title: Linear quantification of lymphoid infiltration of the tumor margin: a reproducible method, developed with colorectal cancer tissues, for assessing a highly variable prognostic factor.
abstract: BACKGROUND
  Lymphoid infiltration is a prognostic marker in solid tumors, such as colorectal, breast and lung carcinomas. However, lymphoid infiltration is heterogeneous and the reproducibility of quantification based on single counts within a tumor is very low. We aimed to develop a reproducible method for evaluating lymphoid infiltration in tumors.
  
  
  METHODS
  Virtual slides were obtained from tissue sections from the localized colorectal carcinomas of 117 patients, stained for CD3 and CD45R0. We assessed the variation of lymphoid cell density by automatic counts in 1 mm-wide, 5 μm-long segments of the invasive front, along an axis 4 mm in length running perpendicular to the invasive front of the tumor.
  
  
  RESULTS
  We plotted curves of the variation of lymphocyte density across the tumor front. Three distinct patterns emerged from this linear quantification of lymphocyte (LQLI). In pattern 1, there was a high density of lymphocytes within the tumor. In pattern 2, lymphocyte density peaked close to the invasive margin. In pattern 3, lymphocytes were diffusely distributed, at low density. It was possible to classify all the tumors studied, and interobserver reproducibility was excellent (kappa =0.9). By contrast, single counts of CD3+ cells on tissue microarrays were highly variable for a given LQLI pattern, confirming the heterogeneity of lymphoid infiltration within individual tumors. In univariate analysis, all pathologic features (stage, metastatic lymph node ratio (LNR), vascular embolism, perineural invasion), CD3+ cell density, LQLI patterns for CD3+ and CD45R0+ cells) were found to have a significant effect on disease-free survival (DFS). In multivariate analysis, only the LQLI pattern for CD3+ cells (HR: 6.02; 95% CI: 2.74-13.18) and metastatic lymph node ratio (HR: 6.14; 95% CI: 2.32-16.2) were associated with DFS.
  
  
  CONCLUSIONS
  LQLI is an automated, reproducible method for the assessment of lymphoid infiltration. However, validation of its prognostic value in larger series is required before its introduction into routine practice for prognostic evaluation in patients with colorectal carcinomas.
  The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9861460717895880.
authors.affiliation: EA4340, Versailles SQY University, Boulogne, France.
authors.forename: Marc-Antoine
authors.initials: MA
authors.lastname: Allard
authors.forename: Jean Baptiste
authors.initials: JB
authors.lastname: Bachet
authors.forename: Alain
authors.initials: A
authors.lastname: Beauchet
authors.forename: Catherine
authors.initials: C
authors.lastname: Julie
authors.forename: Robert
authors.initials: R
authors.lastname: Malafosse
authors.forename: Christophe
authors.initials: C
authors.lastname: Penna
authors.forename: Bernard
authors.initials: B
authors.lastname: Nordlinger
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Emile
mesh_terms: D000368:Aged; D057205:Automation, Laboratory; D014408:Biomarkers, Tumor / Q000032:analysis; D017252:CD3 Complex / Q000032:analysis; D002277:Carcinoma / Q000276:immunology* / Q000401:mortality / Q000473:pathology / Q000601:surgery; D015179:Colorectal Neoplasms / Q000276:immunology* / Q000401:mortality / Q000473:pathology / Q000601:surgery; D018572:Disease-Free Survival; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D053208:Kaplan-Meier Estimate; D017493:Leukocyte Common Antigens / Q000032:analysis; D016014:Linear Models; D018655:Lymphocyte Count / Q000379:methods; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D015999:Multivariate Analysis; D009361:Neoplasm Invasiveness; D015588:Observer Variation; D010363:Pattern Recognition, Automated; D011237:Predictive Value of Tests; D016016:Proportional Hazards Models; D015203:Reproducibility of Results; D012307:Risk Factors; D013997:Time Factors; D046888:Tissue Array Analysis; D016896:Treatment Outcome
chemical_list: D014408:Biomarkers, Tumor; D017252:CD3 Complex; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human