Int J Gynecol Cancer 2008, 18, 1090–1096
Mammaglobin B expression in human endometrial cancer R.A. TASSI*, E. BIGNOTTI*, M. FALCHETTIy, S. CALZAz, A. RAVAGGI*, E. ROSSIy, F. MARTINELLI*, E. BANDIERA*, S. PECORELLI* & A.D. SANTIN*§ *Division of Gynecologic Oncology, yDepartment of Pathology, and zSection of Medical Statistics and Biometry, Department of Biomedical Sciences and Biotechnology, University of Brescia, Brescia, Italy; and §Division of Gynecologic Oncology, University of Arkansas for Medical Sciences, Little Rock, Arkansas
Abstract. Tassi RA, Bignotti E, Falchetti M, Calza S, Ravaggi A, Rossi E, Martinelli F, Bandiera E, Pecorelli S, Santin AD. Mammaglobin B expression in human endometrial cancer. Int J Gynecol Cancer 2008;18:1090–1096. Mammaglobin B (MGB-2) is an uteroglobin gene family member recently found highly differentially expressed in ovarian cancer by gene expression profiling. To evaluate its potential as a novel endometrial cancer biomarker, in this study we quantified and compared MGB-2 expression at messenger RNA and protein levels in endometrial tumors (endometrioid endometrial cancer [EEC]) with different grades of differentiation. MGB-2 expression was evaluated by real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC) in fresh frozen biopsies and paraffin-embedded tissues derived from a total of 70 patients including 50 primary EEC and 20 normal endometria (NECs). High levels of MGB-2 gene expression were detected in 10 of 11 EEC G1 cases (91%), 16 of 17 EEC G2 cases (94%), and 6 of 22 EEC G3 cases (27%) by real-time PCR. In contrast, normal endometrial cells expressed low to negligible levels of MGB-2 by real-time PCR (P ¼ 0.002 EEC vs NEC). Well- and moderately differentiated EECs overexpressed MGB-2 gene at significant higher levels when compared to NECs (P , 0.01). Pairwise differences between both G2 and G1 vs G3 cases for MGB-2 relative gene expression values were also statistically significant (G2 vs G3 P , 0.001, G1 vs G3 P ¼ 0.016). MGB-2 protein expression was detected in 31 (86%) of 36 EEC and 0 of 5 atrophic NEC controls, while seven of eight (88%) of the proliferative/secretory/hyperplastic NECs focally expressed MGB-2 by IHC. MGB-2 is highly expressed in EEC, particularly in well- and moderately differentiated tumors, and may represent a novel molecular marker for EEC. KEYWORDS:
differential expression, endometrioid endometrial cancer, mammaglobin B, tumor marker.
Endometrial carcinoma is the fourth most frequent cancer in women and, typically, represents a disease of postmenopausal women. Two subtypes of endometrial carcinoma have been described based on both clinical and histopathologic variables(1). Type I endometrial cancers, which account for the majority of cases, are usually well differentiated and endometrioid in histology(2). These neoplasms are frequently associAddress correspondence and reprint requests to: Alessandro D. Santin, MD, Department of Obstetrics and Gynecology, UAMS Medical Center, University of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, AR 72205-7199, USA. Email:
[email protected] Supported by grants from the Nocivelli and the Camillo Golgi Foundations, Brescia, and by grants from the Istituto Superiore di Sanita` (Progetto N. 527B/2A/1), Rome, Italy, and the Centre for Innovative Diagnostics and Therapeutics (IDET), Brescia, Italy. doi:10.1111/j.1525-1438.2007.01137.x
ated with a history of unopposed estrogen exposure or other hyperestrogenic risk factors, such as obesity. About 80% of type I cancers are diagnosed at an early stage (FIGO stage I), and typically, these patients have a favorable prognosis with appropriate therapy. In contrast, type II endometrial cancers are poorly differentiated tumors, often with serous papillary or clear cell histology, and are not associated with hyperestrogenic factors. These cancers are more likely to be deeply invasive in the myometrium and/or metastatic at presentation and often recur despite aggressive clinical interventions(3). High-throughput technologies for assessing gene expression such as high-density oligonucleotides and complementary DNA microarrays have recently been applied to endometrial cancer. These studies have identified several genes involved in endometrial cancer tumorigenesis and further highlighted gene # 2007, Copyright the Authors Journal compilation # 2007, IGCS and ESGO
MGB-2 expression in human endometrial cancer
pathways differentially expressed between type I and type II endometrial tumors(4). Consistent with this view, several endometrial cancer biomarkers have recently been described to correlate with tumor differentiation, myometrial invasion(5), and immunophenotypic diversity(6). Importantly, using gene expression profiling analysis, our research group has identified mammaglobin B (MGB-2) as the top differentially expressed gene in primary epithelial ovarian cancer (EOC)(7,8). Of interest, in a later study, the endometrioid subtypes of EOC were found to express the highest levels of MGB-2(7,8). Mammaglobin B (SCGB2A1, lipophilin C, and lacryglobin) is a small protein belonging to the uteroglobin gene family whose expression has been associated with secretory mucosal epithelia (mammary(9), lacrimal glands(10), and uterus(9)) and is likely to be hormonally induced by androgens, as found in human prostate(11), pituitary(12), and ocular tissues(10). Although MGB-2 biological function is still unknown, recent reports have found high expression of MGB-2 in multiple human tumors including primary and metastatic adenocarcinomas of the ovary(7,8,13,14), breast(9,15), and biliary tract carcinomas(16). Importantly, MGB-2 secretion has been reported in human tears as a heterodimer of lipophilin A(10), and because of its secretory nature, it is likely to be also found in serum, as reported in breast cancer patients for the homologous mammaglobin (MGB-1)(17). In this study, we have used a combination of immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (qRT-PCR) to evaluate the potential of MGB-2 as a novel diagnostic and/or therapeutic marker in endometrioid endometrial cancer (EEC).
Materials and methods Tissue specimens and clinicopathologic characteristics Tissues were collected from patients treated at the Division of Gynecologic Oncology of the University of Brescia, Italy, following approval from the Institutional Review Board. All patients signed an informed consent according to institutional guidelines. EEC patients ranged from 42 to 88 years of age (mean SD, 68 11 years), and none of them had received radiation or chemotherapy treatment before surgery. Tumoral stage, grade, and histology were assessed according to FIGO system. All the tumors analyzed in this study were of endometrioid histology and were grouped according to their differentiation grade (Table 1). Flash-frozen tissues were sharply dissected and snap-frozen in liquid nitrogen within 30 min from #
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Table 1. Characteristics of EEC patients from whom tissue biopsies were obtained Histologic grade Variables Surgical stage I II III IV
G1
G2
G3
10 1
13 1 2 1
7 6 8 1
resection and stored at 220°C in RNAlater (Ambion, Austin, TX) before RNA extraction. Tissue fragments from 67 primary EECs were split for histologic confirmation and RNA isolation. Briefly, tumor tissues were embedded in optimal cutting temperature medium and dissected, and the frozen sections were stained with hematoxylin and eosin to analyze microscopically the percentage of benign endometrium, myometrium, or tumor present in the tissues. Only tissue samples containing more than 70% epithelial tumor cells were retained for the study. Fifty cases of EEC met these eligibility criteria (Table 1). In addition, 20 samples of normal endometria (NECs) were obtained from patients (age range, 37–79 years; mean SD, 55 11) undergoing hysterectomy for benign gynecological pathologies. Endometrial specimens were macrodissected to further minimize myometrial contamination and were histologically examined. Total RNA extraction and reverse transcription Total RNA was obtained from 70 samples including 50 primary EECs with different histologic grades (Table 1) and 20 NECs (secretory, n ¼ 5; proliferative, n ¼ 3; dysfunctional, n ¼ 2; atrophic, n ¼ 10). Frozen tissues were sharply dissected and homogenized with a rotary homogenizer (QIAGEN, Valencia, CA). Total RNA was extracted using TRIzol (Invitrogen Life Technologies, Carlsbad, CA) and was purified using the RNeasy clean-up kit (QIAGEN) following the manufacturer’s protocol. RNA purity and quantity were evaluated spectrophotometrically, while the RNA integrity was tested on Agilent 2100 Bioanalyzer. In total, 500 ng of total RNA were reverse transcribed using random hexamers in a final volume of 20 lL according to the SuperScript II RT RNaseH-Reverse Transcriptase protocol (Invitrogen Life Technologies). Quantitative RT-PCR qRT-PCR experiments were performed in duplicate and repeated a minimum of two times by using primer set and probe specific for MGB-2 gene. All the reactions
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were carried out on the ABI PRISM 7000 Sequence Detection System Instrument and software (Applied Biosystems, Applera, UK, and Cheshire, UK) using the TaqMan Universal PCR master Mix and the following Assay on Demand (Applied Biosystems): Hs 00267180_m1 (MGB-2). The endogenous control, glyceraldehyde-3-phosphate dehydrogenase, Hs 99999905_m1 (Applied Biosystems), was used to normalize variations in complementary DNA quantities from different samples. TaqMan reactions for target and internal control genes were performed in separate tubes. The comparative threshold cycle (CT) method was used for the calculation of amplification fold as specified by the manufacturer. TaqMan Gene Expression Assays used in this study span an exon–exon junction, eliminating the possibility of amplifying genomic DNA. One microliter of the reverse transcription volume was used for each PCR reaction in a total volume of 25 lL. The thermal cycling conditions were the following: 10 min at 95°C, 40 cycles of denaturation at 95°C for 15 sec, and annealing–extension at 60°C for 1 min. Mean CT levels of each sample were calculated and used for further analyses. Results were normalized against glyceraldehyde-3-phosphate dehydrogenase and expressed in relation to a calibrator sample to which a relative expression value of 1 was given. Results per PCR reaction were expressed as relative quantification using the delta–delta CT method. IHC on formalin-fixed tissues To evaluate MGB-2 protein expression level, immunohistochemical staining was performed on 49 samples (ie, 36 EECs, 13 NECs) stored in the Department of Pathology at the University of Brescia, Italy. Formalinfixed, paraffin-embedded tissues were cut and stained with hematoxylin and eosin and analyzed by a staff surgical pathologist. Briefly, formalin-fixed, paraffinembedded tissues were cut at 2 lm, mounted on charged slide, and dried. For immunohistochemical analysis, slides were deparaffinized and rehydrated in graded solutions of ethanol and distilled water. Endogenous peroxidase was blocked by incubation with peroxidase-blocking solution (DAKO ChemMate, Carpinteria, CA) for 15 min, followed by rinsing in Tris-buffered saline. Nonspecific staining was blocked by treatment with normal goat serum (1:50) for 5 min. The immunohistochemical method involved sequential application of primary antibody to mammaglobin diluted 1:50 (mammaglobin [clone 31A5] Rabbit Monoclonal Antibody; Zeta Corporation, Sierra Madre, CA) for 45 min, a secondary biotinylated anti-rabbit antibody diluted 1:20 (Menarini, Florence, Italy) for #
15 min, and streptavidin–biotin complex diluted 1:20 (Reagent kit, Menarini) for 15 min. The immunoprecipitate was visualized by treatment with 393-diaminobenzidine (Bio-Optica, Milan, Italy) for 5 min and counterstained by hematoxylin (DAKO). Immunostaining was considered positive for MGB-2 when at least 10% of neoplastic cells were stained. All samples were scored quantitatively and qualitatively in 20 and 40 high-power fields in every section (Eclipse E400; Nikon, Tokyo, Japan). The intensities of MGB-2 expression were blindly scored by three independent pathologists from 0 to 3, with grade 0 indicating no staining, grade 1 weak staining, grade 2 moderate staining, and grade 3 strong staining. Immunostaining extent was also evaluated according to the proportion of cells stained for the antigen in a section and was given a score from 0 to 4, with 0 indicating no positive cell; 1, 1–10% positive cells; 2, 10–50% positive cells; 3, 50–75% positive cells; and 4, .75% positive cells. Statistical analysis Differences in qRT-PCR and IHC values among groups were tested either with a Kruskal–Wallis test or a Mann–Whitney test. Pairwise differences for qRTPCR and IHC values were tested by means of the Behrens–Fisher nonparametrical multiple tests procedure. In all the analyses, a P value was considered significant if less than 0.05. The correlations between MGB-2 expressions measured by qRT-PCR and IHC staining were tested by means of the polyserial correlation coefficient. The polyserial correlation coefficient was computed with the maximum likelihood method, and the P value was calculated with a Wald test.
Results Mammaglobin B gene expression in ovarian cancer tissues Mammaglobin B gene expression was tested by qRTPCR in 50 primary EECs (22 poorly differentiated [G3], 17 moderately differentiated [G2], and 11 well differentiated [G1]) and 20 NECs. Results are shown in Figure 1 and in Table 2. The optimal cutoff point for discriminating normal tissue from EEC, for MGB-2 messenger RNA expression, was determined by means of a receiver operating characteristic curve and was set at relative quantification ¼ 15,722. Estimated qRT-PCR sensitivity was 64% with a 100% specificity (error rate ¼ 0.174, area under the curve ¼ 74.4%). According to this threshold, all the NECs were classified as negative, while 10 of 11 G1 cases (91%), 16 of 17 G2 cases (94%),
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Figure 1. Mammaglobin B expression in well- (G1), moderately- (G2), and poorly differentiated (G3) EECs compared to NECs. The figure shows the box plot of the relative quantification values in natural logarithm (ln) scale. Box plots depict the median (bold line within the box) and the first and the third quartiles (box length, interquartile range). Vertical lines from the box (whiskers) extend to the smallest and the largest observations.
and 6 of 22 G3 cases (27%) were classified as positive. A significantly higher MGB-2 gene expression was found in ECCs compared to NECs (Mann–Whitney test, P ¼ 0.002). Well- and moderately differentiated EECs overexpressed MGB-2 gene at significant higher levels when compared to NECs (G1 EEC vs normal endometrium, P , 0.01; G2 EEC vs normal endometrium, P , 0.01). Pairwise differences between both G2 and G1 vs G3 cases for MGB-2 relative gene expression values were statistically significant (G2 vs G3 P , 0.001, G1 vs G3 P ¼ 0.016). Immunohistochemical staining for mammaglobin B IHC for mammaglobin B was performed on 36 primary EECs (8 G1 cases, 17 G2 cases, and 11 G3 cases) and 13 NECs (5 atrophic, 3 proliferative, 3 secretory, and 2 hyperplastic). MGB-2 immunoreactivity was detected in 31 (86%) of 36 uterine cancers and in 8 of 13
(62%) NECs (Table 2). All five normal atrophic endometria resulted negative at MGB-2 immunostaining (Fig. 2A), while seven of eight (88%) active endometria focally expressed the protein (Table 3) in the cytoplasm of epithelial cells (Fig. 2B, C). As shown in Figure 2, reactive stromal cells surrounding epithelium were found negative both in pathologic and normal samples analyzed. The intensity of staining and the proportion of positive cells were significantly higher in well- and moderately differentiated ECCs compared to NECs (Kruskal–Wallis test, P , 0.001). All the G1 cases were positive, and most of them (5 of 8, 63%) showed diffuse (.50% stained cells) cytoplasmic staining (Table 4) with strong intensity (Table 3; Fig. 2D). The majority of G2 cases (16 of 17, 94%) were also found positive for MGB-2 and showed a staining extent ranging from focal (,10% stained cells) to patchy (10–50% stained cells) (Table 4) and an intensity score ranging from weak to strong (Table 3; Fig. 2E). Some G3 tumors (4 of 11, 36%) were found negative,
Table 2. qRT-PCR assay, IHC assay results and polyserial correlation between the two techniques Relative quantification
IHC
Tissues
No. of tested
Median
IQR
No. of pos (%)
No. of tested
No. of posa (%)
rb (P value)
NECs EECs Grade 1 Grade 2 Grade 3
20 50 11 17 22
4.00 3 103 2.87 3 104 4.24 3 104 4.87 3 104 2.53 3 103
6.83 3 103 5.31 3 104 2.97 3 104 8.00 3 104 1.74 3 104
0 (0) 32 (64) 10 (91) 16 (94) 6 (27)
13 36 8 17 11
8 (62) 31 (86) 8 (100) 16 (94) 7 (64)
0.21 (0.51) 0.54 (,0.001)
IQR, interquartile range; pos, positive. The percentage of positivity in qRT-PCR and IHC is referred to the cutoff used (relative quantification ¼ 15,722; staining presence in more than 10% of the tumoral cellular population). b The polyserial correlation coefficient (r) was computed with the maximum likelihood method, and the P value was calculated with a Wald test. a
#
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Figure 2. Immunohistochemical staining for mammaglobin B in EECs and NECs. A) Atrophic endometrium: please note the absence of mammaglobin B expression in both the epithelial and the stromal components. B) Hyperplastic endometrium: area of simple glandular hyperplasia— cytoplasmatic overexpression of mammaglobin B in the epithelium. C) Secretory phase endometrium: moderate expression of mammaglobin B in the cytoplasm of the epithelial cells. D) Grade 1 endometrioid adenocarcinoma: strong and diffuse cytoplasmatic expression of mammaglobin B. E) Grade 2 endometrioid adenocarcinoma: diffuse cytoplasmatic expression of mammaglobin B of moderate intensity. F) Grade 3 endometrioid adenocarcinoma: focal cytoplasmatic expression of mammaglobin B with strong intensity.
whereas in positive cases (7 of 11, 64%), the protein was localized in few neoplastic areas (Table 4), and weak or intense immunoreactivity was observed in the same percentage of cases (Table 3; Fig. 2F). MGB-2 stain areas were significantly greater in well-differentiated tumors compared to poorly differentiated ones (Kruskal–Wallis test, P , 0.001) (Table 4). In secretory phase endometrium, MGB-2 positivity was exclusive in the secretory epithelium and the protein appeared to be secreted in gland lumens, although immunostaining extent was restricted to few glands (Fig. 2C). A similar MGB-2 expression pattern was observed in pseudostratified cells lining the tubular glands of proliferative endometrium (data not shown). MGB-2 IHC of hyperplastic endometria showed a strong staining intensity, but signal was limited again to a limited number of glands (Fig. 2B). A significant correlation was found between IHC and qRT-PCR data for endometrial cancers overall (r ¼ 0.54, #
P , 0.001), while no significant correlation was found for NECs (r ¼ 0.21, P ¼ 0.51). Finally, protein expression and tumor grade were significantly and negatively correlated (r ¼ 20.52, P ¼ 0.001).
Discussion Endometrial cancer is the most common gynecological malignancy in Western countries and United States(18). The majority of cases are endometrioid in histology and display an association to excessive estrogen exposure in endogenous or pharmacologic form(1). Although advances in genomics have led to the identification of several biomarkers involved in endometrial pathogenesis, there is still a lack of reliable cancerspecific biomarkers for EEC. In this regard, although it has been previously reported that the elevation of CA125 level may be associated with an increase in the incidence of metastatic disease in endometrial tumors
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Table 3. Mammaglobin B immunoreactive staining in EECs and NECs Staining scorea, n (%) Tissue
No. of tested
0
1
2
NECs Atrophic Secretory Proliferative Hyperplastic EECs Grade 1 Grade 2 Grade 3
13 5 3 3 2 36 8 17 11
5 (38.5) 4 (80.0)
5 (38.5) 1 (20.0) 2 (66.7) 2 (66.7)
2 (15.4)
1 (33.3) 5 (13.9)
8 (22.2) 1 (12.5) 4 (23.5) 3 (27.3)
1 (5.9) 4 (36.4)
3 1 (7.6)
1 (33.3) 1 (50.0) 9 (25.0) 3 (37.5) 5 (29.4) 1 (9.0)
1 (50.0) 14 (38.9) 4 (50.0) 7 (41.2) 3 (27.3)
a
The intensity of immunopositive staining of tissue sections with anti-mammaglobin B antibody was rated as follows: 0, no staining; 1, weak staining; 2, intermediate staining; and 3, strong staining.
with endometrioid histology(19,20), this marker appears to have limited utility in monitoring the effects of adjuvant therapy. Moreover, CA125 does not provide adequate diagnostic accuracy for detecting early stage disease(21). Recently, our research group has identified mammaglobin B as a novel promising biomarker for EOC. Because MGB-2 is a small secreted protein of the uteroglobin polypeptide family(9), and in our previous studies, the highest levels of MGB-2 expression in ovarian cancer, both at the gene and protein levels(8), were detected in endometrioid ovarian tumors, in the present report we have evaluated the potential of MGB-2 expression as novel biomarkers in EEC. Our results demonstrated a significant upregulation of MGB-2 gene and protein expressions in EECs when compared to normal tissues by RT-PCR and IHC staining. Indeed, significant MGB-2 gene overexpression was found in well- and moderately differentiated tumors when compared to NECs, while a significant downregulation of MGB-2 transcript was found in grade 3 EECs compared to G1 and G2 tumors. Taken together, these data seem to lead toward the conclusion that, similar to mammaglobin-1 expression in
breast cancer patients(22,23), high levels of MGB-2 transcript may correlate with the higher histologic differentiation of the EEC. This hypothesis is supported by the fact that all the neoplastic specimens tested exhibited a similar pattern of MGB-2 expression both at the gene and protein levels, as suggested by the correlation found between qRT-PCR and IHC results. All atrophic NECs tested were found negative for MGB-2 expression, while a focal distribution of the protein and a slight or intermediate immunoreactivity (scores 11 and 21) in the majority of proliferative/secretory or hyperplastic NECs were observed. Of interest, in these samples, MGB-2 protein expression was restricted to the functional layer of the normal endometrium, whereas the abundant basal layer below was always found negative. Indeed, MGB-2 was localized in the cytoplasm of the epithelial cells lining the tubular glands of endometria in proliferative or secretory phase. Hence, MGB-2 expression is likely to be hormonally regulated in endometrium similar to its regulation in the prostate in males(11). In conclusion, we have demonstrated that MGB-2 is differentially expressed between EEC and normal endometrium at messenger RNA and protein levels
Table 4. Mammaglobin B staining extent in EECs and NECs Staining extent (% stained cells), n (%) Tissue
No. of tested
0%
1–10%
NECs Atrophic Secretory Proliferative Hyperplastic EECs Grade 1 Grade 2 Grade 3
13 5 3 3 2 36 8 17 11
5 (38.5) 4 (80.0)
7 (53.8) 1 (20.0) 3 (100.0) 2 (66.7) 1 (50.0) 13 (36.1) 1 (12.5) 8 (47.0) 4 (36.4)
1 (33.3) 5 (13.9) 1 (5.9) 4 (36.4) #
10–50%
50–75%
75–100%
1 (7.7)
11 (30.6) 2 (25.0) 6 (35.3) 3 (27.2)
1 (50.0) 5 (13.9) 4 (50.0) 1 (5.9)
2 (5.5) 1 (12.5) 1 (5.9)
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and that MGB-2 is expressed at high levels in the majority of EEC. These results, combined with the recent demonstration that recognition of HLAA2-restricted mammaglobin-A epitopes by CD81 cytotoxic T lymphocytes in breast cancer patients is indeed possible(24), may support the use of MGB-2 as a novel therapeutic target in EEC patients harboring recurrent/metastatic disease refractory to standard treatment modalities. More importantly, since mammaglobin B is a secreted protein with limited tissue distribution, its identification in endometrial cancer patients’ serum after surgery may prove useful for early detection of persistent/recurrent disease. To validate this hypothesis, an enzyme-linked immunosorbent assay detection kit for quantification of mammaglobin B in the circulation of EEC patients is under development in our laboratory.
8 9 10
11
12
13 14 15
Acknowledgments We wish to thank Prof. Piergiovanni Grigolato, Prof. Fabio Facchetti, Dr. Carla Donzelli, Chiara Romani, MS, Mrs. Anna Galletti, and Mrs. Lucia Fontana for their excellent technical support to the project. In addition, we wish to thank Prof. Vanio Vannini and Prof. Cesare Danesino, tutors of Dr. E.R. in her PhD program in pathology and genetics.
16 17 18 19 20
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Accepted for publication August 19, 2007
2007 IGCS and ESGO, International Journal of Gynecological Cancer 18, 1090–1096
