Tumor‑infiltrating lymphocytes in tissue material combined with systemic lymphocyte inflammation in patients with colorectal cancer

  • Authors:
    • Katarzyna Jakubowska
    • Mariusz Koda
    • Małgorzata Grudzińska
    • Karolina Lomperta
    • Waldemar Famulski
  • View Affiliations

  • Published online on: March 12, 2021     https://doi.org/10.3892/mco.2021.2259
  • Article Number: 97
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Abstract

The efficacy of cancer immunotherapy has been actively explored in the treatment of various malignant neoplasms of the gastrointestinal tract. In light of recent reports, the present study aimed to investigate the combination of the absolute lymphocyte count (ALC), percentage of tumor‑infiltrating lymphocyte (TILs) and tumor progression status in patients with colorectal cancer (CRC) who underwent surgery. The medical records of 160 patients diagnosed with CRC were retrospectively reviewed. TILs were determined as a percentage of mononuclear inflammatory cells in the total intratumoral or stromal area as determined in five high power fields (magnification, x200‑400), at the invasive front and in the centre of the tumour. Blood samples were obtained within 3 days prior to and 7 days following the surgical treatment. The assessment of the TIL percentage was performed in the tissue at the invasive front and in the centre of the primary tumour mass in combination with the determination of ALC in whole blood samples. The samples were obtained prior to and after surgery from patients with CRC, and the tumour progression status was also assessed (TILs/ALC/tumour progression status). A significant association was observed between the percentage of TILs in the main mass of tumour and the tumour size (P=0.031), the pT stage (P=0.049) and the incidence of necrosis (P=0.037) following surgery. The histological type was associated with the evaluated combined parameters prior to surgery (P=0.046). Lymph node pouch invasion was associated with TILs at the invasive front of tumour and with ALC prior to and after surgery (P=0.006 and P=0.037). Furthermore, the data indicated that the percentage of TILs located on the invasive front and centre of the tumour, and the ALC prior to and following surgery correlated with the treatment status (P=0.032, P=0.018, P≤0.001 and P≤0.001). A significant association was noted between eight features and evaluated combined parameters following surgery. These included the tumour size (P=0.021), TNM stage (P<0.001), tumour deposits (P=0.001), incidence of necrosis (P=0.042) and lymph node metastasis (P<0.001). Furthermore, the degree of invasion of venous (P<0.001), lymphatic (P<0.001) and perineural (P<0.001) sites was also significantly associated with TILs, ALC obtained after surgical treatment and tumor progression status. The data demonstrated that local and systemic chronic inflammation was associated with tumour progression in patients with CRC.

Introduction

Cancer immunotherapy has been actively explored in the treatment of various malignant neoplasms of the gastrointestinal tract (1,2). Recently, nanotechnology-mediated delivery approaches have attracted considerable attention in colorectal cancer (CRC) immunotherapy. Nanoparticle-based immunotherapy exhibits higher specificity and efficacy in comparison with conventional immunotherapy (3). Those immune-related transmitters can locally deliver immune components to the antigen presenting cells with high efficiency and modulate the tumor immune microenvironment. In addition, they act as an adjuvant that can boost immune reactions to antigens (4). CRC is a heterogeneous group of malignant lesions with different types of immune responses (5). Previous studies have focused on the morphological assessment and molecular changes that occur during CRC, which includes mutational status and chromosomal instability, somatic copy number variation, immune infiltration and metabolic regulation. Based on this evidence CRC can be divided into five groups of tumors as follows: Microsatellite instability-associated, immune-associated, canonical, metabolic and mesenchymal (6). The proposed division of cancerous tumors is closely associated with tumor stage and survival time. Recent studies have shown that patients with a high status of microsatellite instability respond better to immunotherapy (7). Therefore, systemic immune response is associated with infiltration of immune cells in CRC tumors, which appear to be separated from the tumor microenvironment (8). The immune cells infiltrating CRC tumors are macrophages, dendritic cells, neutrophils and lymphocytes. Immunotherapy is focused on the ability of lymphocytes to infiltrate the cancerous tissue. It has been shown that lymphocytes infiltrates exhibit considerable variation with regard to their biological characteristics in the different parts of the tumor (9). Laghi et al (9) demonstrated that the infiltrate of T cells in the invasive margin of CRC tumors could improve disease prognosis compared with cells examined at the center of the tumor. In addition, Bindea et al (10) indicated that the amount of T cells present in the tumor was decreased over time, while the number of B cells and innate immune cells was increased. Moreover, local immune response in the tumor tissue was closely associated with systemic exposure to circulating cancer cells in the bloodstream. Therefore, the analysis of hematological parameters appears to be an important indicator of immune response to cancer. Currently, several studies using hematological factors, such as absolute lymphocyte count (ALC), absolute neutrophil count, neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) have been conducted (11-13). Tanio et al (11) indicated that the combined absolute number of lymphocytes, monocytes and neutrophils in the preoperative whole blood can be a useful prognostic factor in CRC patients (11). Kozak et al (12) proposed that the elevated NLR independently predicts worse overall survival in patients with CRC. In addition, Yang et al (13) demonstrated that an increased PLR value was associated with poor overall survival. In light of these reports, the present study aimed to investigate the combination of the ALC, tumor-infiltrating lymphocyte (TIL) percentage and tumor progression status in patients with CRC who underwent surgery.

Patients and methods

Patients and tissue specimens

A total of 160 patients who were diagnosed with CRC were retrospectively reviewed (96 male, 64 female; median age, 72 years). In the present study, the paraffin-embedded tissues were obtained from the Comprehensive Cancer Centre between April 2014 and December 2016. The patients who were diagnosed with CRC underwent surgery in the Division of Surgical Oncology. All of the tissue samples were processed by an identical dissection protocol. The tissues obtained from surgery were fixed in 4% buffered formalin and subsequently small sections of tissues were embedded in paraffin. The sections (4 µm-thick) were cut from paraffin blocks and stained with haematoxylin and eosin (H&E). The slides were deparaffinised in an oven at 60˚C for 5 min. Subsequently, the slides were rehydrated in xylene (three washes) and graded ethanol (100, 95, 85 and 75%). Histological assessment was performed by two pathologists, which were blinded to the study protocol. The following parameters were recorded from each patient: Type of tumour growth, Tumour-Node-Metastasis (TNM) stage, tumour size, histological type, percentage of mucinous components and grade of malignancy. The clinical stage of CRC was evaluated according to the TNM classification (14). Venous, perineural and lymphatic invasion (the number of resected and invaded lymph nodes, the presence of micro- and macro-metastases, invasion of the pouch lymph node, presence of distant metastases and the size of metastases) were also assessed. The deposits of cancer (their number and size) were also included (15).

The patients enrolled in the present study presented similar symptoms. The most common symptoms were abdominal pain, vomiting, rectal bleeding, constipation or diarrhoea. The medical history demonstrated that some patients suffered from hypertension, diabetes (type II), coronary heart disease or osteoarthritis. However, none of the patients had received anti-inflammatory or immunosuppressive therapy. All of the patients underwent routine diagnostic tests prior to surgery (blood tests, electrocardiography and spirometry). The diagnostic examinations, such as a chest computerized tomography and chest X-ray were utilized to identify CRC distal metastasis. The patients with neoplasm in the rectum received preoperative therapy (n=53): Radiotherapy (n=39), chemotherapy (n=7) and radio-chemotherapy (n=7). The radiation dose used was 25 Gy, in fractions of 5 Gy during one week in the pelvic area. According to the Response Evaluation Criteria in Tumours (16), the response to preoperative therapy was evaluated. A total of 26 patients were recruited with a stable disease and 27 patients exhibited partial response.

The inclusion criteria were as follows: i) Pathologically confirmed CRC; ii) treatment with radical resection; and iii) lack of anti-inflammatory therapy. The exclusion criteria were the following: i) Incomplete clinicopathological and follow-up data; and ii) presence of haematological disorders, such as anaemia.

The present study was performed in conformity with the Declaration of Helsinki for Human Experimentation and the protocol was approved by the Bioethics Committee of the Medical University of Bialystok (no. R-I-002/353/2016).

Assessment of the percentage of TILs in CRC tissues

The analysis of TILs was described in our previous study (17). Briefly, tissue material obtained from routine histopathological diagnosis was stained with H&E and used to assess the TIL populations located in the tumor stroma at the invasive front and centre of the tumour by light microscopy (magnification, x200-400; Leica DM6 B; Leica Microsystems, Inc.). The analysis was evaluated by two independent pathologists who were blinded to the clinical information of the study. TILs in the stroma were identified according to recommendations by the International TILs Working Group, 2014(18). They were determined as a percentage of mononuclear inflammatory cells in the total intratumoral or stromal area as counted in 5 high power fields (HPF; magnification, x200-400), at the invasive front and in the centre of the tumour, with the exception of tumour areas with crush artefacts, necrosis or regressive hyalinization. The invasive front of the tumour was defined as the percentage of most progressed cancer cells on the advanced edge of the tumour. For statistical analysis, the percentage of stromal TILs was scored as follows: 1, weak (0-10% stromal TILs); 2, moderate (20-40% stromal TILs); and 3, strong (50-90% stromal TILs). The study population was divided into two groups based on the stromal TIL score as follows: 1, score 1; and 2, score 2 or 3. Representative images of TILs are shown in Fig. 1.

Blood sample examination

Blood samples were obtained within 3 days prior to and 7 days following surgical treatment. Venous blood samples were also obtained from 42 healthy control subjects (female-21, male-21; mean age 45 years old; min-max 25-65 years old). The differential white blood cell count was counted using an XN-1000 automated haematology analyzer (Sysmex America, Inc.). Normal total lymphocyte count ranges were between 0.90 and 4.00x10³ cells/µl (mean 2.25x10³ cells/µl). The total lymphocyte count in CRC patients ranged from 0.47 to 4.06x10³ cells/µl (mean 1.05x10³ cells/µl).

Combination of TIL percentage and ALC

The percentage of TILs was examined in the CRC tissue at the invasive front and centre of the primary tumour mass in combination with the ALC and in whole blood samples obtained prior to and following surgery from patients with CRC. The classification of the immune response system included: i) TILs at the invasive front and ALC prior to surgery; ii) TILs in the centre of the tumour mass and ALC prior to surgery; iii) TILs at the invasive front and ALC following surgery, iv) TILs in the centre of the tumour mass and ALC following surgery. All groups included 4 subgroups: i) High TILs (moderate or strong stromal TILs) and high ALC (≥4.0x10³ cells/µl); ii) high TILs (moderate or strong stromal TILs) and low ALC (≤0.9x10³ cells/µl); iii) low TILs (weak stromal TILs) and high ALC (≥4.0x10³ cells/µl); iv) low TILs (weak stromal TILs) and low ALC (≤0.9x10³ cells/µl).

In the present study, the combination of TIL percentage and ALC was also examined with regard to the tumor progression status (TILs/ALC/tumour progression status). The tumour progression status was determined on the basis of 3 parameters as follows: i) Lymphatic vessel invasion; ii) lymph node involvement and iii) distant metastasis. Based on the tumour progression status the study population was divided into 3 groups as follows: i) patients with local disease; ii) patients with lymphatic vessel invasion and positive lymph node status without distant metastasis; iii) patients that exhibited cancer cell invasion to all the aforementioned structures. The local and systemic response of lymphocytes prior to and following surgical treatment was investigated. Taken together, the patients were divided into 4 groups as follows: i) Negative status of tumour progression with high percentage of TILs at the invasive front and in the centre of the primary tumour mass and high ALC, ii) negative status of tumour progression with high percentage of TILs as the invasive front and in the centre of the primary tumour mass and low ALC, iii) positive status of tumour progression (group 2 or 3) with high percentage of TILs at the invasive front and in the centre of the primary tumour mass and high ALC and iv) positive status of tumour progression (group 2 or 3) with high percentage of TILs at the invasive front and in the centre of the primary tumour mass and high ALC.

Statistical analysis

All calculations were performed using the statistical software STATISTICA 13.0 (StatSoft, Inc.). The enumeration data of examined parameters were analysed using the χ2 test. The comparisons among multiple groups (examined parameters and clinicopathological features) were analysed using one-way ANOVA. Fisher's Least Significant Difference post hoc test was used to analyze multiple comparisons of TILs, ALC, progression status and histopathological factors following the ANOVA test (data not shown). Tukey's test was used to compare more than three groups. P<0.05 was considered to indicate a statistically significant difference.

Results

Patient characteristics and distribution of the combination index including TIL percentage and ALC in CRC patients

The median age was 67.5 years (range, 32 to 88 years) and 60% of the patients were male. The rectum was the most common tumour location (51.25%). The pathological staging was the following: Stage I in 1.88% of patients, stage II in 38.75% of patients, stage III in 56.88% of patients and stage IV in 2.5% of patients. The patients developed adenocarcinoma (81.25%) more frequently than mucinous CRC (18.75%). The distribution of TILs was evaluated based on several factors, such as invasion of cancer cells, distant metastasis or status of tumour progression. The data indicated that the distribution of the combination index including TIL percentage and ALC differed significantly among the groups (Table I). However, statistical significance was not obtained for the distribution of the TILs/ALC/progression tumour status (Table II).

Table I

Distribution of the combined parameters of TILs and ALC.

Table I

Distribution of the combined parameters of TILs and ALC.

 Groups 
ParameterHigh TILs/high ALC, nHigh TILs/low ALC, nLow TILs/high ALC, nLow TILs/low ALC, nP-value
TILs at the invasive front and ALC before surgery3911629<0.001
TILs at the invasive front and ALC after surgery757241 
TILs in the main mass of primary tumor and ALC before surgery3912127<0.001
TILs in the main mass of primary tumor and ALC after surgery746934 

[i] TILs, tumor-infiltrating lymphocytes; ALC, absolute lymphocyte count.

Table II

Distribution of the TILs/ALC/tumor progression status.

Table II

Distribution of the TILs/ALC/tumor progression status.

 Groups 
Parameter1, n2, n3, n4, nP-value
TILs at the invasive front, ALC and tumor progression status124828720.164
TILs in the main mass of the primary tumor, ALC and tumor progression status29304754 

[i] TILs, tumor-infiltrating lymphocytes; ALC, absolute lymphocyte count.

Correlation of the combination index including TIL percentage and ALC with the clinicopathological parameters of CRC patients

The percentage of TILs and the ALC prior to and following surgery were significantly associated with the tumour growth in the main mass (Tables III and IV, P=0.001, P=0.007). A significant correlation was observed in the percentage of TILs with the tumour size (P=0.031), pT stage (P=0.049) and necrosis percentage (P=0.037) following surgery. The histological type correlated with the combination index prior to surgery (P=0.046). Lymph node pouch invasion was associated with the percentage of TILs at the invasive front of the tumour and with ALC prior to and following surgery (P=0.006, P=0.037). Moreover, the percentage of TILs exanimated at the invasive front and centre of the tumour, and the ALC prior to and following surgery correlated with the treatment status (P=0.032, P=0.018, P≤0.001, P≤0.001). The patients with neoadjuvant therapy (status treatment group 1 and 2) exhibited better local and systemic chronic inflammatory response. Furthermore, no significant difference was observed in the remaining evaluated parameters (Tables III and IV).

Table III

Associations between combined parameters of TILs and absolute lymphocyte count obtained before surgery, and clinicopathological features of patients with CRC (n=160).

Table III

Associations between combined parameters of TILs and absolute lymphocyte count obtained before surgery, and clinicopathological features of patients with CRC (n=160).

  TILs and absolute lymphocyte count before surgery
 Invasive frontMain mass
ParameterNo.1, n2, n3, n4, nP-value1, n2, n3, n4, nP-value
Age, years           
     <6040530050.420532030.420
     ≥601202969220 2970219 
Sex           
     Female6412410110.69612410110.696
     Male962258214 2261211 
Localization           
     Right-side20811100.407412130.561
     Transverse144406 1409 
     Left-side151509 15010 
     Sigmoid2911107 88013 
     Rectum821754110 1654012 
Tumor growth           
     Expanding13328822210.10928822210.001
     Infiltrate2761703 62001 
Tumor size, cm           
     <2.527615150.412617130.181
     2.5-5.01062663017 2564116 
     >5.02722113 32103 
Histological type           
     Muc30425010.322822000.046
     Adca1303074224 3079219 
Mucinous component, %           
     10-3015010050.392010050.148
     31-501541100 41100 
TNM stage           
     1+27310491130.5181153090.417
     3+4872450112 2349213 
Grade of malignancies           
     214832932210.81232962180.482
     3122604 2604 
pT stage           
     1+26513391120.15913411100.154
     3+4952160113 2161112 
Venous invasion           
     Absent11322701200.64223730170.083
     Present46122914 112924 
Lymphatic invasion           
     Absent12124751210.95224780190.961
     Present38102413 102422 
Perineural invasion           
     Absent14330892220.41530922190.478
     Present1741003 41003 
Lymph node metastasis           
     Absent811660140.350166212 
     Present791839121 18401200.405
Lymph node pouch invasion           
     Absent411792130.0061051313 
     Present39173019 1426170.775
Distant metastasis           
     Absent14328912220.91629951180.873
     Present176803 5714 
Tumor deposits           
     Absent13324842230.56525871200.474
     Present27101403 91412 
Tumor budding           
     Absent9420571160.43620591140.945
     Present66144219 144318 
Necrosis           
     Absent456290100.632531180.538
     Focal61134107 133909 
     Moderate3692025 112410 
     Extensive186903 5805 
Fibrosis           
     Absent1165000.60364100.917
     Focal72144819 1349010 
     Moderate4372619 82816 
     Extensive3472007 72007 
Treatment statusa           
     1361411560.0321420110.018
     22251412 111100 
     3713622121 75915 
     431412213 72103 

[i] a1, RCHT-resection-no treatment; 2, RCHT-resection-RT/RCHT; 3, no treatment-resection-no treatment; 4, no treatment-resection-RT/RCHT. Adca, adenocarcinoma; Muc, mucinous; RCHT, radiochemotherapy; RT, radiotheraphy.

Table IV

Correlations between combined parameters of tumor-infiltrating lymphocytes and absolute lymphocyte count obtained after surgery, and clinicopathological features of patients with colorectal cancer (n=160).

Table IV

Correlations between combined parameters of tumor-infiltrating lymphocytes and absolute lymphocyte count obtained after surgery, and clinicopathological features of patients with colorectal cancer (n=160).

  TILs and absolute lymphocyte count
  After surgery
 Invasive frontMain mass
ParameterNo.1, n2, n3, n4, nP-value1, n2, n3, n4, nP-value
Age, years           
     <60402212060.2452113150.545
     ≥601204747125 4744227 
Sex           
     Female6430200140.98431200130.719
     Male963939117 3737319 
Localization           
     Right-side2087050.320106130.438
     Transverse142219 22010 
     Left-side153309 32010 
     Sigmoid29127010 27020 
     Rectum823730020 3428020 
Tumor growth           
     Expanding13354511270.48953493280.007
     Infiltrate2715804 15804 
Tumor size, cm           
     <2.527912060.426814140.031
     2.5-5.01064838019 4835221 
     >5.02712916 12807 
Histological type           
     Muc301612110.079167070.056
     Adca1305347030 5250325 
Mucinous component, %           
     10-301555140.14854060.081
     31-501511202 11302 
TNM stage           
     1+27333240160.91333271120.651
     3+487363513 3530220 
Grade of malignancies           
     214866541270.61865523280.806
     3123504 3504 
pT stage           
     1+26528220150.12427242120.049
     3+4954137116 4133120 
Venous invasion           
     Absent11348400250.86148403220.300
     Present46211915 201709 
Lymphatic invasion           
     Absent12151440260.93551431260.879
     Present38181514 171425 
Perineural invasion           
     Absent14361531280.80360523280.903
     Present178603 8504 
Lymph node metastasis           
     Absent814231080.7884132260.623
     Present792728123 2725126 
Lymph node pouch invasion           
     Absent4112101180.0372315030.888
     Present39523010 127218 
Distant metastasis           
     Absent14363521270.52663512270.539
     Present176703 5615 
Tumor deposits           
     Absent13352511290.95352502290546
     Present2716803 15714 
Tumor budding           
     Absent9440331200.23039342190.090
     Present662926011 2923113 
Necrosis           
     Absent4517151120.20116161120.037
     Focal61262708 2524111 
     Moderate36151407 171504 
     Extensive1811304 10215 
Fibrosis           
     Absent1174000.68773100.455
     Focal723227112 3224016 
     Moderate431516012 161728 
     Extensive34151207 1312019 
Treatment statusa           
     13627306<0.00126325<0.001
     22217500 17500 
     3711941111 1937114 
     431121900 121900 

[i] a1, RCHT-resection-no treatment; 2, RCHT-resection-RT/RCHT; 3, no treatment-resection-no treatment; 4, no treatment-resection-RT/RCHT. Adca, adenocarcinoma; Muc, mucinous; RCHT, radiochemotherapy; RT, radiotheraphy.

Subsequent correlation analysis was performed between different clinical parameters. Specifically, the percentage of TILs, the ALC, the tumour progression status and the clinicopathological features were compared (Table V). The histopathological type, percentage of mucinous component and lymph node pouch invasion correlated significantly with the evaluated combined parameters prior to and following surgery. The growth of the tumour was associated with the percentage of TILs, the ALC and the tumour progression status prior to surgery (P=0.037). A significant correlation was observed between eight features with evaluated combined parameters following surgery. These were the tumour size (P=0.021), TNM stage (P<0.001), tumour deposits (P=0.001), necrosis (P=0.042) and lymph node metastasis (P<0.001). Moreover, the percentage of invasion of venous (P<0.001), lymphatic (P<0.001) and perineural (P<0.001) sites exhibited a significant correlation with the combination index including the percentage of TILs and the ALC. Moreover, no additional correlations were noted between the percentage of TILs and the ALC, the tumour progression status and the remaining clinicopathological parameters. Furthermore, multiparametric analysis was associated with the treatment status for patients who had received preoperative treatment. Moreover, TILs/ALC/tumour progression status and recurrence/prognosis were not statistically significant (data not shown).

Table V

Associations between combined parameters of tumor-infiltrating lymphocytes, absolute lymphocyte count and tumor progression status, and clinicopathological features of patients with colorectal cancer (n=160).

Table V

Associations between combined parameters of tumor-infiltrating lymphocytes, absolute lymphocyte count and tumor progression status, and clinicopathological features of patients with colorectal cancer (n=160).

  TILs, absolute lymphocyte count and tumor progression status
 Before surgeryAfter surgery
ParameterNo.1, n2, n3, n4, nP-value1, n2, n3, n4, nP-value
Age, years           
     <60402123210.1129613100.688
     ≥6012011342237 25212832 
Sex           
     Female6415178250.35315817160.966
     Male963291733 19192426 
Localization           
     Right-side2036580.28755750.864
     Transverse141144 51453 
     Left-side151913 2422 
     Sigmoid290314 0146 
     Rectum827261130 2032022 
Tumor growth           
     Expanding133103621490.037252333370.605
     Infiltrate2731049 9485 
Tumor size, cm           
     <2.527534120.599446100.021
     2.5-5.01067351934 23202924 
     >5.02719212 7368 
Histological type           
     Mucinous301113140.00184980.002
     Adenocarcinoma13012352244 26233234 
Mucinous component, %           
     10-3015126040.00833220.013
     31-5015129310 5176 
TNM stage           
     1424155140.085138108<0.001
     231211114 7579 
     369517426 13121820 
     41823154 1265 
Grade of malignancies           
     2148134423540.961332639380.659
     3120224 1124 
pT stage           
     1302010.77002100.096
     2626161022 1591615 
     3916271534 19142426 
     441101 0201 
Venous invasion           
     Absent113103514390.51824212726<0.001
     Present463111119 1061016 
Lymphatic invasion           
     Absent121113715430.39727212830<0.001
     Present38291015 761312 
Perineural invasion           
     Absent143124122530.661312436390.001
     Present171535 3353 
Lymph node metastasis           
     Absent8183110340.19623172321<0.001
     Present795461524 11101821 
Lymph node pouch invasion           
     Absent4110331140<0.001251925260.006
     Present393131418 981616 
Distant metastasis           
     Absent143114120560.851341935410.218
     Present172552 0561 
Tumor deposits           
     Absent13393918510.654262332370.001
     Present274677 7494 
Tumor budding           
     Absent9482415350.459151929210.941
     Present665221023 1981221 
Necrosis           
     Absent453135160.40111710110.042
     Focal61718624 12131416 
     Moderate361111013 661112 
     Extensive182445 5163 
Fibrosis           
     Absent11224450.36521530.294
     Focal72511927 19101720 
     Moderate43511617 691112 
     Extensive343069 7787 
Treatment statusa           
     1361261620.3962125170.007
     22251250 14224 
     371452051 25201115 
     43123224 412312 

[i] a1, RCHT-resection-no treatment; 2, RCHT-resection-RT/RCHT; 3, no treatment-resection-no treatment; 4, no treatment-resection-RT/RCHT. Adca, adenocarcinoma; Muc, mucinous; RCHT, radiochemotherapy; RT, radiotheraphy.

Discussion

Multiple studies have emphasized on the importance of the host immunity during the development of CRC. The local inflammation has been previously measured in the tumour mass of CRC patients following surgical resection (19,20). More specifically, these studies were based on the measurement of CD3+ T cells, CD8+ T cells, and/or CD45RO+ memory T cells that were localized in the invasive front and in the centre of the primary tumour mass. The main finding was that the immune score could be used as a superior predictor of CRC recurrence in everyday practice (19,20). Furthermore, in response to the predictive value of the intensive local inflammation caused by high density TILs, the systemic inflammation is also frequently investigated. It has been shown that systemic inflammatory response is associated with a pro-tumour response and poor prognosis for CRC patients (21).

Recent data have suggested that local and systemic inflammatory response in CRC should be assessed in combination. This type of assessment may affect patient prognosis. Park et al (22) demonstrated a significant role of the density of T lymphocytes within the cancer cell areas. Moreover, an increased level of modified Glasgow Prognostic Score (mGPS) was noted in patients with mismatch repair-deficient (dMMR) CRC (22). Another study indicated that the combination of the number of intratumoral CD8-positive T cells with a high level of C-reactive protein (CRP) may possess predictive value for recurrence in CRC patients receiving chemotherapy treatment (23). The present study indicated that local and systemic chronic inflammation was simultaneously observed in 24% of cases. The majority of the cases (~73%) exhibited a predominantly local chronic response (high percentage of TILs and low ALC). Moreover, Turner et al (24) demonstrated the presence of the local chronic response consisting of lymphocytes, plasma cells, macrophages within tumour areas (24). In addition, systemic chronic inflammation was assessed by the expression of neutrophil-to-lymphocyte ratio (NLR) in preoperative blood samples in only 10% of stage II colon cancer cases (24). The authors of that study further indicated that patients with predominantly local response exhibited the best outcomes, while those with systemic inflammatory response exhibited reduced survival and poor prognosis (24).

The present study demonstrated the association between the combination index of TIL percentage and ALC with clinicopathological features. Patients with low local and systemic inflammatory response exhibited higher tumour growth and larger tumour mass associated with necrosis. Moreover, the patients from group 4 who presented with lymph node metastasis indicated higher percentage of cancer cell invasion beyond the lymph node pouch to the local fat tissue. Wu et al (25) demonstrated that the percentage of TILs and the ALC correlated with local recurrence. Moreover, Xiao et al (26) postulated that the density of CD8+ lymphocytes in the biopsy materials could be combined with the circulating lymphocyte ratio in order to predict complete response following neoadjuvant chemoradiotherapy in rectal cancer. Similar observations were found in studies conducted on breast cancer (27,28). The present study further indicated that patients with neoadjuvant therapy (status treatment group 1 and 2) exhibited optimal local and systemic chronic inflammatory response. Unfortunately, the present study contains certain limitations. Initially, the sample size that corresponded to the patients who responded to preoperative treatment was relatively small. In addition, our analysis did not provide complete data regarding the type of chemotherapeutic drugs used and the number of chemotherapy cycles. Despite these limitations, the present study indicated that the measured parameters were associated with tumour progression and neoadjuvant therapy.

The previous study conducted by our group demonstrated that the percentage of stromal TILs present at the invasive tumour front correlated with the histopathological features of tumour progression (17). In the present study, the data demonstrated that approximately half of all patients exhibited a locally advanced tumour with lymph node involvement, whereas the incidence of distant metastases was less common. Therefore, the study group was divided into 3 subgroups including patients without invasion of cancer cells, patients with invasion of cancer cells to lymphatic vessels and local lymph nodes, patients with lack of distant metastasis and patients with invasion of cancer cells to all aforementioned structures. In addition, the current study attempted to select CRC patients whose TILs and lymphocyte count may have an optimal prognostic value. In the current study, the combination index of different parameters (TILs/lymphocyte count/status progression) exhibited the highest diagnostic value in patients of group 4 (high TILS/high lymphocyte count/presence of local or distant metastases). The aforementioned combination of parameters was significantly associated with numerous pathological and clinical variables, such as tumour growth, tumour size, histological type, percentage of mucinous components, TNM stage, venous, lymphatic and perineural invasions, lymph node metastasis, cancer cell invasion beyond the lymph node pouch, tumour deposits and incidence of necrosis. It is important to note that the findings regarding the examined parameters obtained following surgery exhibited the highest diagnostic value.

In light of these observations, the present study demonstrated that the investigation of local and systemic chronic inflammation was associated with tumour progression of CRC patients. Due to this fact, the diagnosis and prognosis of CRC patients should be undertaken on the basis of numerous local and systemic factors.

Acknowledgements

Not applicable.

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Authors' contributions

KJ collected data, performed analysis, wrote the paper, reviewed the literature, acquired the data and contributed to manuscript drafting. MK analysed and interpreted the pathological examination and approved the final version of the article. WF and KL collected and analysed data, and approved the final version of the article. MG wrote the paper, reviewed the literature, acquired the data and contributed to manuscript drafting. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The study was performed in conformity with the Declaration of Helsinki for Human Experimentation. Written informed consent was not required, and informed consent included an opt-out clause approved by the Medical Ethics Committee. The protocol was approved by the Bioethics Committee of the Medical University of Bialystok (NoR-I-002/353/2016).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Jakubowska K, Koda M, Grudzińska M, Lomperta K and Famulski W: Tumor‑infiltrating lymphocytes in tissue material combined with systemic lymphocyte inflammation in patients with colorectal cancer. Mol Clin Oncol 14: 97, 2021
APA
Jakubowska, K., Koda, M., Grudzińska, M., Lomperta, K., & Famulski, W. (2021). Tumor‑infiltrating lymphocytes in tissue material combined with systemic lymphocyte inflammation in patients with colorectal cancer. Molecular and Clinical Oncology, 14, 97. https://doi.org/10.3892/mco.2021.2259
MLA
Jakubowska, K., Koda, M., Grudzińska, M., Lomperta, K., Famulski, W."Tumor‑infiltrating lymphocytes in tissue material combined with systemic lymphocyte inflammation in patients with colorectal cancer". Molecular and Clinical Oncology 14.5 (2021): 97.
Chicago
Jakubowska, K., Koda, M., Grudzińska, M., Lomperta, K., Famulski, W."Tumor‑infiltrating lymphocytes in tissue material combined with systemic lymphocyte inflammation in patients with colorectal cancer". Molecular and Clinical Oncology 14, no. 5 (2021): 97. https://doi.org/10.3892/mco.2021.2259