ORIGINAL ARTICLE
PROGNOSTIC SIGNIFICANCE OF RB1-INDUCIBLE COILED-COIL 1 IN SALIVARY GLAND CANCERS Hitosuke Tameno, MD,1,2 Tokuhiro Chano, MD, PhD,1 Kaichiro Ikebuchi, MD, PhD,1,2 Yasuko Ochi, MD,1,2 Akihito Arai, MD,1,2 Mitsuo Kishimoto, MD,3 Taketoshi Shimada, MD, PhD,2 Yasuo Hisa, MD, PhD,2 Hidetoshi Okabe, MD, PhD1 1
Department of Clinical Laboratory Medicine, Shiga University of Medical Science, Tsukinowa-cho, Seta, Otsu, Japan. E-mail:
[email protected] 2 Department of Otolaryngology–Head & Neck Surgery, Kyoto Prefectural University of Medicine, Kajiicho, Kamigyo-ku, Kyoto, Japan 3 Department of Pathology, Kyoto Prefectural University of Medicine, Kajiicho, Kamigyo-ku, Kyoto, Japan
Accepted 3 March 2011 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/hed.21797
Abstract: Background. No generally agreed-upon method is available for predicting the prognosis of salivary gland cancers. RB1-inducible coiled-coil 1 (RB1CC1) is a positive regulator for the retinoblastoma tumor suppressor (RB1) pathway, and is a suitable marker for evaluating the clinical course of breast cancer. We investigated whether RB1CC1 predicts the prognosis of salivary gland cancers. Methods. Molecules involved in the RB1CC1 pathway, including RB1CC1, RB1, p53, and Ki-67, were evaluated immunohistochemically in 36 cases of salivary gland cancers. The relationships between clinicopathologic features and disease-free-survival intervals were analyzed by a Kaplan–Meier log-rank test and a multivariate Cox proportional hazard regression. Results. Nuclear RB1CC1 loss in the tumors was significantly associated with a worse disease-free survival (log-rank test, chi-square value ¼ 11.644, p ¼ .0006), and was the maximum critical risk (multivariate Cox proportional hazard ratio ¼ 11.112, 95% confidence interval [CI] ¼ 1.776–69.510, p ¼ .0100). Conclusions. Nuclear expression of RB1CC1 predicts a better clinical outcome and is useful in the follow-up of salivary C 2011 Wiley Periodicals, Inc. Head Neck 00: gland cancers. V 000–000, 2011 Keywords: RB1CC1 (RB1-inducible coiled-coil 1); RB1 (retinoblastoma tumor suppressor); p53 (p53 tumor suppressor); prognosis; salivary gland cancer
Salivary gland tumors vary broadly in their histologic features and their clinical courses, and it is difficult to predict the clinical outcomes of individual
Correspondence to: T. Chano Contract grant sponsor: KAKENHI (Grant-in-Aid for Scientific Research on Priority Areas) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan; contract grant sponsor: Japanese Society of Laboratory Medicine Fund for the Promotion of Scientific Research. The first 2 authors contributed equally to this work. C 2011 Wiley Periodicals, Inc. V
RB1CC1 in Salivary Gland Cancers
cases by evaluation of currently available biomarkers.1,2 Therefore, it is important to find novel biomarkers that might help to predict the postoperative clinical behavior of these tumors. The histologic varieties in salivary gland tumors are related to the characteristic histologic features of the gland. The salivary gland partly resembles the mammary gland, and contains myoepithelium under the acinar and ductal epithelium.3 Myoepithelial differentiation is preserved in a subgroup of salivary carcinomas but is lost from the remaining subgroups of carcinomas during the malignant transformation, similar to the events taking place during the formation of breast carcinomas.3,4 RB1-inducible coiled-coil 1 (RB1CC1: the symbol used here, which is approved by the Human Genome Organization [HUGO] Gene Nomenclature Committee; it is also known as FIP200) was identified as a retinoblastoma tumor suppressor (RB1) pathway regulator that in particular enhances RB1 transcription.5–7 It has been suggested that nuclear expression of RB1CC1 in breast cancer is a predictor of better prognosis.7,8 There are some histologic similarities between salivary and mammary glands, and salivary duct carcinoma pathologically resembles mammary ductal carcinoma.3,4 This resemblance caused us to hypothesize that the expression of RB1CC1 might also be suitable as a marker predicting the prognosis of salivary gland cancers. In the present study, we analyzed RB1CC1 expression immunohistochemically in various salivary cancer tissues, with or without myoepithelial differentiation, and evaluated its prognostic value based on the clinical data of individual cases. MATERIALS AND METHODS
A total of 36 patients diagnosed with operable salivary gland cancers treated at Kyoto
Tissue Samples.
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1
Table 1. Summary of clinicopathologic data on salivary gland carcinomas.
Primary site
Tumor classification (TNM)
Radiotherapy GY
Chemotherapy
Histology
Grade
RB1CC1
75 54 64 52 50 76 63 75 37 5 26 25 75 31 56 52 60 75 62 64 71 13 20 56 40 69 60 72 33 70 77 69 69 87
Parotid gland Parotid gland Parotid gland Parotid gland Submandibular gland Parotid gland Parotid gland Parotid gland Parotid gland Parotid gland Parotid gland Parotid gland Parotid gland Parotid gland Parotid gland Parotid gland Parotid gland Parotid gland Parotid gland Parotid gland Parotid gland Parotid gland Parotid gland Parotid gland Submandibular Parotid gland Parotid gland Parotid gland Parotid gland Parotid gland Parotid gland Parotid gland Parotid gland Parotid gland
T2N1 T1N2b T4N2b T3N0 T4N2b T4N0 T2N2b T1N0 T2N0 T1N0 T3N0 T2N0 T4N0 T3N0 T4N0 T4N0 T3N0 T2N0 T2N0 T2N0 T1N0 T1N0 T4N2b T1N0 T3N2c T2N2b T1N3 T2N0 T1N2b T3N2b T2N2b T3N0 T4N2b T1N0
60 60 60 60 60 — 60 60 40 60 70 — — 60 60 60 60 — 60 60 — — — — 44 — — — 70 60 50 — 60 —
— CBDCA CBDCA — CBDCA — CBDCA — UFT — — — — — CBDCA CBDCA — — CBDCA — — — — — CBCDA UFT UFT — — — — — CBCDA —
High High High High Intermediate Intermediate Intermediate Intermediate Low Low Low Low High High Intermediate Intermediate Intermediate Intermediate Intermediate Intermediate Low Low Low Low High High High High High High High High High High
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
F
67
Parotid gland
T1N0
—
—
Intermediate
þ
M
82
Parotid gland
T1N0
—
—
Mucoepidermoid Mucoepidermoid Mucoepidermoid Mucoepidermoid Mucoepidermoid Mucoepidermoid Mucoepidermoid Mucoepidermoid Mucoepidermoid Mucoepidermoid Mucoepidermoid Mucoepidermoid Adenoid cystic Adenoid cystic Adenoid cystic Adenoid cystic Adenoid cystic Adenoid cystic Adenoid cystic Adenoid cystic Acinic cell Acinic cell Acinic cell Acinic cell Salivary duct Salivary duct Salivary duct Myoepithelial Myoepithelial Adeno NOS Adeno NOS Squamous cell Small cell Carcinoma ex pleomorphic adenoma Epithelial– myoepithelial Mucinous adenocarcinoma
Intermediate
þ
Patient no.
Sex
Age, y
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
M M F F M M M M M M F F M F M M M F F F M M F F M M M F F M M M M M
35 36
Abbreviations: Gy, Gray; RB1CC1, RB1-inducible coiled-coil 1; M, male; F, female; CBDCA, carboplatin; UFT, tegafur-uracil; adeno NOS, adenocarcinoma not otherwise specified.
Prefectural University of Medicine of Japan between March 1997 and November 2006 were included in the present study. Of the 36 cases in this study, 13 were women and 23 were men. The mean age of the patients was 56.1 years (range, 5–87 years). The mean follow-up period was 42 months (range, 1–131 months). All patients were surgically treated before adjuvant radiotherapy and/or chemotherapy. Thirteen, 11, or 2 cases were treated only with surgical operation, radiotherapy, or chemotherapy (tegafururacil [UFT], 400 mg/day/body), respectively. Ten cases received both radiotherapy (40 Gray [Gy]) and chemotherapy (carboplatin [CBDCA], 100 mg/week/ body; or UFT, 400 mg/day/body). Patients with advanced-stage or high-grade cancer principally received adjuvant therapy, although several cases could not tolerate it because of their poor physical
2
RB1CC1 in Salivary Gland Cancers
condition (Table 1). Clinical data were collected by 5 head and neck surgeons (Y.O., A.A., K.I., T.S., and Y.H.). One surgical pathologist (M.K.) diagnosed tumors in accord with the World Health Organization (WHO) criteria.9 The tumor samples were composed of mucoepidermoid carcinomas (12 cases), adenoid cystic carcinomas (8 cases), acinic cell carcinomas (4 cases), salivary duct carcinomas (3 cases), myoepithelial carcinomas (2 cases), adenocarcinomas (not otherwise specified, 2 cases), squamous cell carcinomas (1 case), small cell carcinoma (1 case), carcinoma ex pleomorphic adenoma (1 case), epithelial-myoepithelial carcinoma (1 case), and mucinous adenocarcinoma (1 case). Based on WHO9 and Armed Forces Institute of Pathology (AFIP)10 criteria, 2 pathologists (M.K. and H.O.) classified the tumor grades with a reasonable degree of consensus among pathologists and
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clinicians. Referring to the reports,9–15 mucoepidermoid carcinomas were graded into low, intermediate, and high: adenoid cystic carcinomas with tubular-cribriform and with solid patterns were classified to intermediate and high grades, respectively (Table 1). Before the histologic grading, immunohistochemical data were analyzed by another pathological specialist (H.O.), who did not know the ultimate outcome of the patients. Clinical, pathologic, and immunohistochemical data were independently collected, and all the data were statistically analyzed by 2 investigators (H.T. and T.C.) in a blinded fashion. Data were collected from clinical and pathologic records with the written informed consent of individual patients and after approval by the Ethics Committee of the institute. Immunohistochemistry. Surgical specimens were transferred to 10% buffered formalin and fixed overnight. The fixed samples were embedded in paraffin and serially sliced into 4-lm sections. After dewaxing, sections were autoclaved at 120 C for 1 minute in 10 mM sodium citrate buffer (pH 6.0), and immersed in 0.3% H2O2. They were then incubated overnight at 4 C with primary antibodies to RB1CC1, RB1, p53, and Ki-67. The sections were rinsed with 1 phosphate-buffered saline (PBS) and incubated with the secondary antibody (Simple Stain MAX-PO; Nichirei, Tokyo, Japan) at room temperature for 1 hour. The sections were then colorized with 3.30 -diaminobenzidinetetrahydrochloride and counterstained with hematoxylin. Microscopic Evaluation. The immunohistochemical results for RB1CC1 were quantitatively graded as follows: I, negative staining in both cytoplasm and nuclei; II, positive staining only in cytoplasm; and III, positive staining in nuclei with or without cytoplasmic staining (see Figure 1). Our recent study proved that both RB1 and p16 expressions were significantly higher in cases with nuclear RB1CC1 expression (grade III) than in cases without nuclear RB1CC1 (grades I and II).5,7 Therefore, RB1CC1 staining grades I–II and III were defined as RB1CC1-negative () and -positive (þ), respectively, in the present clinical cohort. An abnormal p53 status was defined as a case of salivary gland cancer in which >50% of tumor cells were strongly positive with anti-p53 antibody (FL393; Santa Cruz Biotechnology, Santa Cruz, CA). Normal status and null status for p53 were differentially assessed by the immunostaining with anti-p53 monoclonal antibody (DO-7; Sigma, St. Louis, MO). Absent RB1 status was defined as a case in which 20% tumor cells showed nuclear expression of the antigen.
RB1CC1 in Salivary Gland Cancers
FIGURE 1. Representative cases of immunohistochemical RB1CC1 analysis in salivary gland carcinomas. RB1-inducible coiled-coil 1 (RB1CC1) expression patterns were graded from I to III. Grade I was defined as negative staining in both cytoplasm and nuclei; II, positive staining only in cytoplasm; III, positive staining in nuclei and/or cytoplasm. RB1CC1 staining grades I–II and III were defined as negative() and positive(þ), respectively. Scale bar represents 50 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Statistical Analysis. The disease-specific survival (DSS) interval was defined as the period from the point of diagnosis to the point of death as the result of salivary gland cancer, and the disease-free survival (DFS) interval was defined as the period until cancer recurrence or metastasis. All statistical analyses were carried out using StatView 5.0 for Windows (SAS Institute, Cary, NC). Statistical significance was defined as a p value of
