SET complex in serous epithelial ovarian cancer V eronique Ouellet 1 ,C ecile Le Page 1 , Marie-Claude Guyot 1 , Christian Lussier 2 , Patricia N. Tonin 3–5 , Diane M. Provencher 1,6,7 and Anne-Marie Mes-Masson 1,7 * 1 Centre de Recherche du Centre Hospitalier de l’Universit  e de Montr eal (CHUM), Institut du cancer de Montr eal, Montreal, Canada 2 Department of Pathology, Centre Hospitalier de l’Universit  e de Montr eal (CHUM), Montreal, Canada 3 Department of Human Genetics, McGill University, Montreal, Canada 4 Research Institute of McGill University Health Centre, Montreal, Canada 5 Department of Medicine, McGill University, Montreal, Canada 6 Division of Gynecologic Oncology, Universit  e de Montr eal, Montreal, Canada 7 Department of Medicine, Universit  e de Montr eal, Montreal, Canada With low cure rates but increasing diverse treatment options that provide variable remission times, ovarian cancer is increasingly being recognized as a chronic disease. This reality indicates the need for a better understanding of factors influencing disease pro- gression. In a previous global analysis of gene expression, we iden- tified genes differentially expressed when comparing serous epi- thelial ovarian tumors of low and high malignant potential (grade 0 vs grade 3). In this analysis, 4 out of 5 members of the SET com- plex, SET, APE1, NM23 and HMGB2, were highly expressed in invasive grade 3 tumors. To further investigate the expression of these genes and the fifth member of the SET complex (pp32), we performed immunohistochemistry, on a tissue array composed of 235 serous tumors of different grades and disease stages. A signifi- cant correlation between expression of all SET complex proteins and the tumor differentiation was observed (p < 0.05). When com- bining all tumors, overexpression of Nm23 (p 5 0.04), Set (p 5 0.004) and Ape1 (p 5 0.004) was associated with the clinical stage of the disease. No marker by itself was associated with prognosis. The combination of a high level of Nm23 in the context of a low level of Set compared to all other combinations of these markers did confer a better prognosis (p 5 0.03). When combined, high expression of Hmgb2 and low expression of Ape1 was also associ- ated with patient prognosis (p 5 0.05). These findings suggest that a strategy that sums the activities of different partners within a pathway may be more appropriate in designing nomograms for patient stratification. ' 2006 Wiley-Liss, Inc. Key words: serous epithelial ovarian cancer; low malignant potential/ borderline tumors; diagnostic and prognositic markers; SET complex; immunohistochemistry Among the gynecologic malignancies, epithelial ovarian cancer (EOC) is infrequent but it is the most lethal mainly because of the absence of symptoms at the earliest stages of the disease. 1 The sur- vival at 5 years of patient with an advanced stage disease reaches only 30%. EOC is a complex disease and tumors can be subdi- vided as low malignant potential (LMP) or borderline and invasive (TOV) tumors. Both types of tumors present multilayer prolifera- tion but TOVs show higher level of cellular atypia than LMP tumors. LMP tumors do not possess the capacity to invade the stroma although microinvasion can be observed. 2–4 In EOC, 4 main histopathology types can be observed (serous, endometrioid, mucinous and clear cell) and the most common type is serous. Ma- lignant tumors of EOC can present different degrees of differentia- tion. LMPs are the most differentiated tumors and referred to as either grade 0 (G0) or B, and TOVs can be well (G1), moderately (G2) or poorly (G3) differentiated. Clinical staging in EOC varies from stage I to IV, where stage I represents disease limited to 1 or both ovaries, stage II is associated with pelvic extension, stage III corresponds to spreading within the abdominal cavity and patients with stage IV tumors present liver or distant metastasis. 5,6 We have previously defined, using Affymetrix gene expression microarray analysis, a molecular signature of serous LMPs and TOVs of G3 either in primary culture or in tumor tissue model system. 7 This signature was established using 3 different statistical methods (Signal-to-noise ratio, Mann-Whitney U test and Signifi- cance Analysis of Microarray) and a subset of genes were vali- dated by quantitative-PCR. 7 Based on this analysis, we found 4 out of the 5 members of the SET complex, composed of NM23 (also known as NME1, NDPKA, GAAD, PP2A), pp32 (PHAP1, LANP, ANP32, I1PP2a), Set (I2PP2A, IGAAD, PHAP2), Hmgb2 (HMG2) and Ape1 (APEX, REF1, HAP1). This complex is impli- cated in apoptosis induced by granzyme-A triggered by immune cells (cytotoxic T lymphocytes or NK cells) 8 that recognize either viral infected or tumor cells. Following lymphocyte attack, the tryptase granzyme-A enters cells using membrane holes created by the perforin and cleaves specific substrates including 3 mem- bers of the SET complex, Set, Hmgb2 and Ape1. The cleavage of these proteins leads to release of the nuclease activity of Nm23 which in turn results in single-stranded cleavage of DNA and cas- pase-independent apoptosis. 9 The complex is also implicated in response to oxidative stress and DNA repair. 10–12 In this report, we focused on protein expression of the SET complex proteins in EOC. For this purpose, we used a tissue array composed of 235 serous tumor samples of different grades to eval- uate protein expression. The staining intensity was related to tu- mor grade, disease stage and patient prognosis in order to evaluate their usefulness in the stratification of EOC tumors. Material and methods Patients and tissue specimens Following appropriate consent, tumor samples were collected through the Division of Gynecologic Oncology at the Centre hos- pitalier de l’Universit e de Montr eal (Ho ˆpital Notre-Dame). An in- dependent pathologist reviewed and scored tumor samples accord- ing to the Federation International of Gynecology and Obstetrics (FIGO) criteria for histopathology, grade and stage of the disease. 6 Clinical data including diagnosis, treatment and clinical outcomes such as disease free interval and survival rate were extracted from the Syste `me d’Archivage des Donn ees en Oncologie (SARDO). For the study, we focused on samples of serous histopathology obtained from chemotherapy na € ıve patients. Serous epithelial ovarian cancer tissue array Using a hematoxilin-eosin stained slide to guide appropriate sam- ple selection, we arrayed 2 cores (0.6 mm diameter) from each tis- sue sample. An experienced pathologist reviewed samples. The se- rous tissue array composed of 56 LMPs, and 179 invasive tumors of G1 (n 5 11), G2 (n 5 53) and G3 (n 5 115) was sectioned, stained with hematoxilin-eosin and received a final pathology review. For Grant sponsor: Canadian Institutes for Health Research; Grant number: MOP-36056. *Correspondence to: CR-CHUM/ICM, 1560, rue Sherbrooke est, Mon- treal, Quebec, Canada H2L 4M1. Fax: 1514-412-7703. E-mail: anne-marie.mes-masson@umontreal.ca Received 6 February 2006; Accepted 28 March 2006 DOI 10.1002/ijc.22054 Published online 5 July 2006 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 119, 2119–2126 (2006) ' 2006 Wiley-Liss, Inc. Publication of the International Union Against Cancer