Study Of Association Between Local And Systemic Immunity And Prognosis In PT3N0M0 Stage Esophageal Squamous Cell Cancer

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CD8+/FOXP3+ RATIO AND PD-L1 EXPRESSION ASSOCIATED WITH SURVIVAL IN pT3N0M0 STAGE ESOPHAGEAL SQUAMOUS CELL CANCERBackgroundDespite significant advances in screening,diagnosis and treatment modalities,ESCC patient prognosis remains poor.TNM remains the major prognostic factors,however,clinical outcome for patients with middle range ESCC(generally stage IB,II,and IIIA)can vary significantly,with some achieving long-term survival,while others die following disease recurrence.In the 7th edition of the AJCC manual,tumor location and histologic grade were identified as important for stage grouping.However,the classic AJCC system only describes tumor burden on a macro scale,and does not provide detailed information on the microenvironment in which ESCC proliferates.The revolutionary breakthrough of immune-checkpoint blockers led to more attention to the prognostic importance of tumor-infiltrating lymphocytes(TILs)and immune-checkpoints in many tumor types.However,discussions of TILs and immune-checkpoints with respect to ESCC have been limited and somewhat inconsistent.In this study,we investigated CD8+ or FOXP3+TILs(Treg)densities and programmed death-ligand 1(PD-L1)expression in a cohort of 151 resected pT3N0M0 ESCC patients without neoadjuvant and adjuvant therapy.MethodsPatients with pathological T3N0M0 stage thoracic ESCC who had radical esophagectomy from June 2005 to June 2013 in our hospital were included into the study.Tumor stage was determined according to the American Joint Committee on Cancer(AJCC,2009)TNM(tumor,node,metastases)staging system.No patient exhibited evidence of distant metastases in preoperative examination,none had received any prior anticancer treatments or postoperative adjuvant therapy,and all underwent complete macroscopic tumor removal.All tumor tissues were confirmed as ESCC by hematoxylin and eosin(H&E)staining after surgical resection.The study design was approved by the Ethics Committee of our hospital.IHC analyses of CD8,FOXP3 and PD-L1 using the streptavidin-biotin-peroxidase method with postoperative specimens from all patients.Quantitative evaluations of CD8+ or FOXP3+TILs were performed and counted both in the cancer cell nest and tumor stroma.The CD8+/FOXP3+ ratio was defined as the number of CD8+TILs divided by the number of FOXP3+TILs(Tregs).Cut-off value defined as the median number of infiltrating cells per field.PD-L1 staining intensity was recorded as follows:negative for no staining,positive for membranous or cytoplasmic staining in tumor cells or lymphocytes.Results151 patients with pathological T3N0M0 stage ESCC who underwent radical Ivor-Lewis esophagetomies were included in this study.121 patients(80.1%)had recurrence and 102(67.55%)died during the follow-up period.Median DFS and OS were 37.7 and 49.5 mo,respectively.Stromal infiltration of CD8+TILs and FOXP3+TILs was more frequently observed than diffuse infiltration.CD8+TILs and FOXP3+TILs were moderately correlated(r=0.554,p<0.001).PD-L1 showed a predominantly membranous or cytoplasmic(or both)focal or scattered staining pattern(43.0%of all specimens).A strong positive correlation and concordance was observed in PD-L1 expression(positive and negative expression)detected using immunohistochemistry(IHC)(r=0.853,p=0.002).PD-L1 expression and CD8+TILs densities(r=-0.610,p<0.001)were negatively correlated.Positive tumor-related PD-L1 expression was correlated with older age(>60,p=0.034),longer tumor length(>4cm,p=0.001),poorer histological differentiation(p=0.002)and tumor recurrence(p=0.001).Neither FOXP3+ nor CD8+TILs alone was related to patient prognosis in the Kaplan-Meier analysis.In contrast,CD8+/FOXP3+ ratio and PD-L1 expression were correlated with both DFS(both p<0.001)and OS(both p<0.001)in univariate analyses.Patients with high CD8+/FOXP3+ ratio and negative PD-L1 expression experienced better DFS(p<0.001)and OS(p<0.001)compared to the other three groups.Furthermore,in both well and moderately/poorly differentiated subgroups with similar tumor lengths,patients with positive PD-L1 expression had poorer DFS(p=0.028 and 0.004,respectively)and OS(p=0.003 and 0.008,respectively)compared to patients with negative PD-L1 expression.Moreover,cox regression analyses demonstrated that both PD-L1 expression and CD8+/FOXP3+ ratio were independent predictors of DFS(HR 2.133,95%CI 1.472-3.089,p<0.001;HR 0.620,95%CI 0.426-0.902,p=0.012;respectively)and OS(HR 2.492,95%CI 1.662-3.737,p<0.001;HR 0.562,95%CI 0.375-0.841,p=0.005;respectively).ConclusionsOur results suggest pT3NOMO ESCC clinical outcomes correlate with CD8+ and FOXP3+ TILs densities and PD-L1 levels.Moreover,an intrinsic mechanism for induction of PD-L1 overexpression may be occurring during early tumor oncogenesis.This information may be useful for stratifying patients and guide the application of checkpoint blockade therapy in ESCC.SECTION ? PROGNOSTIC ROLE OF LYMPHOCYTE-TO-MONOCYTE RATIO AND TUMOR-INFILTRATING LYMPHOCYTE TO TUMOR-ASSOCIATED MACROPHAGE RATIO IN PATIENTS WITH STAGE pT3N0M0 ESOPHAGEAL SQUAMOUS CELL CARCINOMABackgroundEsophageal squamous cell cancer(ESCC),which is characterized with high recurrence rates,remains one of the most lethal gastrointestinal malignancies.Despite recent progress in multimodal treatment approaches,prognosis differs even among patients of the same TNM stage,especially those of middle stage.Currently,the relationship between local and systemic immune responses and ESCC prognosis has attracted much recent attention.However,data is poor on ESCC.In this article,we assessed the prognostic significance of,and the relationship between,the pretreatment lymphocyte-to-monocyte ratio(LMR)and the tumor-infiltrating lymphocytes(TILs)/tumor-associated macrophage(TAM)ratio,in patients with ESCC of pathological stage T3N0M0(pT3N0M0).MethodESCC that was histopathologically confirmed and pathologically staged as T3N0M0 after curative esophagectomy who had not undergone neoadjuvant therapy between January 2006 and January 2013 in our hospital were included.Availability of blood test data can be obtained within 1 week prior to surgery.Complete preoperative evaluation including endoscopic esophageal ultrasonography,chest and abdomen computed tomography,brain MRI,and bone scanning were also necessary to rule out metastasis.The study was approved by the Ethics Committee of our hospital.Densities of CD8+TILs,CD4+TILs,CD45RO+TILS and CD68+TAM were assessed by immunohistochemical staining of tissue microarray cores from all patients and evaluated in a medium-power field(200×)as a percentage of stained lymphocytes in the stromal compartment on a semiquantitative scale(results in 5%steps beginning with 0%).Hematological biomarkers including lymphocyte and monocyte counts were obtained from routine preoperative blood test data,and the LMR and TILs/TAM ratios calculated.Results237 patients with pT3N0M0 ESCC who underwent curative esophagectomy were included in this study.Among them,108 patients(45.57%)underwent surgery alone.9 patients had vascular invasion and 6 had perineural invasion.Follow-up ended in June 30,2017.185 patients(78.06%)had recurrence and 191(80.59%)died during the follow-up period.Median DFS and OS were 38.5 and 51.0 mo,respectively.CD45RO+,CD4+,and CD8+TILs,as well as CD68+TAM infiltrating ESCC tissue were evident in the stroma.High CD68+TAM infiltrates correlated with longer tumor length and vascular invasion(p=0.041 and 0.019 respectively).Meanwhile,high AMC was associated with low differentiation and longer tumor length(p=0.003 and 0.047,respectively).No significant correlation was evident between other single immune marker and clinical variables.Also,high CD45RO/CD68 and CD4/CD68 ratios were associated with both tumor differentiation(p=0.026 and 0.016 respectively)and recurrence(p=0.001 and 0.023 respectively).A significant correlation was evident between the CD68 score and the AMC(r = 0.308,p<0.001).Furthermore,the ALC exhibited a weak positive association with CD4 and a negative correlation with CD68 counts(r=0.162 and-0.158;p=0.015 and 0.017,respectively).The LMR was significantly correlated with the CD8/CD68,CD4/CD68,and CD45RO/CD68 ratios(r=0.400,0.308,and 0.232,all p<0.001).Univariate analysis showed that conventional tumor histopathological features,including tumor length,tumor location,extent of differentiation,and vascular invasion were prognostically significant.No type of lymphocyte infiltrating the stroma and no hematological markers alone were of any prognostic significance.In contrast,except for the CD4/CD68 ratio,high CD45RO/CD68 and CD8/CD68 ratios were significantly associated with a favorable DFS(p=0.001,0.018,respectively)and OS(p<0.001,p=0.005,respectively).High LMR were also associated with a favorable OS(p=0.032).Multivariate analysis showed the CD45RO/CD68 ratio was strongly prognostic of DFS,with HRs of 0.886(95%CI 0.798-0.984,p = 0.024)when used as a continuous variable and 0.691(95%CI 0.513-0.931,p =0.015)when employed as a categorical variable.In addition,the ratio was a good indicator of improved OS,with HRs of 0.884(95%CI 0.795-0.983,p = 0.023)when used as a continuous variable(per point of increase)and 0.687(95%Cl 0.510-0.937,p =0.017)when employed as a categorical variable.In contrast,LMR was not an independent factor in multivariate analysis.ConclusionsIn this study,we found the prognostic significance of the CD45RO/CD68 ratio was higher than that of the LMR.The CD45RO/CD68 ratio is a useful independent prognostic marker in patients with pT3NOMO ESCC who have undergone complete resection without neoadjuvant therapy.Large-scale prospective studies are needed to further evaluate the role of CD45RO/CD68 ratio in ESCC.