Neoplasicas, Lima 15038, Peru
Juvenal Sanchez, Yaqueline Bazan, Luis Taxa, Department of Pathology, Instituto Nacional de Enfermedades Neoplasicas, Lima 15038, Peru
Fernando Barreda, Daniel Valdivia, Department of Medical Specialties, Instituto Nacional de Enfermedades Neoplasicas, Lima 15038, Peru
Milagros Abad-Licham, Department of Pathology, Instituto Regional de Enfermedades Neoplasicas del Norte, Trujillo 13001, Peru
Milagros Abad-Licham, Faculty of Human Medicine, Universidad Privada Antenor Orrego, Trujillo 13008, Peru
Claudio Mengoa, Department of Surgery, Instituto Regional de Enfermedades Neoplasicas del Sur, Arequipa 04002, Peru
Hugo Fuentes, Paola Montenegro, Department of Medical Oncology, Instituto Nacional de Enfermedades Neoplasicas, Lima 15038, Peru
Ebert Poquioma, Department of Epidemiology, Instituto Nacional de Enfermedades Neoplasicas, Lima 15038, Peru
Claudio J Flores, Unidad de Investigación Básica y Traslacional, Oncosalud-AUNA, Lima 15038, Peru
Luis Taxa, Faculty of Medicine, Universidad San Martin de Porres, Lima 15008, Peru
ORCID number: Carlos A Castaneda (0000-0001-6200-0856); Miluska Castillo (0000-0002-0111-3176); Luis A Bernabe (0000-0003-1896-7060); Joselyn Sanchez (0000-0002-6764-4180); Matteo Fassan (0000-0001-6515-5482); Katherine Tello (0000-0002-4981-3411); Ignacio Ivan Wistuba (0000-0003-3365-6340); Ivan Chavez Passiuri (0000-0002-3431-3262); Eloy Ruiz (0000-0001-5561-0752); Juvenal Sanchez (0000-0002-9825-8573); Fernando Barreda (0000-0002-7923-6299); Daniel Valdivia (0000-0002-5917-6452); Yaqueline Bazan (0000-0002-7337-2396); Milagros Abad-Licham (0000-0002-3530-6937); Claudio Mengoa (0000-0001-8547-7892); Hugo Fuentes (0000-0002-2747-7381); Paola Montenegro (0000-0003-1734-1840); Ebert Poquioma (0000-0003-4016-6112); Raul Alatrista (0000-0002-7735-1533); Claudio J Flores (0000-0002-3659-4993); Luis Taxa (0000-0002-0914-9149).
Author contributions: Castaneda CA and Castillo M contributed to the conception and design of the study; Castaneda CA, Castillo M and Flores CJ performed data analysis and interpretation; Bernabe LA, Sanchez J, Tello K, Fassan M, Bazan Y, Alatrista R, Poquioma E and Taxa L performed data acquisition, as well as providing technical support; Chavez Passiuri I, Barreda F, Ruiz E, Wistuba II, Abad-Licham M, Mengoa C, Fuentes H, Montenegro P and Valdivia D provided administrative and material support; all authors drafted the article, made critical revisions and approved the final version of the manuscript.
Supported by Ministerio de la Produccion de Peru, No. 317-PNICP-EC-2014, and No. 430-PNICP-PIAP-2014; Consejo Nacional de Ciencia Tecnologia e Innovacion Tecnologica, No. 196-2015-FONDECYT, No. 197-2015-FONDECYT, and No. 204-2015-FONDECYT.
Institutional review board statement: This study was reviewed and approved by the Ethics Committee of the Institutional of Instituto Nacional de Enfermedades Neoplasicas (Approval No. 025-2016-DI-DICON/INEN).
Informed consent statement: All study participants provided informed written consent prior to study enrollment.
Conflict-of-interest statement: The authors have no financial relationships to disclose.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Carlos A Castaneda, MD, MSc, Assistant Professor, Faculty of Health Sciences, Universidad Cientifica del Sur, Panamericana Sur 19, Villa El Salvador, Lima 15038, Peru. ccastaneda@gecoperu.org
Received: February 7, 2024
Revised: April 4, 2024
Accepted: April 11, 2024
Published online: June 15, 2024
Processing time: 135 Days and 14.5 Hours

Abstract
BACKGROUND

The influence of Helicobacter-pylori (H. pylori) infection and the characteristics of gastric cancer (GC) on tumor-infiltrating lymphocyte (TIL) levels has not been extensively studied. Analysis of infiltrating-immune-cell subtypes as well as survival is necessary to obtain comprehensive information.

AIM

To determine the rates of deficient mismatch-repair (dMMR), HER2-status and H. pylori infection and their association with TIL levels in GC.

METHODS

Samples from 503 resected GC tumors were included and TIL levels were evaluated following the international-TILs-working-group recommendations with assessment of the intratumoral (IT), stromal (ST) and invasive-border (IB) compartments. The density of CD3, CD8 and CD163 immune cells, and dMMR and HER2-status were determined by immunohistochemistry (IHC). H. pylori infection was evaluated by routine histology and quantitative PCR (qPCR) in a subset of samples.

RESULTS

dMMR was found in 34.4%, HER2+ in 5% and H. pylori-positive in 55.7% of samples. High IT-TIL was associated with grade-3 (P = 0.038), while ST-TIL with grade-1 (P < 0.001), intestinal-histology (P < 0.001) and no-recurrence (P = 0.003). dMMR was associated with high TIL levels in the ST (P = 0.019) and IB (P = 0.01) compartments, and ST-CD3 (P = 0.049) and ST-CD8 (P = 0.05) densities. HER2- was associated with high IT-CD8 (P = 0.009). H. pylori-negative was associated with high IT-TIL levels (P = 0.009) when assessed by routine-histology, and with high TIL levels in the 3 compartments (P = 0.002-0.047) and CD8 density in the IT and ST compartments (P = 0.001) when assessed by qPCR. A longer overall survival was associated with low IT-CD163 (P = 0.003) and CD8/CD3 (P = 0.001 in IT and P = 0.002 in ST) and high IT-CD3 (P = 0.021), ST-CD3 (P = 0.003) and CD3/CD163 (P = 0.002).

CONCLUSION

TIL levels were related to dMMR and H. pylori-negativity. Low CD8/CD3 and high CD163/CD3 were associated with lower recurrence and longer survival.

Key Words: Lymphocytes, Macrophages, Gastric cancer, Helicobacter pylori, HER2, Mismatch repair

Core Tip: Absence of Helicobacter pylori was associated with high tumor-infiltrating lymphocyte (TIL) levels and CD8 density. Deficient mismatch-repair was associated with high TIL levels, and CD3 and CD8 density. Longer overall survival was associated with a low CD8/CD3 ratio, and high CD3 and CD3/CD163 ratio.


  • Citation: Castaneda CA, Castillo M, Bernabe LA, Sanchez J, Fassan M, Tello K, Wistuba II, Chavez Passiuri I, Ruiz E, Sanchez J, Barreda F, Valdivia D, Bazan Y, Abad-Licham M, Mengoa C, Fuentes H, Montenegro P, Poquioma E, Alatrista R, Flores CJ, Taxa L. Association between Helicobacter pylori infection, mismatch repair, HER2 and tumor-infiltrating lymphocytes in gastric cancer. World J Gastrointest Oncol 2024; 16(6): 2487-2503
  • URL: https://www.wjgnet.com/1948-5204/full/v16/i6/2487.htm
  • DOI: https://dx.doi.org/10.4251/wjgo.v16.i6.2487

INTRODUCTION

Gastric cancer (GC) is one of the most common cancers throughout the world and the second most frequent in Peruvian males, and carries a poor prognosis[1-3]. The pathological features of GC can define tumor behavior and prognosis[4]. Helicobacter pylori (H. pylori) infection, which is highly prevalent in Peru, is an accepted trigger of GC and has recently been suggested to predict a lower effect of immunotherapy with checkpoint immune inhibitors[5].

Microsatellite instability (MSI) defines one of the four molecular GC subtypes[6]. It is usually detected by deficient mismatch-repair (dMMR) and has been associated with high levels of neoantigens, and along with HER2 positive status are biomarkers of response to checkpoint immune inhibitors and trastuzumab treatment, respectively[7-11]. A recent study found that the addition of checkpoint inhibitors to antiHER2 therapy in HER2 positive cases increases clinical response[12].

High levels of tumor-infiltrating lymphocytes (TILs) have been associated with longer survival and greater response to checkpoint inhibitors in different malignancies[13,14]. Information on the type, density and location of TIL subpopulations has been associated with tumor features, such as Epstein-Barr-Virus infection[4] and survival in gastrointestinal malignancies[15,16], and predicts response to checkpoint inhibitors in advanced GC[11,17-19]. Despite the relevance of H. pylori infection, dMMR and HER2 status in GC, few studies have evaluated their association with TIL levels in resected non-metastatic tumors. The studies available differ in the methodologies used to evaluate TIL and very few have included South American populations.

In the present study, TIL levels were determined following the International Immuno-Oncology Biomarkers Working Group: Part 2 recommendations[20] as well as the density of CD3+ T lymphocytes, CD8+ cytotoxic T lymphocytes and CD163+ M2 macrophages. In addition, the relationship of TIL levels with the clinicopathological features, including H. pylori infection, dMMR, HER2 status and survival in resected GC, was evaluated.

MATERIALS AND METHODS
Study population

We included information from 503 GC patients who underwent surgery at the Instituto Nacional de Enfermedades Neoplasicas in Lima, Peru from January 2008 to December 2018 and in whom pathology material was available. Clinical-pathological features were obtained from medical histories and pathology reports of the patients, and hematoxylin and eosin (HE)-stained slides were prospectively reviewed when no specific information was found[21,22].

This single-center retrospective cohort study was approved by the Research and Ethics Committee (Protocol Number 050-2015-CIE/INEN), and the patients provided signed informed consent.

Evaluation of TIL levels and the presence of H. pylori

Several original sections from each primary tumor were re-examined and the most representative tissue block, with a 5 µm thickness and stained in HE was selected. Original and new sections were examined by experienced histopathologists (Sanchez J & Taxa L) for review of the standard pathological features, including the presence of H. pylori, and contrasted with original reports. The level of TILs was estimated avoiding ulcerated or necrotic areas and classified by spatial location [intratumoral (IT), stromal (ST) and invasive-border (IB) compartments (Figures 1 and 2)]. TIL levels above the median (calculated for every compartment) were classified as high[20].

Figure 1
Figure 1 Pathological images of a case of gastric cancer with Helicobacterpylori infection with a low level of tumor-infiltrating lymphocytes. A: Hematoxylin and eosin (HE) staining showing the presence of Helicobacter pylori (yellow arrow) at 100 × magnification; B: HE staining of the intratumoral compartment with a low level of tumor-infiltrating lymphocytes at 20 ×.
Figure 2
Figure 2 Pathological images of a case of gastric cancer with deficient mismatch-repair and a high level of tumor-infiltrating lymphocytes. A: Hematoxylin and eosin (HE) staining of stromal compartment with high level of tumor-infiltrating lymphocytes (TILs) at 20 × magnification; B: Field magnification of HE with high TIL stain at 40 ×; C: CD3 immunohistochemistry (IHC) staining at 40 ×; D: Identification of CD3 density by machine learning-based image processing showing positive (green) and negative (blue) cells; E: CD8 IHC staining; F: Digital identification of CD8 density; G: IHC staining showing absence of MSH6 expression and; H: Absence of PMS2 expression.
Tissue array method and IHC staining

Core tissue biopsies (6 mm in diameter) were taken from tumoral areas with a high density of TIL in every individual paraffin-embedded tumor and 10-12 cores were re-arranged in a new recipient paraffin block (tissue array block) using the Quick-Ray Manual Tissue Microarrayer (Unitma Co., Ltd., Seoul, Korea). Sections (4 mm) were taken from each tissue array block, deparaffinized, and dehydrated[23].

Staining of GC tissue sections was performed using the EnVision FLEX Kit (K8000, Dako Glostrup, Denmark) in paraffin-embedded sections. Briefly, each paraffin section was deparaffinized, followed by antigen retrieval with Epitope Retrieval Solution, Tris/EDTA buffer pH 9 (DM830, Dako, Glostrup, Denmark) in a preheated water bath (95°C, 20 min). Endogenous peroxidase was blocked, and antihuman primary antibodies were applied for 25 to 45 min at 25ºC. Immune cells were evaluated using anti-mouse CD3 antibody (Is503, Dako), anti-human CD8 (IS623, Dako) and CD163 antibodies (clone EP324, Master Diagnostica, Granada, Spain).

The MMR proteins evaluated were mouse anti-human MutL protein homolog 1 (MLH1, ES01 Dako), mouse anti-human MutS protein homolog 2 (MSH2, FE11 Dako), rabbit anti-human MutS protein homolog 6 (MSH6, EP49 Dako) and rabbit anti-human postmeiotic segregation increased 2 (PMS2, EP51 Dako). HER2 was evaluated with the polyclonal rabbit anti-human c-erbB-2 oncoprotein (AO485 Dako).

Thereafter, the sections were incubated with secondary Abs as per the EnVision FLEX Kit, and counter-stained with hematoxylin. Positive staining controls were performed with paraffin sections of normal human tonsil.

Assessment of HER-2 status was performed following standard scoring criteria specific for GC[7,8,24], while dMMR was determined when the expression of at least one of the 4 MMR proteins evaluated was lost (Figure 3)[11,17-19].

Figure 3
Figure 3 Overall survival analyses. A: Kaplan-Meier overall survival curve according to clinical stage; B: Intratumoral CD3/163 ratio; C: Intratumoral CD3/CD8 ratio.
Evaluation of H. pylori gene expression

H. pylori gene expression was determined by the constitutive hspA and UreA genes in DNA obtained from frozen gastric samples by quantitative PCR (qPCR) in the LightCycler 96 Instrument Thermal Cycler (Roche, Mannheim, Germany).

Values were considered positive when ≥ 10 copies/μL were detected. The virulence of the cagA, vacAs, and vacAm genes as well as vacAs1 and vacAm1 alleles was tested by an experienced cancer biologist (NS) in H. pylori-positive patients, as described in a previous study by our group[3].

Quantitative analysis of TIL subpopulations

Immunostained slides were scanned with a digital virtual microscope BX63 Olympus (Tokyo, Japan), and the region with the highest immune cell density was selected. Five high power fields (HPF; or three when there was not enough stained tissue) within the IT and ST compartments were captured at 20 × magnification and analyzed using Visiopharm Tissuemorph Digital Pathology image analysis software (Visiopharm, Hoersholm, Denmark) under the supervision of a pathologist (Sanchez J). The density of the immune cells was calculated by the mean of positive cells in the captured HPF[25] (Figure 3). The optimal cutoff values for defining a higher density of immune cells (CD3, CD8 and CD163) were calculated using the maximally selected rank statistics according to Lausen [in relation to overall survival (OS)][26].

Statistical analysis

The non-paired Student’s t-test was used to examine differences between groups. Correlations between values were evaluated using the non-parametric Spearman rank correlation. The intraclass correlation test was used to compare TIL levels and the density of immune cells in different compartments. OS was calculated from the date of surgery until death or until the date patients were last known to be alive (obtained from National Registry of Identification and Marital Status). Disease-free survival (DFS) was calculated from the date of surgery until relapse (patient records) or last known alive status. The last review of state of life was carried out in May 2022. In the univariate analysis, the survival curves were compared according to clinical characteristics using the log-rank or Breslow test, and in multivariate analysis using the Cox regression model with a stepwise selection method. A P value < 0.05 was considered significant. Analyses were performed using the SPSS statistical software (IBM SPSS Statistical 19) and R program. The statistical methods of this study were reviewed by Flores CJ from Oncosalud-AUNA.

RESULTS
General features

The clinicopathological features of the patients included are described in Table 1. Most cases underwent subtotal gastrectomy (62.4%). Adjuvant chemotherapy was administered in 45.1% and radiation in 13.9%.

Table 1 Clinicopathological features and prognostic value.
Feature
Total (n = 503)
%
Median
5 yr DFS
P value
Median
5 yr OS
P value
Age, yr (19-95 yr)
    < 6023145.94.649.10.0437.052.50.009
    ≥ 6027254.13.039.63.741.6
Sex
    Female25250.13.343.70.5823.845.30.319
    Male25149.94.044.64.548.2
Bormann
    I-II9018.1NA65.4< 0.001NA66.00.002
    III31162.72.940.64.044.4
    IV-V9519.22.636.82.837.6
Lauren (n = 496)
    Intestinal22244.84.339.90.7164.849.30.598
    Diffuse18136.53.046.24.143.9
    Mixed9318.82.342.93.243.2
Grade
    1469.1NA62.20.025NA67.80.044
    216031.84.446.94.549.0
    329759.12.539.73.642.3
ILV
    No14929.66.962.4< 0.0018.166.4< 0.001
    Yes35470.42.436.53.038.5
Antrum
    Yes32364.22.747.90.0643.551.00.104
    No18035.84.538.85.140.8
Clinical stage
    I6412.7NA80.7< 0.001NA83.0< 0.001
    II13827.47.759.67.759.5
    III30159.81.929.32.333.2
Node involvement
    No13927.610.567.0< 0.001NA68.2< 0.001
    Yes36472.42.335.43.038.4
Recurrence
    No32264.0---NA68.5< 0.001
    Yes18136.0--1.712.3
H. pylori HE (n = 415)
    Absent18444.33.842.90.2524.546.00.637
    Present23155.77.348.35.351.2
H. pylori qPCR (n = 234)
    Negative8536.32.536.80.2293.139.10.219
    Positive14963.73.838.34.039.7
dMMR (n = 479)
    No26965.64.345.80.3964.747.90.158
    Yes14134.43.142.6-3.644.7-
HER-2 status (n = 468)
    Negative43495.03.543.40.8684.346.20.892
    Positive2353.139.83.339.3
IT TIL (n = 462)
    < 1018640.32.741.80.7633.743.60.395
    ≥ 1027659.73.744.04.547.1
ST TIL (n = 461)
    < 3025855.92.641.50.4583.845.00.853
    ≥ 3020344.03.844.84.446.3
IB TIL (n = 332)
    < 7017853.63.346.00.9914.447.80.660
    ≥ 7015446.44.446.44.949.4
IT CD3/HPF (n = 453)
    < 5812627.82.434.30.0282.936.50.021
    ≥ 5832772.24.447.05.450.5
IT CD8/ HPF (n = 443)
    < 7023152.14.447.40.0604.849.90.142
    ≥ 7021247.92.640.44.043.8
IT CD163/HPF (n = 205)
    < 24018389.32.842.70.0023.845.30.003
    ≥ 2402210.71.40.01.50.0
IT CD8/CD3 ratio (n = 432)
    < 0.6321048.64.549.1<0.0016.052.90.001
    ≥ 0.6322251.42.336.02.838.5
IT CD3/CD163 (n = 179)
    < 813776.52233.90.0042.636.60.002
    ≥ 84223.5NA59.5NA64.3
ST CD3/HPF (n = 363)
    < 9518049.62.636.60.0142.938.70.003
    ≥ 9518350.45.250.77.054.6
ST CD8/HPF (n = 341)
    < 6821663.33.843.50.5784.045.00.382
    ≥ 6812536.73.643.04.547.5
ST CD8/CD3 ratio (n = 328)
    < 1.227784.54.345.90.0054.649.10.002
    ≥ 1.25115.51.926.12.026.6

H. pylori (+) was found in 231 of 415 (55.7%) cases when evaluated in HE staining and in 149 of 234 (63.7%) cases when evaluated with qPCR. The cagA gene was detected in 87.2% (130/149) of H. pylori (+) patients identified by qPCR, while the vacAs gene was detected in 79.1% (117/148) and vacAm in 75.2% (112/149) of H. pylori (+) patients. VacAs1 and vacAm1 alleles were detected in 47.9% (56/117) and 70.5% (79/112) of H. pylori (+) patients, respectively, and concurrent presence was found in 40.2% (39/97).

The presence of dMMR was found in 141 (34.4%; negative in 269 cases and not conclusive staining in 69), HER2-positive status in 23 (5%; negative in 434 and equivocal in 11 cases) and both features were found in 8 (2.2%) cases (Table 1). dMMR was associated with grade 1 (P = 0.028). HER2 overexpression was associated with an intestinal subtype (P < 0.001), grade 3 (P < 0.001) and low stage (P = 0.009).

Regarding survival analysis, the median follow-up was 6.4 years (95%CI 5.9-6.8 years), and recurrence and death were found in 181 cases and 270 cases, respectively. The clinicopathological features related to survival are described in Table 1.

Correlation between TILs and clinicopathological features

The median TIL level in the IT compartment was 10% (1%-80%) (n = 462), being 30% in the ST (1%-95%; n = 461) and 70% (1%-95%; n = 332) in the IB compartments, with a significant correlation among the three compartments [IT vs ST, intraclass correlation coefficient (ICC) = 0.613; IT vs IB, ICC = 0.340; IB vs ST, ICC = 0.726]. A high IT TIL level was associated with grade 3 (P = 0.038), lymphovascular invasion+ (P = 0.028) and stage 2 (P = 0.016). High ST TIL levels were associated with age ≥ 60 years (P < 0.001), non-antrum location (P = 0.049), intestinal histology (P < 0.001), grade 1 (P < 0.001), stage 2 (P = 0.001) and no recurrence (P = 0.003). Lastly, a high IB TIL level was associated with intestinal histology (P = 0.002), lymph node negative (P = 0.029) and earlier stages (P = 0.001; Table 2).

Table 2 Relationship between level of infiltrating lymphocytes and clinicopathological features.
Features
IT TILs
P value
ST TILs
P value
IB TILs
P value
Median> 10%> 30%> 70%
Age0.762< 0.0010.279
    < 6059.133.343
    ≥ 6060.454.848.9
Sex0.3820.0740.678
    Female57.739.845.1
    Male61.748.147.4
Gastric region location0.4500.0490.160
    No antrum625051.1
    Antrum58.440.543.3
Bormann0.2210.3790.094
    154.55044.4
    247.336.438.8
    363.347.652.2
    46138.333.3
    5505028.6
Lauren histology0.891< 0.0010.002
    Intestinal58.758.7453.1
    Diffuse58.920.428.6
    Mixed61.65042.6
Grade0.038< 0.0010.716
    15058.352.8
    253.555.545.6
    364.635.645.8
Lymphovascular invasion0.0280.5200.968
    No51.941.746.2
    Yes62.944.946.5
Lymph node involvement0.4030.7140.029
    Yes60.943.542.5
    No56.645.555.6
Pathology stage0.0160.0010.001
    I422841.5
    II65.455.860.4
    III60.341.538.9
Recurrence 0.2300.0030.369
    No61.949.548
    Yes56.335.242.7
H. pylori HE0.0090.4450.411
    Absent65.947.647.5
    Present52.443.642.5
H. pylori qPCR0.0020.0470.010
    Absent67.148.650.9
    Present43.734.129.9
CagA/H. pylori+0.7610.2750.002
    CagA-4046.764.3
    CagA+44.132.424.1
VacAs/H. pylori+0.3320.7190.045
    VacAs-51.93747.6
    VacAs+41.433.325
VacAs1/H. pylori+0.5300.8870.894
    VacAs1-38.532.724.4
    VacAs1+44.73425.7
VacAm/H. pylori+0.8240.5350.265
    VacAm-41.938.738.5
    VacAm+44.232.626.8
VacAm1/H. pylori+0.1540.0930.508
    VacAm1-55.244.821.7
    VacAm1+39.427.329.2
VacAs1+m1+/H. pylori+0.9370.9530.527
    No VacAs1+m1+44.232.724.4
    VacAs1+m1+43.366.731.8
dMMR0.8750.0190.010
    No58.738.938.6
    Yes59.651.554.2
HER2 positive0.4980.4200.55
    No60.144.147.2
    Yes52.447.635.3

There was poor correlation between IT TIL levels and the densities of CD3+ (ICC = 0.106), CD8+ (ICC = 0.151) and CD163+ (ICC = 0.065) cells.

The mean densities of IT CD3+ (n = 453) and CD8+ (n = 443) cells/HPF were 108.6 (0.2-925.2) and 66.6 (1-777.4), respectively, while the mean density of IT CD163+ was 43 (0-519)/HPF (n = 205), and those of ST CD3+ (n = 363) and CD8+ (n = 341) cells/HPF were 95.2 (0.2-858.6) and 45.8 (0.5-581.8), respectively (Table 1). There was almost perfect agreement between the densities of CD3+ and CD8+ cells in the IT (ICC = 0.692) as well as in the ST compartments (ICC = 0.606). There was moderate agreement between the density of IT CD163+ and both CD3+ (ICC = 0.327) and CD8+ cells (ICC = 0.259) in the IT compartment.

A high density of IT CD3+ cells were associated with diffuse histology (P = 0.03), grade 3 (P < 0.001), absence of recurrence (P = 0.02) and longer DFS and OS (P = 0.028 and 0.021, respectively). A high density of IT CD8+ cells were associated with grade 3 (P < 0.001; Table 3) and a high density of IT CD163+ cells were associated with a non-antrum location (P = 0.031), grade 3 (P = 0.02) and shorter DFS and OS (P = 0.002 and P = 0.003, respectively). Cases with a high IT CD3/CD163 ratio presented a longer DFS (P = 0.004) and OS (P = 0.002; Table 1 and Figure 3).

Table 3 Relationship between density of infiltrating immune cells and clinico-pathological features.
Features
IT CD3
P value
ST CD3
P value
IT CD8
P value
ST CD8
P value
IT CD163
P value
Median> 58> 95> 70> 68> 240
Age0.7140.2750.0260.2240.162
< 60734742.132.97.4
≥ 6071.552.852.739.313.5
Sex0.0390.5570.8140.6990.354
Female76.55248.437.78.7
Male67.84947.335.712.7
Gastric region location0.5030.5850.7950.9010.031
No antrum74.152.448.736.216.7
Antrum71.149.447.436.97.1
Bormann0.6480.4410.3380.0060.462
I-II70.447.441.822.65.3
III73.655.650.74311.7
IV-V68.952.946.13013
Lauren histology0.030.0030.3080.0030.038
Intestinal66.257.443.740.38.2
Diffuse7836.65022.217.8
Mixed75.950.852.746.32.9
Grade<0.0010.311<0.0010.0830.02
15955.326.820.53.8
262.954.240.738.24.3
379.546.455.339.216.4
Lymphovascular invasion0.6020.2410.0920.1170.865
No73.955.241.830.411.3
Yes71.548.450.539.310.5
Lymph node involvement0.7230.9830.4870.1410.252
Yes72.650.448.939.112.1
No7150.545.230.66.3
Pathology stage0.9420.1010.6740.0580.197
I70.95042.9209.5
II73.25947.2414.1
III7246.349.237.813.3
Recurrence 0.020.0790.4480.4750.389
No7653.946.635.29.3
Yes65.944.45039.113.2
H. pylori HE0.5470.8170.3560.970.637
Absent71.151.449.13613.4
Present73.952.844.236.211.1
H. pylori qPCR0.1430.3350.0010.0010.105
Absent83.155.962.749.126.1
Present7448.13722.814.1
CagA/H. pylori+0.2540.6030.0230.540.729
CagA-82.942.957.521.214.3
CagA+74.24837.226.617.2
VacAs/H. pylori+0.9920.8430.0710.8550.823
VacAs-76.845.851.724.415.2
VacAs+76.947.637.325.916.9
VacAs1/H. pylori+0.3970.4320.9420.2340.543
VacAs1-79.253.536.418.414.7
VacAs1+72.244.7373020.8
VacAm/H. pylori+0.10.2480.2550.8840.865
VacAm-69.440.447.624.517.1
VacAm+80.650.638.725.615.8
VacAm1/H. pylori+0.1450.0980.2420.8220.877
VacAm1-89.765.248.123.816.7
VacAm1+7744.835.426.315.2
VacAs1+m1+/H. pylori+0.1130.2890.9130.2530.345
No VacAs1+m1+8657.537.721.612.9
VacAs1+m1+72.24438.934.623.5
dMMR0.3930.0490.0950.050.169
No75.447.645.532.311.5
Yes71.359.554.743.95.1
HER2 status0.060.2040.0090.4040.835
Negative74.452.449.937.711.1
Positive56.538.121.728.69.1

A high density of ST CD3+ cells was associated with an intestinal subtype (P = 0.003), and longer DFS and OS (P = 0.014 and P = 0.003, respectively), while a high density of ST CD8+ cells was associated with Bormann III GC (P = 0.006) and an intestinal subtype (P = 0.003; Table 3).

Patients with a low CD8/CD3 ratio in the IT and ST compartments had a longer DFS (P < 0.001 and P = 0.005, respectively) and OS (P = 0.001 and P = 0.002, respectively; Table 1 and Figure 3).

Multivariate analysis showed that age, tumor grade, stage as well as a high density of IT CD3+ cells and a low IT CD8/CD3 ratio were associated with a longer DFS and OS (Table 4).

Table 4 Multivariate analysis of factors associated with disease-free survival and overall survival.
FeaturesDFS
OS
HR
P value
HR
P value
Age
    < 60Reference-Reference-
    > 601.8 (1.2, 2.5)0.0021.9 (1.3, 2.8)< 0.001
Histological grade
    DifferentiatedReferenceReference
    Undifferentiated1.7 (1.01, 2.7)0.0432.0 (1.2, 3.3)0.007
WHO classification
    1-2ReferenceReference
    3-42.5 (1.6, 3.8)< 0.0012.4 (1.5, 3.7)< 0.001
Clinical stage
    I-IIReferenceReference
    III2.1 (1.4, 3-2)< 0.0011.9 (1.2, 2.9)0.004
IT CD3/HPF
    High (> 58)ReferenceReference
    Low (< 58)1.5 (1.02, 2.2)0.0371.6 (1.1, 2.4)0.018
IT CD8/CD3 ratio
    < 0.63ReferenceReference
    > 0.631.7 (1.2, 2.5)0.0021.6 (1.1, 2.3)0.008
Non-significant variables
    Sex-0.939-0.51
    Cardia-fundus-0.398-0.212
    Bormann-0.328-0.315
    Lauren-0.311-0.291
    Lymph nodes-0.836-0.663
    lymphovascular invasion-0.972-0.277
    H. pylori-0.123-0.140
    Deficient mismatch-repair-0.973-0.365
    HER2-0.222-0.114
    IT TIL -0.940-0.547
    Stromal TIL-0.818-0.806
    IT CD8/HPF-0.077-0.340
Correlation between TIL levels and H. pylori infection

A high IT TIL level was associated with absence of H. pylori (P = 0.009) when evaluated by HE in the whole series. When evaluated by qPCR (n = 234), H. pylori (-) was associated with high IT (P = 0.002), ST (P = 0.047) and IB (P = 0.01) TIL levels. High IB TIL levels were associated with infection by H. pylori+ cagA- (P = 0.002) and vacA- (P = 0.045; Table 2).

High densities of IT CD8+ (P = 0.001) and ST CD8+ (P = 0.001) cells were associated with H. pylori (-) and a high density of IT CD8+ cells were associated with H. pylori+ cagA- (P = 0.023; Table 3).

Correlation between TIL and both dMMR and HER2 expression

High ST (P = 0.019) and IB (P = 0.01) TIL levels were associated with dMMR. High densities of ST CD3+ (P = 0.049) and CD8+ (P = 0.05) cells were associated with dMMR (Table 3) and a high density of IT CD8+ cells were associated with HER2-negative (P = 0.009; Tables 2 and 3).

DISCUSSION

Our series shows that the association between TIL levels and clinical-pathological features varies according to the tumor compartments in which TILs are determined. This is the first study to describe that high TIL levels in the IT compartment are associated with the absence of H. pylori infection, while high TIL levels in both the IB and ST compartments are associated with dMMR, and ST TIL levels are also associated with low disease recurrence. High densities of CD3+ and CD8+ T lymphocytes were associated with dMMR in the ST compartment, and CD8+ T lymphocytes with HER2 negative in the IT compartment.

We found that patients with tumors with a high density of CD3+ T lymphocytes in the IT and ST compartments, a low CD8/CD3 ratio in the IT and ST compartments, a low density of CD163+ macrophages, and a high CD3/CD163 ratio had greater survival than the remaining patients.

H. pylori infection detected by 2 methodologies was consistently associated with low TIL levels in the IT compartment. In addition, a low TIL level in the IB compartment was associated with H. pylori detected by qPCR, as well as with strains without strong virulence factors (CagA- and VacA-). Furthermore, the high density of CD8+ T lymphocytes in the IT and ST compartments was also associated with the absence of H. pylori (determined by qPCR). Different studies find that the intestinal microbiota modulates the activity of the immune system against cancer and even the activity of checkpoint inhibitors[27,28]. However, to our knowledge, this is the first time that a strong association between H. pylori and immune activity against cancer in GC has been described. Our results need further validation and suggest that H. pylori status evaluated by qPCR should be analyzed in clinical trials evaluating checkpoint inhibitors in GC.

Different series have evaluated the association between MSI and TIL, and Angell et al[29] found that 18.9% of GC cases were MSI- high and were associated with a high density of CD3+ and CD8+ T lymphocytes, as well as a better OS in a series including 380 cases of GC[30-33]. We found that the association between high levels of TIL and dMMR depends on the compartment evaluated, since the association was found in the ST and IB compartments but not in the IT compartment. Similarly, high densities of CD3+ and CD8+ T lymphocytes were associated with dMMR in the ST compartment but not in the IT compartment.

We found that a low density of CD8+ T lymphocytes in the IT compartment was associated with HER2 positivity. Similarly, Lv et al[34] reported a HER2 positive rate of 14% and an inverse relationship with the density of CD8+ T lymphocytes in a series of 120 patients with GC. However, other studies have described different findings[29].

The TIL levels were highest in the IB and ST compartments and were associated with less aggressive clinicopathological features (grade 1 and intestinal subtype for IB and ST, and negative lymph nodes for IB). The association between increased survival and a strong lymphocyte infiltrate has been described by different studies including up to 400 GC cases; however, these studies used different and non-standard methodologies (reporting rates of strong lymphocyte infiltration of 14% to 47%)[35-37].

On the other hand, aggressive features such as grade 3 were associated with high TIL levels in the IT compartment but low TIL levels in the ST compartment. Similarly, grade 3 was associated with high density of CD3 in the IT compartment but not in the ST compartment. This different activity of TILs depending on their spatial location in relation to malignant cells could be explained by a higher percentage of anergic immune cells in the IT compartment[38].

We found that a high density of CD3+ T lymphocytes in the IT and ST compartments, as well as tumors with a low CD8/CD3 ratio in the IT and ST compartments, were associated with increased survival in univariate and multivariate analyses. In addition, as densities were calculated in small tumor cores (tissue microarray-stained samples), we expect that the prognostic value of the density of CD3+ T lymphocytes and the CD8/CD3 ratio would be maintained when evaluated in samples obtained from gastroscopies or conservative surgery. Therefore, we recommend that these densities be evaluated in prospective GC trials.

The poor prognosis associated with the CD8/CD3 ratio has also been described by other groups and could be related to an anergic status of tumor-infiltrating CD8+ T lymphocytes[16,39]. Recent studies suggest that 30% to 38% of cases with a high CD8 T- cell density also have high levels of positive PD-L1[40], and Wang et al[41] found that the presence of both stains was associated with a shorter survival in a series of 147 GC cases.

In addition, our finding of a poor prognosis associated with high levels of CD163+ M2 macrophages has also been described by other groups and confirms the protumoral activity of these immune cells. The high ratio of CD3/CD163 was also associated with a favorable prognosis even in the smaller population size in which it was tested. Nonetheless, further studies on the ratio of immune cells in GC are necessary to confirm these findings[42,43].

Finally, this is the largest South American series evaluating molecular markers in GC, reporting dMMR and HER2 overexpression rates of 29% and 4.7% in early GC, respectively. The prevalence of both biomarkers is similar to what has previously been described in Caucasian series (5%-33% for dMMR and less than 18% for HER2)[10,44-46].

CONCLUSION

The levels of TILs are significantly related to dMMR and a H. pylori-negative status. However, the association of TIL levels with tumor features depends on the tumor compartment evaluated. Low CD8/CD3 and high CD163/CD3 values were strongly associated with a lower rate of recurrence and longer survival.

ACKNOWLEDGMENTS

The authors thank the Universidad Cientifica del Sur, Grupo de Estudios Clinicos Oncologicos del Peru (GECO Peru) and the Instituto Nacional de Enfermedades Neoplasicas for the administrative support provided to carry out this research and language revision by a native English reviewer.

Footnotes

Provenance and peer review: Unsolicited article; Externally peer reviewed.

Peer-review model: Single blind

Specialty type: Oncology

Country/Territory of origin: Peru

Peer-review report’s classification

Scientific Quality: Grade B

Novelty: Grade B

Creativity or Innovation: Grade B

Scientific Significance: Grade B

P-Reviewer: Zhang JW, China S-Editor: Lin C L-Editor: A P-Editor: Cai YX

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