Prognostic implications of lymphocyte/C-reactive protein ratio with tumor-inltrating lymphocytes in resected intrahepatic cholangiocarcinoma

Background Lymphocyte/C-reactive protein (CRP) ratio (LCR), is a promising prognostic factor that can reect tumor inammation and systemic patient condition. The aim of this study was to investigate whether preoperative LCR can be a prognostic factor for post-surgical outcomes among patients with intrahepatic cholangiocarcinoma (IHCC). We also investigated the relationship between LCR and tumor inltrating lymphocytes (TILs) to clarify whether systemic host immune parameters reect local tumor immunity. Methods We enrolled 45 patients who had undergone hepatectomy for IHCC. Patients were divided into low LCR and high LCR groups, according to reactive operating characteristic curve (Cut-off value: 8780). We analyzed their overall survival (OS) and disease-free survival (DFS) with respect to LCR and other clinicopathological factors. We also investigated stromal TILs and numbers of CD8 + TILs in surgical specimens, and the relationship between LCR and TILs. Results Twenty-one patients (46.7%) were associated with low LCR. Low LCR was signicantly correlated with older age, high CRP and advanced disease stage, and was a prognostic factor for OS and DFS. And multivariate analysis revealed that low LCR was an independent prognostic factor for worse OS (HR: 2.81 P<0.04). Regarding the relationship between LCR and local tumor immunity, while LCR and levels for stromal TILs were signicantly related, LCR and levels for CD8 + TILs were signicantly related. Conclusions Preoperative LCR levels could predict the post-surgical prognosis of patients with IHCC, and reected numbers of intra-tumoral CD8 + TILs.

Reportedly, the preoperative platelet/lymphocyte ratio (PLR), systemic immune in ammation index (SII; platelets × NLR), and lymphocyte/monocyte ratio (LMR) are signi cantly associated with outcomes in patients with IHCC who undergo curative resection [9][10][11]. We have already focused on the prognostic effects of some proteins in resected IHCC specimens or preoperative image ndings of IHCC patients. High expression of Heat Shock Protein 1 (HSF-1), and loss of Secreted Frizzled-Related Protein-1 (SFRP-1) and F-box and WD repeat domain-contain 7 (FBXW7) in surgical specimens were associated with poor prognosis in IHCC patients [12][13][14]; as were low apparent diffusion coe cient of diffusion-weighted image magnetic resonance imaging, and central hypo-enhancement in hepatic arterial phase of dynamic computed tomography in preoperative images [15,16].
After Okugawa et al. reported lymphocyte/C-reactive protein (CRP) ratio (LCR), a new immune-nutritional index, to be a strong prognostic factor for patients with colorectal cancer [17], LCR was also found to indicate prognosis of gastric cancer, pancreatic cancer, rectal cancer treated with chemoradiotherapy, and breast cancer. [18][19][20][21].
Whereas LCR and other immune-nutritional parameters represent the patient's systemic condition, tumorin ltrating lymphocytes (TILs) may re ect local tumor immunity. Lymphocyte-predominant breast cancer (LPBC), which has high TIL levels, has a good prognosis [22]. Lee  This study investigated the relationships between preoperative LCR and postsurgical outcomes in patients with IHCC, and between LCR levels and intra-tumoral TILs, to clarify whether systemic immune indicators re ect local tumor immunity.

Patients
We enrolled 45 patients who underwent primary hepatectomy for IHCC at Tokushima University Hospital between July 2002 and May 2015 in this study. We excluded patients who had undergone chemotherapy or radiation prior to surgery, or whose blood tests including cell fractions, were not measured within 4 weeks before surgery. We obtained data on other clinicopathological parameters from the medical database. The patients' median follow-up period was 2.5 years (range: 0.17-17.08). This study was approved by Tokushima University Hospital ethics committee and with the approval of corresponding regulatory agencies, and all the experiments were carried out in accordance with the approved guidelines (Tokushima Clinical Trial Management System Number; 3215). All the patients involved in this study signed informed consent forms and agreed to participate.
Treatment strategy for IHCC Patients were treated according to a previously described strategy for resectable IHCC [15,27]. Brie y, limited hepatectomy was performed without typical lymph node dissection or extrahepatic bile duct resection for peripheral tumors. For tumors in the perihilar region, or in the peripheral region with hilar in ltration, anatomical hepatectomy and regional lymph node dissection were performed. Extrahepatic bile duct resection and reconstruction were performed if needed for surgical margins.
Preoperative immune parameters Blood samples were taken prior to hepatectomy. LCR was calculated by dividing total lymphocyte count (TLC) (/µL) by serum CRP (mg/dL). The optimal cutoff value for LCR was determined by using receiver operating characteristics (ROC) curves on mortality two years after surgery to assess early postoperative death. The cutoff value of LCR determined by ROC curve was 8780 (1-Speci city, Sensitivity: 0.296, 0.772; area under curve: 0.765).

Assessment of TILs
TILs were evaluated according to the standardized methodology proposed by the International TIL Working Group [28]. In summary, rather than counting the number of lymphocytes, we assessed stromal TILs by calculating the ratio of TILs to stromal areas, excluding tumor cells, in hematoxylin and eosin (HE)-stained samples (Fig. 1a). We excluded the tumor cell areas (Fig. 1b) and calculated stromal and TILs areas (Fig. 1c) by using the image analysis software ImageJ (National Institute of Health). Assessments were performed in areas within the borders of the invasive tumor, and tumor zones with crush artifacts, necrosis, regressive hyalinization were excluded. We scored all mononuclear cells, including lymphocytes and plasma cells, but polymorphonuclear leukocytes or broblasts were excluded. After a full assessment of average TILs in the tumor border area, we assessed the stromal TILs averaged among three selected high-power elds (400 × magni cation), not focused on hotspots. Thus, we evaluated TILs for 38 patients whose HE slides were available, under the guidance of two expert pathologists who were blinded to clinical features.

Assessment of CD8 + TILs
Anti-CD8 antibody (dilution 1:100, M7103; Dako) was used as the primary antibody. We used immunohistochemistry procedures in our department that were previously reported by Ishikawa et al [29].
Selecting average regions within the tumor borders, we manually counted CD8 + TILs in three high-power elds (400 × magni cation) per patient and calculated the mean number of CD8 + TILs, for the 31 patients whose samples were available. Figure 2 showed representative cases of high (Fig. 2a) and low (Fig. 2b) CD8 + TILs. These procedures were conducted under the guidance of two expert pathologists.

Statistical analysis
Cutoff values for immune-nutritional parameters and TILs were determined with ROC curves. We used the unpaired Mann-Whitney U-test or the χ2 test to compare clinicopathological variables between two groups. Overall survival (OS) and disease-free survival (DFS) curves were created using Kaplan-Meier method; differences were analyzed by log-rank test. The Cox proportional hazard regression model was used for multivariate analysis. P < 0.05 was considered signi cant.

Results
Preoperative LCR levels can accurately re ect post-surgical prognosis Patients were divided into the high LCR (LCR High ) group and the low LCR (LCR Low ) group. Median values indicated that the LCR Low group was signi cantly older, and had higher CRP levels, longer surgeries, and more advanced disease than the LCR High group (Table 1). Other host, surgical, and tumor factors did not signi cantly differ between the two groups. Five-year OS rates after hepatectomy were signi cantly worse in the LCR Low group (15.2%) than in the LCR High group (57.5%; P < 0.01; Fig. 3a). In univariate analysis, advanced disease stage (III/IV), lymph node metastasis (LNM), low LCR, and high CA19-9 were associated with worse OS. In multivariate analysis, LNM, low LCR, and high CA19-9 were independent prognostic factors ( Table 2). Three-year DFS rates after hepatectomy were also signi cantly worse in the LCR Low group (0%) than in the LCR High group (31.8%; Fig. 3b). Univariate analysis associated age ≥ 75 years old, advanced disease stage, LNM, portal vein invasion, and low LCR with poor prognosis, but none of these factors were found to be independent prognostic factors in multivariate analysis (Table 3).  (Fig. 4a) LCR and stromal TILs were not signi cantly associated.

Discussion
The present study suggests that preoperative LCR can indicate the postsurgical prognosis for patients who undergo resection for IHCC. Furthermore, as LCR was associated with CD8 + TILs, it indirectly re ects local tumor immune activity. This is the rst report to nd a correlation between a systemic immune marker and CD8 + TILs in patients with IHCC.
LCR is already reported to be a prognostic marker for colorectal, gastric, pancreatic, and breast cancers [17][18][19][20][21]. For patients with IHCC who undergo curative resection, NLR, LMR, PLR, and SII are reportedly signi cant prognostic factors [4,[9][10][11]. Many papers have especially suggested NLR as a strong prognostic marker for IHCC. In our result, NLR tended to be a prognostic factor in OS, but not signi cantly so, whereas LCR was found to be an independent prognostic factor. LCR may be stronger prognostic factor than NLR in this setting.
LCR is the combination of TLC and CRP. CRP is a well-established marker for in ammation. Elevated preoperative CRP level is widely reported to be associated with poor prognosis in various cancers, including IHCC [30][31][32]. CRP is synthesized by hepatocytes in response to IL-6, TNF and IL-1β, which can activate cancer cell proliferation. CRP may re ect systemic in ammation, and cancer cell proliferation and protection from apoptosis. [33,34]. TLC also re ect host immune status; several papers have associated low TLC with poor prognosis [35,36].
However, the mechanism that links LCR to patients' long-term prognosis has been unclear. In the present study, we focused on the relationship between LCR and TILs, which are indicators of local tumor immunity. TILs have attracted recent attention with the development of immune checkpoint inhibitors. In addition, there were some reports TILs had the relationship between peripheral blood cells, such as TLC and absolute neutrophil count (ANC) [23,24].
Interestingly, although this study found no signi cant relationship between LCR and stromal TILs, LCR and CD8 + TILs, speci cally, were associated. TILs volume is reportedly correlated with tumor mutation burden (TMB) [37]. TMB is the number of gene mutations cancer cells have, and is associated with predicted immunotherapy response [38]. In breast cancer, which is reported to have relatively high TMB and TILs, patients who have more than 50% TILs are often de ned as LPBC; <10% TILs are considered minimal [28]. In the present study, the median TIL percentage in all patients was 0.43% (IQR: 0.26-0.77), which was much lower than for breast cancer. Biliary cancer is also reported to have relatively low TMB [39]. Thus, TILs in IHCC were expected to be low, and our result was consistent with that. Low TIL percentage in IHCC may be why LCR and TILs did not correlate. Furthermore, the TIL evaluation method included all mononuclear cells-not only lymphocyte. As IHCC apparently has low TILs, possibly cells other than lymphocytes were affected.
TILs include various types of lymphocytes, such as CD3 + , CD4 + , CD8 + cells; CD8 + T cells are considered especially important among them. Tumor in ltration by CD8 + T cells has been strongly associated with Page 12/25 survival of cancer patients [26,28]. Yoshida et al. also reported that CRP inhibited proliferation, activation, and function of CD8 + T cells in patients with melanoma [40]. Thus, CD8 + TILs may be more strongly associated with LCR, which is a robust prognostic factor.
This study had several limitations. First, it is a retrospective analysis from a single center, with a relatively small study cohort. Second, although lymphocytes come in various types, we assessed only CD8 + T cells.
A larger study, and an assessment of non-CD8 + lymphocytes are issues for the future.

Conclusions
In conclusion, LCR can predict prognosis of patients with IHCC via correlation with CD8 + TILs.

Consent for publication
All the patients involved in this study signed informed consent forms and agreed to participate.

Availability of data and material
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.