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Linear quantification of lymphoid infiltration of the tumor margin: a reproducible method, developed with colorectal cancer tissues, for assessing a highly variable prognostic factor

Marc-Antoine Allard12, Jean Baptiste Bachet136, Alain Beauchet4, Catherine Julie15, Robert Malafosse12, Christophe Penna127, Bernard Nordlinger12 and Jean-François Emile15*

Author Affiliations

1 EA4340, Versailles SQY University, Boulogne, France

2 Department of Digestive and Oncological Surgery, Ambroise Paré Hospital, APHP, Boulogne, France

3 Department of Digestive Oncology, Ambroise Paré Hospital, APHP, Boulogne, France

4 Department of Clinical Research, and Ambroise Paré Hospital, APHP, Boulogne, France

5 Department of Pathology, Ambroise Paré Hospital, APHP, 9 Av. Charles de Gaulle, Boulogne F-92104, France

6 present address: Hôpital de la Pitié, APHP, Paris, France

7 present address: Hôpital de Bicêtre, APHP, Bicêtre, France

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Diagnostic Pathology 2012, 7:156  doi:10.1186/1746-1596-7-156

Published: 13 November 2012



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.


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.


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.


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.

Virtual slides

The virtual slide(s) for this article can be found here: webcite

Tumor infiltration; Lymphocytes; Invasive margin; Linear quantification; Colorectal cancer; Image analysis; Automated count