Abstract
Background:
Colorectal carcinoma (CRC) is the third most common malignancy worldwide. Human endogenous retrovirus H long terminal repeat-associating protein 2 (HHLA2) is a novel immune checkpoint molecule. The association between HHLA2 expression and clinicopathological features and its prognostic significance in CRC patients are still controversial. The aim of this study is to evaluate the prognostic value of immunohistochemical (IHC) expression of HHLA2 and CD8 in CRC.
Material and methods:
This retrospective study included 134 cases diagnosed with primary CRC at the Gastrointestinal Surgery Center (GISC) department, Mansoura Faculty of Medicine, during the period from December 2014 to December 2018. Clinicopathological and survival data were collected. IHC for HHLA2 and CD8 was performed, and they were correlated with clinicopathological parameters and patient prognosis.
Results:
Among 134 CRC cases, high HHLA2 expression was detected in 73 (54.5%). High HHLA2 expression was significantly related to the depth of invasion (P = 0.005*), lymph node metastasis (P = 0.01*), tumor stage )P = 0.002*), and distant recurrence )P = 0.012*). Multivariate analysis spotted HHLA2 high expression as an independent prognostic predictor for OS in CRC (P = 0.03*) and DFS (P = 0.008*). CD8 shows a significant correlation with tumor infiltrating lymphocytes (TILs) (P ≤ 0.001*), absence of metastasis ((P = 0.029*), absence of tumor deposits (P=0.014*). However, CD8 shows no significant association with survival or HHLA2.
Conclusion:
HHLA2 is an independent prognostic factor for the overall survival and disease free survival of CRC patients and can predict poor prognosis in CRC patients.
Key Words: HHLA2, CD8, Colorectal carcinoma, Immunotherapy
Introduction
Colorectal carcinoma is the third most common cancer globally. Despite improvements in CRC prevention, diagnosis, and therapy, the disease still has a poor prognosis, and a large majority of CRC patients experience both local recurrence and distant metastasis (Sung et al., 2021). Immunotherapy, in particular immune checkpoint inhibitors, is seen as a very promising treatment approach. Its objective is to stimulate and enhance the anticancer immune response. The major effectors of antitumor immunity are cytotoxic CD8+ TILs. Immuno-checkpoints, especially the B7/CD28 immuno-checkpoint family, are significant molecules that either stimulate or inhibit the function of T cells. Targeting inhibitory receptors on immune effector cells, immune checkpoint inhibitors (ICIs) reactivate the immunological response (Golshani and Zhang, 2020).
HHLA2 has been considered a newly discovered ligand of the B7 family. It is expressed on some normal tissue, such as the epithelial cells of the stomach, kidney, gall bladder, and placenta, as well as on antigen-presenting cells like macrophages and activated dendritic cells. It is widely expressed in many types of malignant tumors, including pancreas, esophagus, stomach, colon, ovary, bladder, and lung cancers. Also, it is expressed in triple-negative breast cancer, glioma, cholangiocarcinoma, osteosarcoma, renal cell carcinoma (RCC), prostate cancer, and melanoma (Li et al., 2022).
HHLA2 has a dual role in the immune response in various tumors, as it can act as co-stimulatory or co-inhibitory according to the receptors to which it binds. Overall, the studies demonstrate that HHLA2 predominantly functions as a T-cell co-inhibitory molecule with negative effects on TCR-mediated CD4+ and CD8+ T cell proliferation. Also, it suppresses their cytokine production. So HHLA2 is participating in tumor immune escape (Niu et al., 2022).
Some studies demonstrated a significant association between HHLA2 expression and CD8+ T cell infiltration in tumor microenvironments (Zhu and Dong, 2018; Yan et al., 2020). The value of HHLA2 expression in CRC remains indefinite and needs more investigation. Furthermore, the reports on HHLA2 are often conflicting. The current study assesses the IHC expression of HHLA2 and CD8 in CRC and evaluates their correlation with the patient’s prognosis.
Materials and Methods
This is a retrospective study carried out on formalin-fixed, paraffin-embedded (FFPE) tissue blocks for primary CRC obtained from resection specimens of 134 CRC cases. Patients were diagnosed and operated at GISC at our institute during the period from December 2014 to December 2018. Two hundred and fifty cases were diagnosed as CRC (116 cases were excluded, including 26 cases received pre-operative adjuvant therapy, 30 cases with unavailable blocks in the archive, 11 cases with repetitive tissue loss during the antigen retrieval procedure and 49 cases had lost follow up). So the final included cases were 134 cases.
The demographic and clinicopathological data of the enrolled cases were retrospectively retrieved from the electronic medical records of the patients in Clinical Oncology and Nuclear medicine department, Mansoura university hospital and the pathology database of the Surgical Pathology Laboratory at the GISC. In addition, the clinical outcomes of the patients were followed up in terms of overall survival and disease-free survival. The follow-up period started on the date of diagnosis and ended in March 2023. DFS was considered the period from the date of primary radical surgery to the date of the first treatment failure. Overall survival (OS) was measured from the diagnosis date to the end of the follow-up period or death. PFS is the time from the end of first-line treatment until disease recurrence or progression.
The tumors were classified histopathologically basing on the most recent WHO classification of colorectal carcinoma tumors (Nagtegaal et al., 2019). Each tumor was assigned a stage according to the latest American Joint Committee on Cancer (AJCC) TNM staging criteria, 8th edition. As regards TILs, the density of TILs was estimated based on the recommendations of the International TILs Working Group (ITWG). TILs are described as the mean percentage of the invasive tumor area occupied by lymphocytes and plasma cells. So, the tumors comprised in the current study were divided into 3 grades: low TILs (0–10%), intermediate TILs (20–40%), and high TILs (50–90%). Areas with necrosis, hemorrhage, or crush artifacts were ruled out during the TIL assessment (Fuchs et al., 2020).
The tissue microarray blocks (TMA) were designed by utilizing a fully manual-validated approach (Foda, 2013). Three tissue cores were extracted from three different sites from each donor block of resected colorectal carcinoma.
Immunohistochemical Staining
Sections from FFPE tissue blocks were deparaffinized and hydrated by standard approaches. We used HHLA2 antibodies (rabbit polyclonal, 1:100 dilutions, IgG; Abclonal, Inc. catalog number A13262) and anti-CD8 antibodies (mouse monoclonal primary antibody, clone C8/144B, ready to use; catalogue number IR623) according to the manufacturer’s instructions with proper positive and negative controls.
Immunohistochemical evaluation
slides were scored in an independent manner by two pathologists who were blinded to the patients’ data. HHLA2 is expressed in cytoplasmic and membranous reactions. Based on the ratio of positively stained cells and staining intensity, HHLA2 staining was quantified. According to the following metrics, the percentage of positive cells is graded: score 0 (0%), score 1 (1%–5%), score 2 (6%–30%), score 3 (31%–60%), and score 4 (61%–100%). The intensity of HHLA2 staining was recorded as negative (0), mild (1), moderate (2), and strong (3). The final H-score was calculated by the equation: H-score = percentage × intensity. The median score was utilized to detect the cut off value of high or low HHLA2 expression (Klua et al., 2023). The interpretation of CD8 staining reaction was based on the study of Zhu et al 2018 by counting the number of CD8 positive cells in each core of TMA. The cutoff point of high or low expression could be detected on the median of total scores (Zhu and Dong, 2018),
Statistical analysis
SPSS software version 25 (SPSS Inc., PASW statistics for Windows version 25) carried out the data analysis. Chicago: SPSS Inc. Qualitative data were described using numbers and percentages. For non-normally distributed data, the median (the middle number between the lowest and highest values) was used to define the data. For normally distributed data, the mean±SD was used after the Kolmogorov-Smirnov test was used to check for normality. The significance of the obtained results was judged at the (≤0.05) level. Chi-Square, Fischer exact test, and Monte Carlo tests were used to compare qualitative data between groups as appropriate. Mann-Whitney U and Kruskal-Wallis tests were utilized to compare between two studied groups and more than two groups, respectively, for non-normally distributed data. • Kaplan-Meier test: utilized to calculate OS and disease-free survival by utilizing log-rank tests to detect the effect of predisposing factors affecting survival. Cox regression was used to assess predictors of survival with the calculation of the hazard ratio.
Results
Patients’ clinicopathological characteristics are described in Table 1. According to the aforementioned criteria for HHLA2 and CD8 IHC evaluation, The H-score of HHLA2 ranged from 0 to 12, with a median of 6. H score ≥ 6 was defined as a high HHLA2 expression, and an H-score <6 indicated a low HHLA2 expression. In 134 CRC tissues, 45.5% (61/134) cases of low HHLA2 expression (Figure 1 A, B) and 54.5% (73/134) cases of high HHLA2 expression (Figure 1 C, D) were found.
Table 1.
Clinicopathologic Characteristics of Studied Patients
| Clinicopathologic characteristics |
The studied group (n=134) Number (%) |
|---|---|
| Age classes | |
| <60 y | 88 (65.7%) |
| ≥60 y | 46 (34.3%) |
| Sex | |
| Male | 75 (56.0%) |
| Female | 59 (44.0%) |
| Site | |
| Right | 49 (36.6%) |
| Left | 85 (63.4%) |
| Size | |
| Mean ± SD | 6.23±2.73 |
| Min-Max | 1.50-19.00 |
| Histological type | |
| Adenocarcinoma NOS | 110 (82%) |
| Mucinous adenocarcinoma | 18 (13.4%) |
| Signet ring carcinoma | 6 (4.5%) |
| Adenocarcinoma grade | |
| Low | 107 (97.27%) |
| High | 3 (2.7) |
| TILs | |
| Low | 50 (37.3%) |
| Moderate | 51 (38.1%) |
| High | 33 (24.6%) |
| Tumor budding | |
| Low | 90 (67.2%) |
| Moderate | 24 (17.9%) |
| High | 20 (14.9%) |
| Tumor deposits | |
| Present | 15 (11.2%) |
| Absent | 119 (88.8%) |
| Lympho-vascular emboli (LVI) | |
| Present | 79 (59.0%) |
| Absent | 55 (41.0%) |
| Perineural invasion (PNI) | |
| Present | 37 (27.6%) |
| Absent | 97 (72.4%) |
| Tumor depth of invasion | |
| T2 | 17 (12.7%) |
| T3 | 102 (76.1%) |
| T4 | 15 (11.2%) |
| Distant metastasis | |
| M 0 | 126 (94.0%) |
| M 1 | 8 (6.0%) |
| AJCC stage | |
| Stage I | 12 (9.0%) |
| Stage II | 63 (47.0%) |
| Stage III | 51 (38.1%) |
| Stage IV | 8 (6.0%) |
| Clinicopathologic characteristics |
The studied group (n=134) Number (%) |
| Local recurrence | |
| Yes | 31 (23.1%) |
| No | 103 (76.9%) |
| Distant recurrence | |
| Yes | 31 (23.1%) |
| No | 103 (76.9%) |
| Patient fate | |
| Survived | 38 (28.4%) |
| Died | 96 (71.6%) |
Figure 1.
Immunohistochemical Staining of HHLA2 in Various Cases of CRC: Low cytoplasmic expression (A, B). High cytoplasmic expression (C, D). Original magnification: 400X)
As regards CD8, the median value of CD8 was 60. Sixty-seven cases (50%) had a high CD8 T-cell count (≥60) (Figure 2 A, B), and 67 cases (50%) were low (<60) (Figure 2 C, D).
Figure 2.
CD8 IHC of CD8 in Different Cases of CRC. High CD8 expression (A, B). Low CD8 expression (C, D). (Original magnification: A, C 400×; B, D 200×).
As demonstrated in Table 2, there was a statistically significant association between higher HHLA2 expression and depth of invasion (P = 0.005*), advanced N stage (P = 0.01*), higher tumor stage P = 0.002*), and distant recurrence P = 0.012*). There were no observed associations between HHLA2 expression and other clinicopathological parameters. CD8 expression showed a statistically significant positive association between CD8 and TILs (P≤0.001*), M stage (P=0.029*), absence of tumor deposits (P=0.014*), and margin status (P=0.016*). There was no association between CD8 and other clinicopathological parameters and HHLA2.
Table 2.
Association between HHLA2, CD8 and Clinicopathologic Parameters
| Clinicopathologic parameters |
Total | HHLA2 | χ2 | CD8 | χ2 | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Low (n=61) | High (n= 73) | (p value) | <60 (n = 67) | ≥60 (n= 67) | (p value) | ||||||||||
| Age classes | |||||||||||||||
| <60 y | 88 | 41 (46.6%) | 47 (53.4%) | χ2 =0.118 | 44 (50%) | 44 (50.0%) | χ2 =0.0 | ||||||||
| ≥60 y | 46 | 20 (43.5%) | 26 (56.5) | p=0.731 | 23 (50 %) | 23 (50 %) | P=1.0 | ||||||||
| Sex | |||||||||||||||
| Male | 75 | 33 (44.0%) | 42 (56.0%) | χ2 =0.159 | 44 (58.7%) | 31 (41.3%) | χ2 =5.12 | ||||||||
| Female | 59 | 28 (47.5%) | 31 (52.5%) | p=0.690 | 23 (39.0%) | 36 (61.0%) | P=0.024* | ||||||||
| Site | |||||||||||||||
| Right | 49 | 23 (46.9%) | 26 (53.1%) | χ2 =0.062 | 29 (59.2%) | 20 (40.8%) | χ2 =2.61 | ||||||||
| Left | 85 | 38 (44.7%) | 47 (55.3%) | p=0.803 | 38 (44.7%) | 47 (55.3%) | P=0.106 | ||||||||
| Size | |||||||||||||||
| ≤6 | 79 | 37 (46.8%) | 42 (53.2%) | χ2 =0.134 | 38 (48.1%) | 41 (51.9%) | χ2 =0.278 | ||||||||
| >6 | 55 | 24 (43.6%) | 31 (56.4%) | P=0.715 | 29 (52.7%) | 26 (47.3%) | P=0.598 | ||||||||
| Histological type | |||||||||||||||
| Adenocarcinoma NOS | 110 | 50 (45.5%) | 60 (54.5%) | MC=0.057 | 50 (45.5%) | 60 (54.5%) | MC | ||||||||
| Mucinous adenocarcinoma | 18 | 8 (44.4%) | 10 (55.6%) | P=0.972 | 11 (61.1%) | 7 (38.9%) | P=0.015* | ||||||||
| Signet ring carcinoma | 6 | 3 (50%) | 3 (50%) | 6 ( 100%) | 0 ( 0%) | ||||||||||
| Adenocarcinoma NOS grade (n= 110) | |||||||||||||||
| Low | 107 | 50 (46.7%) | 57 (53.3%) | FET=2.57 | 49 (45.79%) | 58 (54.2%) | FET | ||||||||
| High | 3 | 0 (0%) | 3 (100%) | P=0.249 | 1 (33.3%) | 2 (66.7%) | P=1.0 | ||||||||
| TILs | |||||||||||||||
| Low | 50 | 19 (38%) | 31 (62%) | χ2 =2.27 | 35 (70.0%) | 15 (30.0%) | χ2 =21.38 | ||||||||
| Moderate | 51 | 27 (52.9%) | 24 (47.1%) | P=0.321 | 26 (51.0%) | 25 (49.0%) | P≤0.001* | ||||||||
| High | 33 | 15 (45.5%) | 18 (54.5%) | 6 (18.2%) | 27 (81.8%) | ||||||||||
| Tumor budding | |||||||||||||||
| Low | 90 | 44 (48.9%) | 46 (51.1%) | χ2 =1.28 | 40 (44.4%) | 50 (55.6%) | χ2 =3.58 | ||||||||
| Moderate | 24 | 9 (37.5%) | 15 (62.5%) | P=0.527 | 14 (58.3%) | 10 (41.7%) | P=0.167 | ||||||||
| High | 20 | 8 (40%) | 12 (60%) | 13 (65.0%) | 7 (35.0%) | ||||||||||
| Tumor deposits | |||||||||||||||
| Present | 15 | 6 (40%) | 9 (60%) | χ2 =0.208 | 12 (80.0%) | 3 (20.0%) | χ2 =6.08 | ||||||||
| Absent | 119 | 55(46.2%) | 64 (53.8%) | P=0.649 | 55 (46.2%) | 64 (53.8%) | P=0.014* | ||||||||
| LVI | |||||||||||||||
| Present | 79 | 31(39.2%) | 48 (60.8%) | χ2 =3.06 | 45 (57.0%) | 34 (43.0%) | χ2 =3.73 | ||||||||
| Absent | 55 | 30 (54.5%) | 25 (45.5%) | P=0.08 | 22 (40.0%) | 33 (60.0%) | P=0.053 | ||||||||
| PNI | |||||||||||||||
| Present | 37 | 19 (51.4%) | 18 (48.6%) | χ2 =0.700 | 20 (54.1%) | 17 (45.9%) | χ2 =0.336 | ||||||||
| Absent | 97 | 42 (43.3%) | 55 (56.7%) | P=0.403 | 47 (48.5%) | 50 (51.5%) | P=0.562 | ||||||||
| Tumor depth of invasion | |||||||||||||||
| T2 | 17 | 13 (76.5%) | 4 (23.5%) | χ2MC =10.58 | 10 (58.8%) | 7 (41.2%) | χ2 =0.635 | ||||||||
| T3 | 102 | 45 (44.1%) | 57 (55.9%) | P=0.005* | 50 (49.0%) | 52 (51.0%) | P=0.728 | ||||||||
| T4 | 15 | 3 (20%) | 12 (80%) | 7 (46.7%) | 8 (53.3%) | ||||||||||
| Lymph node metastasis | |||||||||||||||
| N0 | 77 | 43 (55.8%) | 34 (44.2%) | χ2 =9.17 | 33 (42.9%) | 44 (57.1%) | χ2 =5.45 | ||||||||
| N1 | 31 | 12 (38.7%) | 19 (61.3%) | P=0.01* | 16 (51.6%) | 15 (48.4%) | P=0.066 | ||||||||
| N2 | 26 | 6 (23.1%) | 20 (76.9%) | 18 (69.2%) | 8 (30.8%) | ||||||||||
| Distant metastasis | |||||||||||||||
| M 0 | 126 | 60 (47.6%) | 66 (52.4%) | χ2 =3.74 | 60 (52.4%) | 66 (52.4%) | χ2 =4.78 | ||||||||
| M 1 | 8 | 1 (12.5%) | 7 (87.5%) | P=0.053 | 7 (87.5%) | 1 (12.5%) | P=0.029* | ||||||||
| AJCC stage | |||||||||||||||
| Stage I | 12 | 10 (83.3%) | 2 (16.7%) | χ2MC =14.69 | 5 (41.7%) | 7 (58.3%) | MC | ||||||||
| stage II | 63 | 33 (52.4%) | 30 (47.6%) | P=0.002* | 27 (42.9%) | 36 (57.1%) | P=0.086 | ||||||||
| Stage III | 51 | 17 (33.3%) | 34 (66.7%) | 28 (54.9%) | 23 (45.1%) | ||||||||||
| Stage IV | 8 | 1 (12.5%) | 7 (87.5%) | 7 (87.5%) | 1 (12.5%) | ||||||||||
| Local recurrence | |||||||||||||||
| Yes | 31 | 15 (48.4%) | 57 (55.3%) | χ2 =0.133 | 14 (45.2%) | 17 (54.8%) | χ2 =0.378 | ||||||||
| No | 103 | 46 (44.7%) | 16 (51.6%) | P=0.837 | 53 (51.5%) | 50 (48.5%) | P=0.539 | ||||||||
| Distant recurrence | |||||||||||||||
| Yes | 31 | 8 (25.8%) | 23 (74.2%) | χ2 =6.32 | 20 (64.5%) | 11 (35.5%) | χ2 =3.39 | ||||||||
| No | 103 | 53 (51.5%) | 50 (48.5%) | P=0.012* | 47 (45.6%) | 56 (54.4%) | P=0.065 | ||||||||
| Fate of patient | χ2 =0.588 | ||||||||||||||
| Died | 96 | 38 (39.6%) | 58 (60.4%) | χ2 =4.82 | 46 (47.9%) | 50 (52.1%) | P=0.443 | ||||||||
| Survived | 38 | 23 (60.5%) | 15 (39.5%) | P=0.028* | 21 (55.3%) | 17 (44.7%) | |||||||||
| CD8 | |||||||||||||||
| <60 | 67 | 33(49.3%) | 34 (50.7 %) | χ2 =0.752 | |||||||||||
| ≥60 | 67 | 28(41.8%) | 39 (58.2%) | P=0.386 | |||||||||||
χ2, Chi-Square test; FET, Fisher's Exact Test; MC, Monte carlo test P, Probability value; *, statistically significant (P<0.05).
The median OS was 49.32 months (44.05–54.58). It is demonstrated that high HHLA2 expression has low OS in patients with CRC (Figure 3A, P = <0.001*). The median DFS was 45.80 months (40.96–50.65). High HHLA2 expression had a lower DFS (Figure 4A, P = <0.0001*). The median PFS was 44.25 months (39.17–49.33). The high HHLA2 expression had a lower PFS (Figure 5A, P = 0.003*). CD8 shows no significant association with survival (Figures 3B, 4B, and 5B).
Figure 3.
Kaplan-Meir Survival Curves for HHLA2, CD8 with OS of Colorectal Carcinoma Cases. Significantly lower OS in patients with high HHLA2 expression (A). No statistically significant association between CD8 and OS(B)
Figure 4.
Kaplan-Meir Survival Curves for HHLA2, CD8 with DFS of Colorectal Carcinoma Cases. Significantly lower DFS in patients with high HHLA2 expression (A). No statistically significant association between CD8 DFS (B)
Figure 5.
Kaplan-Meir Survival Curves for HHLA2, CD8 with PFS of Colorectal Carcinoma Cases. Significantly lower PFS in patients with high HHLA2 expression (A). No statistically significant association between CD8 and PFS (B)
Univariate and multivariate analyses on OS and DFS were conducted to detect the prognostic value of HHLA2 expression and other clinicopathological variables (Table 3). In the univariate analysis, HHLA2 expression demonstrated a significant correlation to the OS of cases with CRC (p = 0.039*). Meanwhile, histological grade (p = 0.03*), TILS (P = 0.009*), depth of invasion (P = 0.025*), lymph node (LN) metastasis (P = 0.02*), and tumor stage (P = 0.04*) were also found to be associated with OS. HHLA2 expression demonstrated a significant correlation with the DFS of patients with CRC (p=0.005*). Histological grade (P=0.026*) and tumor budding (P = 0.04*) were also associated with DFS.
Table 3.
Univariate and Multivariate Survival Analysis of OS and DFS in CRCs
| Overall survival | Disease free survival | ||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Variables | Univariate analysis | Multivariate analysis | Univariate analysis | Multivariate analysis | |||||||||||||||||
| Median OS | Hazard ratio (95% CI) | P value | Hazard ratio (95% CI) | P value | Median DFS | Hazard ratio (95% CI) | P value | Hazard ratio (95% CI) | P value | ||||||||||||
| Age classes | |||||||||||||||||||||
| <60 y (R) | 38 | 1 | 48 | 1 | |||||||||||||||||
| ≥60 y | 45 | 0.943 (0.615-1.45) | 0.787 | 51 | 1.24 (0.683-2.25) | 0.478 | |||||||||||||||
| Sex | |||||||||||||||||||||
| Male (R) | 43 | 1 | 52 | 1 | |||||||||||||||||
| Female | 38 | 1.095 (0.729-1.64) | 0.662 | 48 | 1.27 (0.587-2.75) | 0.544 | |||||||||||||||
| Site | |||||||||||||||||||||
| Right (R) | 45 | 1 | 40 | 1 | |||||||||||||||||
| Left | 40 | 0.893 (0.582-1.37) | 0.603 | 48 | 0.723 (0.369-1.41) | 0.343 | |||||||||||||||
| Size | |||||||||||||||||||||
| <6 (R) | 37 | 1 | 42 | 1 | |||||||||||||||||
| >6 | 51 | 0.756 (0.497-1.152) | 0.193 | 53 | 0.900 (0.497-1.63) | 0.727 | |||||||||||||||
| Histological type | |||||||||||||||||||||
| Adenocarcinoma NOS | 43 | 1.007 (0.548-1.85) | 0.981 | 51 | 1.29 (0.681-2.47) | 0.429 | |||||||||||||||
| Mucinous adenocarcinoma | 34 | 1.48 (0.599-3.67) | 0.394 | 55 | 2.13 (0.764-5.95) | 0.148 | |||||||||||||||
| Signet ring carcinoma (R) | 27 | 1 | 32 | 1 | |||||||||||||||||
| Adenocarcinoma grade | |||||||||||||||||||||
| Low | 43 | 0.265 (0.078-0.901) | 0.03* | 0.333 (0.095-1.17) | 0.086 | 51 | 0.171 (0.036-0.809) | 0.026* | 0.451 (0.117-1.73) | 0.246 | |||||||||||
| High(R) | 12 | 1 | 1 | 12 | 1 | 1 | |||||||||||||||
| TILs | |||||||||||||||||||||
| Low (R) | 29 | 1 | 1 | 33 | 1 | ||||||||||||||||
| Moderate | 53 | 0.517 (0.315-0.849) | 0.009* | 1.24 (0.703-2.2) | 0.454 | 54 | 0.834 (0.325-2.14) | 0.706 | |||||||||||||
| High | 53 | 0.598 (0.340-1.05) | 0.074 | 0.825 (0.428-1.59) | 0.565 | 54 | 0.654 (0.256-1.67 | 0.374 | |||||||||||||
| Tumor budding | |||||||||||||||||||||
| Low (R) | 48 | 1 | 51 | 1 | 1 | ||||||||||||||||
| Moderate | 34 | 0.742 (0.418-1.318) | 0.309 | 34 | 2.37 (1.04-5.43) | 0.04* | 1.79 (0.911-3.54) | 0.091 | |||||||||||||
| High | 30 | 1.35 (0.705-2.55) | 0.364 | 38 | 0.708 (0.188-2.66) | 0.609 | 0.945 (0.391-2.29 | 0.9 | |||||||||||||
| LVI | |||||||||||||||||||||
| Absent (R) | 54 | 1 | 54 | 1 | |||||||||||||||||
| Present | 34 | 1.04 (0.565-1.91) | 0.903 | 43 | 0.986 (0.496-1.96) | 0.968 | |||||||||||||||
| PNI | |||||||||||||||||||||
| Absent (R) | 48 | 1 | 50 | 1 | |||||||||||||||||
| Present | 34 | 0.946 (0.580-1.54) | 0.823 | 48 | 1.18 (0.576-2.43) | 0.646 | |||||||||||||||
| T (depth of invasion) | |||||||||||||||||||||
| T2 | 59 | 1.93 (0.809-4.60) | 0.078 | 1.71 (0.418-7.02) | 0.457 | 55 | 0.796 (0.134-4.72) | 0.805 | |||||||||||||
| T3 | 39 | 4.43 (1.20-16.35) | 0.025* | 0.895 (0.416-1.93) | 0.778 | 48 | 0.455 (0.161-1.29) | 0.139 | |||||||||||||
| T4(R) | 49 | 1 | 1 | 53 | 1 | ||||||||||||||||
| N (lymph node stage) | |||||||||||||||||||||
| N0 | 54 | 1.815 (0.171-19.23) | 0.799 | 54 | 0.14 (0.08-7.2) | 0.99 | |||||||||||||||
| N1 | 27 | 1.41 (0.460-4.32) | 0.621 | 37 | 4.56 (0.850-24.51) | 0.08 | |||||||||||||||
| N2(R) | 32 | 1 | 1 | ||||||||||||||||||
| M (Metastasis stage) | |||||||||||||||||||||
| M 0 | 45 | 0.427 (0.144-1.27) | 0.125 | 50 | 7.90 (3.1-9.8) | 0.99 | |||||||||||||||
| M 1(R) | 24 | 1 | 50 | 1 | |||||||||||||||||
| AJCC stage | |||||||||||||||||||||
| stage I | 52 | 2.9 (0.28-13.7) | 0.067 | 7.9 (0.25-12.69) | 0.881 | 56 | 0.897 (0.059-13.52) | 0.937 | |||||||||||||
| stage II | 53 | 0.069 (0.005-0.893) | 0.04* | 8.2 (0.15-10.44) | 0.876 | 53 | 0.962 (0.087-10.60) | 0.975 | |||||||||||||
| Stage III | 34 | 0.302 (0.03-2.75) | 0.288 | 9.1 (0.14-13.19) | 0.867 | 34 | 1 | ||||||||||||||
| Stage IV ® | 24 | 1 | 1 | ||||||||||||||||||
| CD8 | |||||||||||||||||||||
| <60 | 40 | 0.769 (0.470-1.26) | 0.298 | 51 | 0.643 (0.316-1.31) | 0.222 | |||||||||||||||
| >60 (R) | 43 | 1 | 48 | 1 | |||||||||||||||||
| HHLA2 | |||||||||||||||||||||
| Low (R) | 57 | 1 | 1 | 55 | 1 | 1 | |||||||||||||||
| High | 32 | 1.74 (1.03-2.96) | 0.039* | 1.81 (1.06-3.07) | 0.03* | 29 | 2.06 (1.24-3.42) | 0.005* | 1.90 (1.18-3.06) | 0.008* | |||||||||||
Kaplan-Meier test, used to calculate OS and DFS to detect effect of risk factors on survival. Cox regression was used to assess predictors of survival with calculation of Hazard ratio
The independent prognostic value was detected by multivariate analysis. HHLA2 was independent prognostic factors with OS (P=0.03*) and DFS (P=0.008*).
Discussion
The B7 family immune checkpoint molecule HHLA2 is very important in the tumor microenvironment and could be a good target for human cancer therapy. The high HHLA2 expression is associated with worse prognosis in various malignancies. These include prostate cancer, lung cancer, osteosarcoma, hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma, bladder urothelial carcinoma, stomach cancer, breast carcinoma, colorectal cancer, and esophageal cancer. On the other hand, it is associated with a better prognosis for cervical adenocarcinoma, ampullary tumors, and pancreatic cancer (Bhatt et al., 2021).
There has been debate in the few studies that have been done on HHLA2 expression in CRC. In our study, 54% of cases had high HHLA2 expression. This result was slightly higher than Zhu et al., (2018) (47%). In contrast, two more investigations indicated that the HHLA2 gene was downregulated in colorectal cancer (Ying et al., 2022). This variation can result from the use of different monoclonal antibodies, a different study population, or a different scoring system.
In the current study, HHLA2 expression level was significantly associated with depth of invasion, LN metastasis, distant metastasis, and advanced tumor stage, which was consistent with HHLA2 expression in many cancers. According to Zhu and Dong (2018), HHLA2 was substantially associated with the invasion depth in CRC cases. According to Janakiram et al., (2015), elevated expression of HHLA2 was substantially correlated with local LN metastases and advanced stage at diagnosis in triple-negative breast cancer. Furthermore, advanced clinical stage, tumor invasion, lymph node metastasis, and distant metastasis were all positively linked with increased HHLA2 expression in gastric cancer tissues (Wei et al., 2019), bladder urothelial carcinoma (Lin et al., 2019), and intrahepatic cholangiocarcinoma (Jing et al., 2019). Additionally, in cases with clear cell RCC, HHLA2 overexpression in tumor tissues exhibited a positive link with a number of clinicopathological parameters like tumor size, clinical stage, and histologic grade (Chen et al. 2019; Zhou et al., 2020). Furthermore, Ding et al., (2022) observed that high HHLA2 was associated with a higher grade and stage of hepatocellular carcinoma.
In contrast, other studies demonstrate that HHLA2 has no significant correlation with clinico-pathologic parameters in CRC cases (Kula et al., 2023) and in pancreatic adenocarcinoma cases (Yan et al., 2019). In cervical adenocarcinoma (AC), Byun et al., (2021) observed that HHLA2 expression demonstrated a significant negative link with lymph node metastases but did not demonstrate a significant correlation with stage, tumor grade, LVI, tumor size, or invasion depth.
In our study, patients with CRCs who had a high HHLA2 expression had the worst OS and DFS. HHLA2 was found by multivariate analysis to be an independent predictor of OS and DFS in CRC patients. This finding supports a prior study by Zhu and Dong (2018) that found HHLA2 to be an independent predictive factor of the OS in CRC patients. Also, it was in agreement with preceding research in stomach cancer (Wei et al., 2019), lung adenocarcinoma (Chen et al., 2020; Farrag et al., 2021), osteosarcoma (Koirala et al., 2016), bladder cancer (Lin et al., 2019), intrahepatic cholangiocarcinoma (Jing et al., 2019), clear cell RCC (Chen et al., 2019; Zhou et al., 2020), hepatocellular carcinoma (Ding et al., 2022), spinal chordoma (Xia et al., 2022), and medullary thyroid carcinoma (Niu et al., 2022). On the other hand, some research suggested that HHLA2 was associated with better outcomes in various malignancies such as CRCs (Khan et al., 2021), cervical AC (Byun et al., 2021), ovarian cancer (Xu et al., 2021), gastric cancer (Shimonosono et al., 2018), and hepatocellular carcinoma (Liao and Zhang, 2022). Additionally, elevated HHLA2 expression was also associated with an improved post-surgical prognosis in pancreatic and ampullary cancers (Boor et al., 2020).
In our study, the relationship between HHLA2 and TILs and CD8+ T cells is not statistically significant. This is in line with the findings of a study by Kula et al. (2023), which found no relation between HHLA2 and CD8. According to Chen et al.’s (2019) research, there was no statistically significant correlation between HHLA2 overexpression and the quantity of CD8+ T cells in RCC.
In CRCs, on the other hand, the HHLA2 high-expression group had a lot fewer CD8+ cells than the HHLA2 low-expression group (Zhu and Dong, 2018). On the other hand, positive HHLA2 expression had higher levels of CD8+ T cells in pancreatic cancer (Yan et al., 2020) and in RCC (Zhou et al., 2020). The current study demonstrated that there is a statistically significant correlation between CD8 expression and gender and certain favorable prognostic factors such as absence of tumor deposits, TILs, lack of metastasis, and histological type. However, CD8 does not significantly correlate with other clinico-pathologic parameters or survival. These findings concur with those of Ko and Pyo (2019), who found a substantial correlation between CD8 and the absence of metastasis. However, a significant association was reported between CD8 and depth of invasion, lymph node stage, LVI, PNI, and histological grade.
According to Zhu and Dong (2018) and Barbosa et al. (2023), there is no correlation between CD8 and tumor site and OS, but Barbosa et al., (22,023 recorded a significant association between CD8 and lymph node status and depth of invasion. Furthermore, Yin et al., (2022) discovered that there was a strong link between CD8 and survival but not between CD8 and tumor site, histological grade, or depth of invasion.
Our study had some limitations as factors influencing HHLA2 expression, which include other T-cell subsets, and the expression of other co-inhibitory molecules, such as PD-L1, were not assessed. Additional research is needed to determine the biological significance, detect mechanisms of high HHLA2 expression in CRC, and explain its contributions to tumor immune escape.
In conclusion, this study suggests that HHLA2 is an independent prognostic predictor for the survival of CRC patients, associating with higher stage, advanced depth, and nodal stage. Therefore, HHLA2 could be considered a promising prognostic and therapeutic target in the CRC.
Author Contribution Statement
All authors contribute equally; all authors share in the writing, data processing, and collecting of data, and they reviewed the whole work and approved the final version of the manuscript.
Acknowledgements
Compliance with Ethical Standards
The present study was carried out after obtaining approval from the committed Institutional Research Board (IRB) at the Faculty of Medicine, Mansoura University, Egypt (Code Number: MDP/20.06.41). The study was processed under the ethical standards of the Helsinki Declaration.
Availability of data and material
All the clinical, radiological, and pathological data used in this manuscript is available on No scientific organization had approved this research, and it was not a component of any accepted student thesis. Mansoura University medical system (Ibn Sina Hospital management system) http://srv137.mans.edu.eg/mus/newSystem/.
Conflict of interest statement
The authors declare no relevant financial affiliations or conflicts of interest.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
All the clinical, radiological, and pathological data used in this manuscript is available on No scientific organization had approved this research, and it was not a component of any accepted student thesis. Mansoura University medical system (Ibn Sina Hospital management system) http://srv137.mans.edu.eg/mus/newSystem/.