Predictive value of different proportion of lesion HLA-G expression in colorectal cancer

Differential expression of HLA-G has been observed among cancer types and tumors from individuals with the same type of cancer; however, its clinical significance is rather limited. In this study, expression and predictive relevance of HLA-G expression in 457 primary colorectal cancer (CRC, ncolon = 232, nrectal = 225) patients was investigated. Data showed 70.7% (323/457) of the CRC were HLA-G expression when the above 5% (HLA-GLow) was considered as positive, which wasn't associated with patient survival (p = 0.109). However, HLA-G expression above 55% (HLA-GHigh) was associated with a worse prognosis of CRC patients (p = 0.042). Furthermore, a shorter survival was found for the female (p = 0.042) and elder (p = 0.037) patients whose HLA-G expression was above HLA-GLow level. HLA-G expression above HLA-GHigh level showed a worse prognosis for female (p = 0.013), elder (p = 0.023), colon cancer (p = 0.016), advanced tumor burden (T3+4, p = 0.018), regional lymph node status (N1+2, p = 0.044), and advanced clinical stage patients (AJCC III+IV, p = 0.037). In conclusion, our results demonstrated for the first time that combination of differential lesion HLA-G expression notably improved the value of traditional survival prediction for CRC patients.


INTRODUCTION
Colorectal cancer (CRC) occurs in an estimated 376,300 new cases and 191,000 deaths in 2015 in China [1].Tumor progression is dictated by the intimately continuous interaction between malignant cells and the tumor microenvironment such as immune effector molecules and immune regulatory factors, and infiltration of various immune cells [2,3].Unfortunately, tumor cells have developed various strategies to avoid recognition and destruction by the host immune milieus, and the resistant variants eventually results in cancer [4].
The immune suppressive molecule human leukocyte antigen G (HLA-G), is rarely observed in normal adult tissues.However, it is found frequently neoexpressed in most tumor cells as cell membrane-bound or soluble forms [5].Amounts of in vitro and in vivo evidence showed HLA-G could directly interact with its receptors expressed on almost all types of immune cells or by the pathway of "trogocytosis", revealing a broad immune inhibiting function on both innate and adaptive immune responses [6].In clinical settings and animal models, earlier studies have demonstrated the aberrant neoexpressed HLA-G in various types of cancers was related to advanced tumor grade, more aggressive behavior and worse disease outcome [7].
To be mentioned, we recently found that levels of peripheral soluble HLA-G in CRC patients were strongly related to prognosis, and it could improve the prognostic value by traditional prognosticators [8].Indeed, different proportion of lesion HLA-G expression has been found between different cancer types and also between tumors from individuals with the same type of cancer, and the prognostic significance of the different degree of HLA-G expression remains rather limited.With the combination of lesion HLA-G expression percentages, we also aim to evaluate whether it could improve the prognostic value of traditional clinical prognosticators.
In the current study, HLA-G expression in 457 primary colorectal cancer lesions was analyzed with immunohistochemistry, and the differential lesion HLA-G expression for prognostic stratification with traditional prognosticators was analyzed.

HLA-G expression in primary CRC lesions
Different proportion of HLA-G expression was found and its expression in CRC lesions was from negative to 99% (Figure 1).Overall, 70.7% (323/457) of primary CRC samples were HLA-G positive, which was positive in 76.7% of the colon (178/232) and 64.4% of the rectal carcinoma lesions (145/225), respectively (Table 1).
Kaplan-Meier survival analysis was performed to determine the minimum proportion of HLA-G expression which reaches statistic significance to patient survival.Data showed that, with the percentage at 5% as the cutoff value, HLA-G expression wasn't associated with the patient survival (p = 0.109), while HLA-G expression above 55% reached significantly to the patient survival (p = 0.042; Table 2).Base on this data, the different proportion of HLA-G expression in CRC lesions was divided into two groups as HLA-G Low (cut-off = 5%) and HLA-G High (cut-off = 55%) in this study.

Association between and HLA-G expression and clinical parameters
Data showed that (Table 1), a higher proportion of HLA-G expression was observed in colon carcinoma than that in rectal carcinoma lesions [HLA-G Low :76.7% (178/232) vs. 64.4% (145/225), p = 0.004; HLA-G High :72.4% (168/232) vs. 58.2%(131/225), p = 0.001], while no significance was found between the HLA-G expression and patient sex, age, primary tumor burden (T), regional lymph node status (N), distant metastases (M), and clinical disease stage.Also, no significant difference was observed for the relationship between the HLA-G expression and the clinical parameters either in colon or in rectal carcinoma patients (Supplementary Table 1).

Significance of HLA-G status on the prognostic value of clinical parameters in CRC patients
Furthermore, we analyzed the prognostic significance of HLA-G status with stratification of clinical parameters in CRC patients.Briefly, The tumor histological type was stratified to colon and rectal carcinoma, patient sex to male and female, age to below and above the median age (66 years), categories T to T 1 , T 2 ,T 3 and T 4 ; N to N 0 , N 1 and N 2 ; M status to M 0 and M 1 , and clinical stage to I, II, III and IV, respectively.
Kaplan-Meier survival analysis revealed that both HLA-G Low and HLA-G High status could significantly affects the CRC patient survival when clinical parameters were stratified.To be noted, HLA-G Low status (Table 4) was less powerful than the HLA-G High status (Table 5) in affecting the patient survival between stratified clinical parameters.Data showed female patients whose HLA-G above the cut-off 5% (HLA-G Low ) have a marked worse survival than those below the 5% level (mean: 63.5 months vs. 82.6 months, p = 0.042; Figure 4Ab).Similarly, the elder patients have worse survival with HLA-G above 5% (HLA-G Low ) than those HLA-G expression was lower (mean: 61.3 months vs. 77.9months, p = 0.037; Figure 4Bb).Furthermore, patients with HLA-G expression above the cut-off 55% (HLA-G High ) have dramatically worse survival than those HLA-G expression was lower among the female (mean: 59.1 months vs 83.5 months, p = 0.013; Figure 4Ad), the elder patients (mean: 60.0 months vs. 78.2months, p = 0.023; Figure 4Bd), and patients with colon carcinoma (mean: 68.1 months vs 84.8 months, p = 0.016; Figure 4Cd ), stage of T 3+4 (mean: 62.5 months vs. 74.8months, p = 0.018; Figure 4Dd), N 1+2 (mean: 52.7 months vs. 65.4months, p = 0.044; Figure 4Ed), and disease stage III+IV (mean: 52.7 months vs. 65.7 months, p = 0.037; Figure 4Gd ).

DISCUSSION
In some cancers, patients within the same TNM stage but their clinical outcomes varied significantly, such as rapid disease progression and cancer-related death with early stage; however, advanced stage cancer can remain stable for years in some patients is often seen [9].The major reason for the limited predictive power of the traditional staging system is that relies only on the tumor cell characteristics but ignores the effects of the host immune response against the cancers [10].Indeed, increasing evidence has documented host immune contexture in tumor microenvironment play a critical role in the prediction of prognosis, such as the prognostic value of CD3+, CD8+ and CD45RO+ T cell infiltration in CRCs [11,12].
Among various factors of host immune contexture in tumor immunology, induction of an immunotolerant HLA-G expression by tumor cells has been observed in numerous tumoral tissues [13].HLA-G have multiple  immune inhibitory effects such as impairing T cell functions by inhibiting of proliferation and cytotoxicity, induction of apoptosis and expansion of regulatory T cells [5].Moreover, HLA-G expression was found inversely related to the frequency of intratumoral lymphocyte infiltration such as CD8+ T cells or the numbers of peripheral activated T cells (CD8+CD28+ T cells) [14,15].Previous studies showed that tumor HLA-G expression was associated with advanced stage or worse clinical outcome and its potential as a prognostic biomarker has been intensively investigated [5].In CRC patients, Guo et al. [16] evidenced HLA-G was significantly related to the overall survival of CRC patients and it could be an independent prognosticator, similar findings were obtained in a study by Ye et al. [17].Recently, Kuppen and colleagues [18,19] found that absence of HLA-G expression indicated a better survival for colon cancer patients and weak expression of HLA-G revealed a worse survival in rectal cancer patients.
Our findings in the current study revealed that different proportion of HLA-G expression in CRC lesions is of significance to the patient survival or prognosis.Analogously, in our recent study, patients with higher levels of peripheral sHLA-G had a significantly worse survival than those with lower levels, and sHLA-G was considered as an independent prognostic factor for CRC patients.Moreover, with stratification of clinical parameters in survival by sHLA-G low and sHLA-G high , could improve the prognostic power by traditional prognosticators in CRC patients [8].Our previous in vitro studies had reported that inhibition of NK cytolysis is dependent on the proportion of HLA-G expression.In these studies, data showed that the power of HLA-G in NK cell cytotoxicity was dependent on the level of both HLA-G1 and HLA-G5 expression, and HLA-G1 and HLA-G5 have an additive effect on the NK cell cytolysis suppression [20].Importantly, a significant inhibition would be reached when the HLA-G expression was more than 60% [21].Thus, it's reasonable to speculate that the different proportion of HLA-G expression in tumor lesions could influence disease progression and clinical outcome.
In consistent with our mentioned above findings, CRC lesion HLA-G expression status also could significantly affect the CRC patient survival with the stratified clinical parameters.Applying HLA-G expression above the cut-off level at 5% (HLA-G Low ) as positive which were commonly used in previous studies, HLA-G Low was not significantly related to the CRC patient survival; however, when HLA-G expression above the level of 55% (HLA-G High ), HLA-G High reaches a statistic significance point to a worse survival, which echoes a study by Kirana et al. [22] that high, but not negative and moderate local HLA-G expression was closely linked to the CRC patient survival.Degree of HLA-G expression could be influenced by multiple factors and mechanisms are complex.HLA-G genetic variation such as polymorphisms in 5′upstream regulatory region and 3′untranslated regions affects the affinity of targeted gene for transcriptional or posttranscriptional factors [23], epigenetic pathways through DNA methylation and histone modifications [24], posttranscriptional mechanisms by and microRNAs [25], as well as environmental factors including various cytokines, growth factors and hormones [26].However, mechanisms underlying the individual or tumor-specific expression of HLA-G are required to be explored.Moreover, different proportion of lesion HLA-G expression has been found between different cancer types, between tumors from individuals with the same type of cancer, and even between the different intra-tumor areas within a single sample.In this context, Rouas-Freiss et al. recently reported that HLA-G and other immune molecules such as PD1/ PDL1,ILT2/4 were heterogeneous expressed in the various areas of the same tumor [27].These findings further highlights more detail and comprehensive evaluation of the intra-and inter-heterogeneity of HLA-G expression is necessary for explore the clinical significance the HLA-G in tumor biology.
Finally, our data showed that HLA-G High status was powerful in affecting the patient survival when clinical parameter was stratified.Among female patients, the elder patients, colon carcinoma patients, stage of T 3+4 , N 1+2 , and stage III+IV whose HLA-G expression above the cut-off 55% (HLA-G High ) have dramatically poor survival than those with lower HLA-G expression.In serous ovarian carcinoma, a study by Andersson et al. [14] revealed that HLA-G expression was correlated to a significant worse prognosis in patients with the genotype HLA-A*02.These data indicated that HLA-G expression status together with other clinical parameters, tumor microenvironment factors is possible to discriminate subpopulations and identify patients with even worse prognosis.
Our study demonstrated that different proportion of HLA-G expression in CRC patients influence the patient survival and a combination of HLA-G expression status with traditional clinical risk factors could refine the prediction of specific clinical outcomes of the subpopulations of CRC patients.Taken together, lesion HLA-G expression percentages in patients with CRC could be another prognostic factor which contributes an additional significance to the classical cancer TNM classification system.Abbreviations: 95% CI = 95% confidence interval; TNM, lymph-node-metastasis and stage according to the TNM classification.

Figure 1 :
Figure 1: Immunohistochemistry analysis of HLA-G expression in primary CRC lesions.Representative staining of negative (A and B), and positive expression (C and D; E and F) of HLA-G in CRC lesions.Original magnification: A, C, E (100×) and B, D, F (400×).

Figure 2 :
Figure 2: Kaplan-Meier survival analysis of HLA-G expression in CRC patients.Comparison of the overall survival (A) between HLA-G negative and HLA-G positive patients with the cut-off = 5% (HLA-G Low ; p = 0.109), and (B) between HLA-G negative and HLA-G positive patients with the cut-off 55% (HLA-G High ; p = 0.042).

Figure 4 :
Figure 4: Kaplan-Meier survival analysis of stratified clinical parameters in survival by lesion HLA-G expression (HLA-G Low; A~G-a and -b) and (HLA-G High ; A~G-c and -d) in CRC patients, respectively.Stratified clinical parameters (A) male and female patients; (B) patients with age above and below the median of 66 years; (C) tumor histological types of colon and rectal cancer; (D) primary tumor status T 1+2 and T 3+4 ; (E) regional lymph node status N 0 and N 1+2 ; (F) tumor metastasis status M 0 and M 1 ; and (G) disease stages I + II and III + IV of CRC patients.