[CANCER RESEARCH 60, 822– 828, February 15, 2000] Advances in Brief FasL:Fas Ratio–A Prognostic Factor in Breast Carcinomas 1 Toralf Reimer, 2 Christina Herrnring, 2 Dirk Koczan, Dagmar Richter, Bernd Gerber, Dieter Kabelitz, Klaus Friese, and Hans-Ju ¨ rgen Thiesen 3 Department of Obstetrics and Gynecology [T. R., C. H., D. R., B. G., K. F.], Institute of Immunology [D. Ko., H-J. T.], University of Rostock, 18055 Rostock, and Department of Immunology, Paul-Ehrlich-Institute, 63225 Langen [D. Ka.], Germany Abstract Programmed cell death (apoptosis) is primarily mediated by Fas ligand (FasL; CD95L) and the Fas receptor (Fas; CD95). In this study, FasL was detected by immunohistochemical analysis in 85% of breast carcinomas and 14% of fibroadenomas randomly chosen, indicating that high expres- sion of FasL might play a role in tumor pathology. FasL and Fas levels as well as FasL:Fas ratios were further ascertained in 215 human breast tumors, including 199 invasive ductal carcinomas, by real-time quantita- tive reverse transcription-PCR and compared with expression levels and ratios found in 25 normal human tissues, in 37 fibroadenomas, and in 5 normal breast tissues. Among breast carcinomas, high FasL mRNA ex- pression seems to be positively correlated with histological grading (n  212; P < 0.0001). A ratio of FasL:Fas mRNA transcripts >1 is found to be significantly associated with decreased patient’s disease-free survival (n  211; P < 0.03) and increased mortality (n  211; P  0.19). A FasL:Fas ratio >1 is related to tumor progression scored by histological grading (n  212; P < 0.02). The selection process leading to highly aggressive breast tumor variants might be enhanced by FasL-mediated tumor fratricide, eventually a possible target for novel therapeutic strategies. Introduction Breast cancer is the most common cancer of women in many parts of the world. Despite intensive research over the past decades, mor- tality from this disease has hardly decreased (1). Although TILs 4 are capable of infiltrating the tumor, their functional cytotoxic activities seem to be hampered (2, 3). Different mechanisms, for example alterations in the expression of the MHC of the tumor (4), lack of costimulatory molecules like B7.1 (CD 80; Ref. 5), or secretion of soluble tumor-derived inhibitory factors (6), have been considered to play a role in this immune escape of the tumor. Recently, it has been shown that an additional mechanism might play an important role in the immune escape of tumors. A variety of apoptosis-inducing ligands like the tumor necrosis factor, FasL, and the tumor necrosis factor-related apoptosis-inducing ligand have been found to induce apoptosis in target cells that express the correspond- ing receptors. Fas, the receptor of FasL, trimerizes upon activation by FasL and binds to an intracellular death domain containing protein called Fas-associated death domain protein, thereby activating a cas- cade of caspases perpetuating the apoptotic process of cell killing (7). Expression of Fas has been detected in numerous different solid tissues (8) and in the hematopoietic system. Elevated FasL expressions have been found in tumor cells of colon (9), esophageal (10), stomach (11, 12), lung (13), and ovarian cancer (14). In esophageal cancer, areas of the tumor that expressed high FasL levels showed a reduction in the number of TILs and an increased apoptosis of TILs (10). FasL expressed by tumor cells seemed to induce apoptosis in Fas-sensitive TILs. In another study, FasL-positive hepatoma cells displayed reduced Fas expression, in accordance with loss of sensitivity to Fas-mediated apoptosis (15). To assess FasL and Fas expressions in malignant human tissues in comparison with normal human tissues, FasL and Fas levels as well as FasL:Fas ratios were determined in mRNA pools derived from 25 normal human tissues and correlated with results obtained from the analysis of 257 human breast specimens. Absolute copy numbers of FasL and Fas mRNAs were determined by quantitative RT-PCR, and their distribution within the tumor was visualized by immunohisto- chemical analysis. Expression and distribution of FasL and Fas were analyzed in breast carcinoma (n = 215), fibroadenoma (n = 37), and normal breast tissue (n = 5) at the mRNA and partly at the protein level. The present study was designed to address the role of the apoptosis-inducing ligand FasL and its receptor Fas in breast carci- noma as a possible mechanism for the immune escape of the tumor. Materials and Methods Human body screening of Fas and FasL was done by using human total RNAs commercially available from Clontech (Palo Alto, CA). Total RNA samples from 25 different human tissues were analyzed by Fas- and FasL real-time quantitative RT-PCR (TaqMan analysis; see below). The RNA samples were pooled from tissues of the following origins (see the data sheet from Clontech). In brief, RNA of heart came from 2 female Caucasians, ages 16 and 36; pancreas RNA from 18 male/female Caucasians, ages 17– 69; liver RNA from 2 male/female Caucasians, ages 15 and 35; and RNA from cerebellum from a 64-year old male Caucasian (death by acute heart failure). Small intestine RNA was pooled from 11 male/female Caucasians, ages 15– 60; stomach RNA from 15 male/female Caucasians, ages 23– 61; spleen RNA from 5 male Caucasians, ages 22– 48; RNA of bone marrow from 3 male/female Caucasians, ages 24 –59; and fetal brain RNA from 8 male/female spontaneously aborted Cau- casian fetuses, ages 20 –34 weeks. Fetal liver RNA came from spontaneously aborted Caucasian fetuses, ages 15–24 weeks; adrenal gland RNA from 6 male Caucasians, ages 32–50; kidney RNA from 8 male/female Caucasians, ages 24 –55; brain RNA from 2 female Caucasians, ages 16 and 36, and salivary gland RNA from 43 male/female Caucasians, ages 13–78. Lung RNA was pooled from 5 male/female Caucasians, ages 14 – 40; thyroid RNA from 4 male/female Caucasians, ages 10 – 46; trachea RNA from 84 male/female Caucasians, ages 17–70; mammary gland RNA from 8 female Caucasians, ages 23– 47; colon RNA from 2 male Caucasians, ages 35 and 50; prostate RNA from 23 male Caucasians, ages 26 – 64; and spinal cord RNA from 31 male/female Caucasians, ages 17–72. Skeletal muscle RNA came from 10 male/female Caucasians, ages 23–56; thymus RNA from 13 male/female Caucasians, ages 17–37; testis RNA from 25 male Caucasians, ages 28 – 64; and placenta RNA from normal afterbirth of 15 female Caucasians, ages 22– 41. Patients and Tissue Collection. Tissue samples of 215 unselected primary breast carcinomas, 37 fibroadenomas, and 5 normal breast tissues were col- lected during surgery at the Department of Obstetrics and Gynecology of the Received 9/14/99; revised 12/1/99; accepted 1/4/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the general research fund of the Department of Obstetrics and Gyne- cology, University of Rostock (to T. R., C. H., D. R., B. G., and K. F.). 2 T. Reimer and C. Herrnring contributed equally to this work. 3 To whom requests for reprints should be addressed, at Institute of Immunology, Post Office Box 10 08 88, D-18055 Rostock, Germany. 4 The abbreviations used are: TIL, tumor-infiltrating lymphocyte; FasL, Fas ligand; Fas, Fas receptor; RT-PCR, reverse transcription-PCR; CI, confidence interval. 822 Research. on December 11, 2021. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from