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

Diagnostic Pathology

Jean-François Emile⁴  Bernard Nordlinger³  Christophe Penna⁵  Robert Malafosse³  Catherine Julie⁴  Alain Beauchet²  Jean Baptiste Bachet¹  Marc-Antoine Allard³ 

[1] present address: Hôpital de la Pitié, APHP, Paris, France;Department of Clinical Research, and Ambroise Paré Hospital, APHP, Boulogne, France;Department of Digestive and Oncological Surgery, Ambroise Paré Hospital, APHP, Boulogne, France;Department of Pathology, Ambroise Paré Hospital, APHP, 9 Av. Charles de Gaulle, Boulogne F-92104, France;present address: Hôpital de Bicêtre, APHP, Bicêtre, France

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

Others  :  807858
DOI  :  10.1186/1746-1596-7-156

 received in 2012-08-20, accepted in 2012-11-04,  发布年份 2012

【 摘 要 】

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.

Conclusion

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:http://www.diagnosticpathology.diagnomx.eu/vs/9861460717895880 webcite

【 授权许可】

   
2012 Allard et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Dunn GP, Bruce AT, Ikeda H, Old LJ, Schreiber RD: Cancer immunoediting: from immunosurveillance to tumor escape. Nat Immunol 2002, 3:991-998.
• [2]Shankaran V, Ikeda H, Bruce AT, White JM, Swanson PE, Old LJ, Schreiber RD: IFNgamma and lymphocytes prevent primary tumour development and shape tumour immunogenicity. Nature 2001, 410:1107-1111.
• [3]House AK, Watt AG: Survival and the immune response in patients with carcinoma of the colorectum. Gut 1979, 20:868-874.
• [4]Jass JR: Lymphocytic infiltration and survival in rectal cancer. J Clin Pathol 1986, 39:585-589.
• [5]Pagès F, Berger A, Camus M, Sanchez-Cabo F, Costes A, Molidor R, Mlecnik B, Kirilovsky A, Nilsson M, Damotte D, Meatchi T, Bruneval P, Cugnenc P-H, Trajanoski Z, Fridman W-H, Galon J: Effector memory T cells, early metastasis, and survival in colorectal cancer. N Engl J Med 2005, 353:2654-2666.
• [6]Sato E, Olson SH, Ahn J, Bundy B, Nishikawa H, Qian F, Jungbluth AA, Frosina D, Gnjatic S, Ambrosone C, Kepner J, Odunsi T, Ritter G, Lele S, Chen Y-T, Ohtani H, Old LJ, Odunsi K: Intraepithelial CD8+ tumor-infiltrating lymphocytes and a high CD8+/regulatory T cell ratio are associated with favorable prognosis in ovarian cancer. Proc Natl Acad Sci USA 2005, 102:18538-18543.
• [7]Zingg U, Montani M, Frey DM, Dirnhofer S, Esterman AJ, Went P, Oertli D: Tumour-infiltrating lymphocytes and survival in patients with adenocarcinoma of the oesophagus. Eur J Surg Oncol 2010, 36:670-677.
• [8]Wang B, Xu D, Yu X, Ding T, Rao H, Zhan Y, Zheng L, Li L: Association of intra-tumoral infiltrating macrophages and regulatory T cells is an independent prognostic factor in gastric cancer after radical resection. Ann Surg Oncol 2011, 18:2585-2593.
• [9]Droeser R, Zlobec I, Kilic E, Güth U, Heberer M, Spagnoli G, Oertli D, Tapia C: Differential pattern and prognostic significance of CD4+, FOXP3+ and IL-17+ tumor infiltrating lymphocytes in ductal and lobular breast cancers. BMC Cancer 2012, 12:134. BioMed Central Full Text
• [10]Shimizu K, Nakata M, Hirami Y, Yukawa T, Maeda A, Tanemoto K: Tumor-infiltrating Foxp3+ regulatory T cells are correlated with cyclooxygenase-2 expression and are associated with recurrence in resected non-small cell lung cancer. J Thorac Oncol 2010, 5:585-590.
• [11]Laghi L, Bianchi P, Miranda E, Balladore E, Pacetti V, Grizzi F, Allavena P, Torri V, Repici A, Santoro A, Mantovani A, Roncalli M, Malesci A: CD3+ cells at the invasive margin of deeply invading (pT3-T4) colorectal cancer and risk of post-surgical metastasis: a longitudinal study. Lancet Oncol 2009, 10:877-884.
• [12]Salama P, Phillips M, Grieu F, Morris M, Zeps N, Joseph D, Platell C, Iacopetta B: Tumor-infiltrating FOXP3+ T regulatory cells show strong prognostic significance in colorectal cancer. J Clin Oncol 2009, 27:186-192.
• [13]Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pagès C, Tosolini M, Camus M, Berger A, Wind P, Zinzindohoué F, Bruneval P, Cugnenc P-H, Trajanoski Z, Fridman W-H, Pagès F: Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 2006, 313:1960-1964.
• [14]Devy L, Blacher S, Grignet-Debrus C, Bajou K, Masson V, Gerard RD, Gils A, Carmeliet G, Carmeliet P, Declerck PJ, Nöel A, Foidart J-M: The pro- or antiangiogenic effect of plasminogen activator inhibitor 1 is dose dependent. FASEB J 2002, 16:147-154.
• [15]André T, Boni C, Mounedji-Boudiaf L, Navarro M, Tabernero J, Hickish T, Topham C, Zaninelli M, Clingan P, Bridgewater J, Tabah-Fisch I, de Gramont A: Oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment for colon cancer. N Engl J Med 2004, 350:2343-2351.
• [16]Schrag D, Somerfield MR, Cohen AM, Figueredo AT, Flynn PJ, Krzyzanowska MK, Maroun J, McAllister P, Van Cutsem E, Brouwers M, Charette M, Haller DG: American Society of Clinical Oncology recommendations on adjuvant chemotherapy for stage II colon cancer. J Clin Oncol 2004, 22:3408-3419.
• [17]Julié C, Trésallet C, Brouquet A, Vallot C, Zimmermann U, Mitry E, Radvanyi F, Rouleau E, Lidereau R, Coulet F, Olschwang S, Frébourg T, Rougier P, Nordlinger B, Laurent-Puig P, Penna C, Boileau C, Franc B, Muti C, Hofmann-Radvanyi H: Identification in daily practice of patients with Lynch syndrome (hereditary nonpolyposis colorectal cancer): revised Bethesda guidelines-based approach versus molecular screening. Am. J. Gastroenterol 2008, 103:2825-2835. quiz 2836.
• [18]Sargent DJ, Wieand HS, Haller DG, Gray R, Benedetti JK, Buyse M, Labianca R, Seitz JF, O’Callaghan CJ, Francini G, Grothey A, O’Connell M, Catalano PJ, Blanke CD, Kerr D, Green E, Wolmark N, Andre T, Goldberg RM, De Gramont A: Disease-free survival versus overall survival as a primary end point for adjuvant colon cancer studies: individual patient data from 20,898 patients on 18 randomized trials. J Clin Oncol 2005, 23:8664-8670.
• [19]Berger AC, Sigurdson ER, LeVoyer T, Hanlon A, Mayer RJ, Macdonald JS, Catalano PJ, Haller DG: Colon cancer survival is associated with decreasing ratio of metastatic to examined lymph nodes. J Clin Oncol 2005, 23:8706-8712.
• [20]Halama N, Michel S, Kloor M, Zoernig I, Benner A, Spille A, Pommerencke T, von Knebel DM, Folprecht G, Luber B, Feyen N, Martens UM, Beckhove P, Gnjatic S, Schirmacher P, Herpel E, Weitz J, Grabe N, Jaeger D: Localization and density of immune cells in the invasive margin of human colorectal cancer liver metastases are prognostic for response to chemotherapy. Cancer Res 2011, 71:5670-5677.
• [21]Halama N, Zoernig I, Spille A, Westphal K, Schirmacher P, Jaeger D, Grabe N: Estimation of immune cell densities in immune cell conglomerates: an approach for high-throughput quantification. PLoS One 2009, 4:e7847.
• [22]Prall F, Dührkop T, Weirich V, Ostwald C, Lenz P, Nizze H, Barten M: Prognostic role of CD8+ tumor-infiltrating lymphocytes in stage III colorectal cancer with and without microsatellite instability. Hum Pathol 2004, 35:808-816.
• [23]Rizzardi AE, Johnson AT, Vogel RI, Pambuccian SE, Henriksen J, Skubitz AP, Metzger GJ, Schmechel SC: Quantitative comparison of immunohistochemical staining measured by digital image analysis versus pathologist visual scoring. Diagn Pathol 2012, 7:42. BioMed Central Full Text
• [24]Parc Y, Gueroult S, Mourra N, Serfaty L, Fléjou J-F, Tiret E, Parc R: Prognostic significance of microsatellite instability determined by immunohistochemical staining of MSH2 and MLH1 in sporadic T3N0M0 colon cancer. Gut 2004, 53:371-375.
• [25]Ribic CM, Sargent DJ, Moore MJ, Thibodeau SN, French AJ, Goldberg RM, Hamilton SR, Laurent-Puig P, Gryfe R, Shepherd LE, Tu D, Redston M, Gallinger S: Tumor microsatellite-instability status as a predictor of benefit from fluorouracil-based adjuvant chemotherapy for colon cancer. N Engl J Med 2003, 349:247-257.
• [26]Michael-Robinson JM, Biemer-Hüttmann A, Purdie DM, Walsh MD, Simms LA, Biden KG, Young JP, Leggett BA, Jass JR, Radford-Smith GL: Tumour infiltrating lymphocytes and apoptosis are independent features in colorectal cancer stratified according to microsatellite instability status. Gut 2001, 48:360-366.
• [27]Naito Y, Saito K, Shiiba K, Ohuchi A, Saigenji K, Nagura H, Ohtani H: CD8+ T cells infiltrated within cancer cell nests as a prognostic factor in human colorectal cancer. Cancer Res 1998, 58:3491-3494.
• [28]Kayser G, Schulte-Uentrop L, Sienel W, Werner M, Fisch P, Passlick B, Zur Hausen A, Stremmel C: Stromal CD4/CD25 positive T-cells are a strong and independent prognostic factor in non-small cell lung cancer patients, especially with adenocarcinomas. Lung Cancer 2012, 76:445-451.
• [29]Coca S, Perez-Piqueras J, Martinez D, Colmenarejo A, Saez MA, Vallejo C, Martos JA, Moreno M: The prognostic significance of intratumoral natural killer cells in patients with colorectal carcinoma. Cancer 1997, 79:2320-2328.
• [30]Ladoire S, Mignot G, Dabakuyo S, Arnould L, Apetoh L, Rébé C, Coudert B, Martin F, Bizollon MH, Vanoli A, Coutant C, Fumoleau P, Bonnetain F, Ghiringhelli F: In situ immune response after neoadjuvant chemotherapy for breast cancer predicts survival. J Pathol 2011, 224:389-400.