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Cancer. Author manuscript; available in PMC 2011 Feb 15.
Published in final edited form as:
PMCID: PMC2819580
NIHMSID: NIHMS158487

Serum Amyloid A (SAA): a Novel Biomarker for Endometrial Cancer

Abstract

Background

We investigated the expression of Serum-Amyloid-A (SAA) in endometrial endometrioid carcinoma (EEC), and evaluated its potential as a serum biomarker.

Methods

SAA gene and protein expression levels were evaluated in EEC and normal endometrial tissues (NEC), by real time-PCR, immunohistochemistry (IHC) and flow cytometry. SAA concentration in 194 serum samples from 50 healthy-women, 42 women with benign diseases and 102 patients including 49 grade-1, 38 grade-2 and 15 grade-3 EEC was also studied by a sensitive bead-based-immunoassay.

Results

SAA gene expression levels were significantly higher in EEC when compared to NEC (mean-copy-number by RT-PCR = 182 vs 1.9; P=0.001). IHC revealed diffuse cytoplasmic SAA protein staining in poorly differentiated EEC tissues. High intracellular levels of SAA were identified in primary EEC cell lines evaluated by flow cytometry and SAA was found to be actively secreted in vitro. SAA concentrations (μg/ml) had medians of 6.0 in normal healthy females and 6.0 in patients with benign disease (P=0.92). In contrast, SAA values in the serum of EEC patients had a median of 23.7 significantly higher than those of the healthy group (P=0.001) and benign group (P=0.001). Patients harboring G3 EEC were found to have SAA concentrations significantly higher than G1/G2 patients.

Conclusions

SAA is not only a liver-secreted-protein but is also an EEC-cell product. SAA is expressed and actively secreted by G3-EEC and it is present in high concentration in the serum of EEC patients. SAA may represent a novel biomarker for EEC to monitor disease recurrence and response to therapy.

Keywords: Endometrial carcinoma, Serum Amyloid A, Biomarkers, Tumor markers

INTRODUCTION

Cancer of the uterine corpus is the most prevalent gynecologic tumor in women, with an estimated 40,100 cases and 7470 deaths in the United States in 2008 (1). Based on both clinical and histopathological variables, two subtypes of endometrial carcinoma (EC), namely Type I and Type II tumors, have been described (2). Type I ECs, which account for the majority of cases, are estrogen-related tumors usually well differentiated and endometrioid in histology. Typically these patients have a favorable prognosis with appropriate therapy. In contrast, Type II ECs include poorly differentiated endometrioid tumors (G3-EEC), serous papillary and clear cells ECs. These tumors are not associated with hyperestrogenic factors and they are more likely to have deep myometrial invasion and/or metastases at presentation and often recur despite aggressive clinical interventions (3). G3-EEC accounts for the majority of Type II ECs and unfortunately, to date, no good marker for screening or disease monitoring for these biologically aggressive cancers is available. In this regard, CA125 is often used in clinical practice to monitor EC patients (4). However, this marker appears to have limited utility in monitoring the effects of adjuvant therapy or in the prediction of tumor recurrence (5). The discovery of novel diagnostic and therapeutic markers against this aggressive subset of endometrial cancers remains a high priority.

Human serum Amyloid A (SAA), is an HDL-associated lipoprotein known to play a major role as a modulator of inflammation and in the metabolism and transport of cholesterol (6). Of interest, SAA has been recently proposed as a potentially useful biomarker to monitor patients harboring human tumors including gastric and nasopharyngeal cancer (7,8). Moreover, in lung cancer patients, using mass spectrometry and proteomic technologies, SAA was identified as the top differentially expressed protein able to differentiate the serum of patients from the serum of healthy individuals (9). One major problem with the use of SAA, an acute phase reactant, as a potential serum marker in human cancer patients, is the fact that its elevation in the serum of patients is suggested to be of liver origin rather than a tumor-cell product (10). Indeed, SAA level in the blood may elevate up to 1000-fold in response of the body to various injuries including trauma and various inflammations in addition to neoplasia (10). Importantly, however, extrahepatic SAA expression has been previously demonstrated in several histologically normal tissues, predominantly by their epithelium (11,12). Unfortunately, only scant information regarding SAA expression in malignant human tissues has been so far reported and, to our knowledge, no studies have yet addressed a potential direct secretion of SAA by human endometrial tumors. This report represents the first investigation examining SAA expression and secretion in human endometrial carcinoma.

PATIENTS AND METHODS

Primary Tumors

Snap frozen tumor biopsies and tumor samples were derived from primary specimens staged according to the F.I.G.O. (1988) surgical staging system. Only specimens with > 75% tumor content were used in the RT-PCR experiments. Briefly, fresh tumor biopsies from 18 EEC [obtained from 5 FIGO grade 1 (G1), 10 FIGO grade 2 (G2) and 3 FIGO grade 3 (G3) EEC patients (age 63± 9: mean ± SD)] were obtained under approval of the Institutional Review Board at the time of surgery and analyzed for SAA expression. Patients from which fresh tumor biopsies were obtained included 9 stage I, 5 stage II and 4 stage III patients. Total abdominal hysterectomy, bilateral salpingo-oophorectomy, lymph node dissection and washings were performed in all endometrial cancer patients. Normal endometrial control cell samples (NEC) were obtained from biopsies of benign hysterectomy specimens obtained from women of similar age. Three primary EEC cell lines derived from patients harboring G2/G3 endometrioid carcinomas (i.e., EEC-ARK-1, EEC-ARK-2 and EEC-ARK-3), were also established as short term cultures following previously reported standard tissue culture techniques (13). Briefly, tumor tissues obtained from cancer patients were mechanically minced and enzymatically dissociated with 0.14% collagenase Type I (Sigma, St. Louis, MO) and 0.01% DNAse (Sigma, 2000 KU/mg) in RPMI 1640 media, as described previously by Santin et al, (13). After 1–2 hrs incubation with enzyme on a magnetic stirring apparatus at 37°C in an atmosphere of 5% CO2, the resulting suspension was collected by centrifugation at 100 g for 5–10 minutes and washed twice with RPMI 1640 medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS, Gemini, Woodland, CA). The final pellet was then placed in RPMI 1640 (Invitrogen) containing 10% FBS, 200 U/ml penicillin, and 200 μg/ml streptomycin in tissue culture flasks or Petri dishes (Corning, Acton, MA). The epithelial nature and the purity of EEC cultures was verified by immunohistochemical staining and flow cytometric analysis with antibodies against cytokeratin and vimentin as previously described (13). Only primary cultures which had at least 90% viability and contained >99% epithelial cells were used for SAA quantification by a sensitive bead-based immunoassay, as described below.

RNA extraction and quantitative real-time PCR

RNA isolation from all primary snap frozen samples including eighteen EEC as well as three normal endometrial cell controls was performed using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. Quantitative PCR was done with a 7500 Real Time PCR System using the manufacturer’s recommended protocol (Applied Biosystems)to evaluate expression SAA in all the samples. Each reaction was run in triplicate. Briefly, 5 μg of total RNA from each sample was reverse transcribed using SuperScript III first-strand cDNA synthesis (Invitrogen). Five microliters of reverse transcribed RNA samples (from 500 μL of total volume) were amplified by using the TaqMan Universal PCR Master Mix (Applied Biosystems) to produce PCR products specific for SAA. The primers for SAA were obtained from Applied Biosystems (Assay ID Hs00761940_s1). The comparative threshold cycle (CT) method (Applied Biosystems) was used to determine gene expression in each sample relative to the value observed in the lowest nonmalignant endometrial epithelial cell sample, using glyceraldehyde-3-phosphatedehydrogenase (Assay ID Hs99999905_m1) RNA as internal controls.

Intracellular Flow cytometry

The mouse anti-human anti-SAA monoclonal antibody (i.e., clone mcl, DAKO Corporation; Carpinteria, CA), was used for our flow cytometry study. Briefly, freshly established EEC cell lines and control cells were fixed with 2% paraformaldehyde in PBS, washed and permeabilized by incubation in PBS plus 1% BSA and 0.5% saponin (S-7900, Sigma) for 10 min at room temperature. Tumor cells were stained with anti-SAA MAb and isotype-matched controls (DAKO Corporation; Carpinteria, CA). After staining, cells were washed twice with PBS plus 0.5% BSA. Secondary goat-anti-mouse antibody (IgG1-FITC, cat# 349031, Beckton Dickinson) was then added for 30 min at 4 °C. Cells were then washed twice with PBS plus 0.5% BSA. Analysis was conducted with a FACScalibur utilizing CellQuest software (Beckton Dickinson).

SAA Immunostaining of Formalin-fixed Tumor Tissues

Formalin-fixed paraffin-embedded normal endometrial control tissues and tumor tissues were evaluated by standard immunohistochemical staining for SAA expression. Study blocks (i.e., 10 samples including 5 EEC and 5 NEC) were selected after histopathologic review by a surgical pathologist. The most representative block was selected for each specimen. Briefly, deparaffinized and rehydrated sections were treated according to the manufacturer’s instructions (DAKO). The antibody was diluted 1:20 in DAKO Antibody Diluent (DAKO Corporation) and incubated at pH 9.0 for 1/2 hr at room temperature. DAKO Envision system was used for secondary detection and color was developed using DAB chromogen (DAKO) for 5 min followed by counterstaining with hematoxylin. The anti-SAA monoclonal antibody used (i.e., clone mcl, DAKO Corporation; Carpinteria, CA), was directed against AA-amyloid fibril protein. The preparation and specificity of this antibody has been previously described and demonstrated (14). Negative controls included replacement of the primary antibodies by PBS and by normal mouse isotype matched IgG (IgG2a, kappa; DAKO Corporation). Liver sections were used as positive controls.

Analysis of SAA secretion in tumor samples

To evaluate the potential secretion of SAA by primary EEC, supernatants obtained from EEC-ARK-1, EEC-ARK-2 and EEC-ARK-3 as well as multiple control cell lines including normal human fibroblasts, EBV-transformed B cells (LCL) and cervical carcinoma cell lines were evaluated by a sensitive bead-based immunoassay (Millipore, Corp. Danvers, MA). Briefly, tumor supernatants to be tested for SAA secretion were collected by primary tumor cell lines seeded at a density of 1 × 105 cells/ml in tissue culture Petri dishes (Corning) in RPMI 1640 media, supplemented with 10% FBS (i.e., EEC and human fibroblasts), or serum-free keratinocyte medium (KFSM, i.e., cervical cancer cell lines). After 72 hrs incubation at 37°C, supernatants were aspirated, rendered cell-free by centrifugation at 1,500 rpm for 10 minutes, and stored at −20°C before being analyzed for SAA by a bead-based immunoassay (see below).

Measurement of SAA concentration in serum samples

SAA concentration was quantified in the serum of 50 apparently healthy women, 42 women with benign diseases (i.e., 22 uterine fibroids, 8 ovarian cysts and 12 endometrial polyps), and 102 women with histologically proven primary EEC, by a commercially available bead-based immunoassay (Lincoplex kit, acute phase proteins, Millipore, Corp. Danvers, MA). Patient characteristics are described in Table 1. All samples tested were derived from patients who had provided written informed consent and the study was approved by the local institutional review boards. In brief, the assay is based on conventional sandwich assay technology. The antibody specific to SAA is covalently coupled to Luminex microspheres. The microspheres are incubated with standards, controls, and samples (25 μl) in a 96-wellmicrotiter filter plate for 1 h at room temperature. After incubation, the plate is washed to remove excess reagents, and detection antibody is added. After 30-min incubation at room temperature, streptavidin-phycoerythrin is added for an additional 30 min. After a final wash, the beads are resuspended in buffer and read on a Bio-Rad Luminex100 Instrument to determine the concentration of SAA. All specimens were tested in replicate wells. Results are reported as the mean of the replicates. Serum samples from all patients were collected before surgery and stored at −80°C until analysis. Detailed information about this assay is available at: http://www.millipore.com/userguides.nsf/dda0cb48c91c0fb6852567430063b5d6/018e9e0e26525c1185257259004e227e/$FILE/hcvd2-67bk.pdf.

Table 1
Characteristics of the patients from whom serum samples were obtained

Statistical Analysis

For q-RT-PCR data, the right-skewing was removed by taking copy-number ratios relative to the lowest-expressing NEC sample (“relative copy numbers”), log2-transforming them to ΔCTs, and comparing the results via unequal-variance t-test for the EEC-versus-NEC difference. The analyses of differences among supernatants obtained from tumor cultures with different histologies, and among expression levels measured by flow cytometry and IHC, were performed using the Wilcoxon-Mann-Whitney (WMW) test. SAA serum concentrations among the different groups of patients (i.e., healthy controls, benign gynecologic diseases, and EEC with different degree of differentiation) were summarized as medians and ranges, and compared for pairwise differences via the WMW test. SPSS 15 (SPSS Inc., Chicago, IL) was used for statistical analysis. A 5% significance level was used for all statistical comparisons.

RESULTS

SAA Expression in Snap Frozen EEC by Quantitative Real-Time PCR

To minimize the risk of contamination of EEC RNA with that of normal cells or endometrial tumor cells with different histology (i.e., clear cells or uterine serous tumors), we extracted RNA to be evaluated for SAA expression by RT-PCR from eighteen pure EEC containing a minimum of 75% tumor cells. A comparison of the q-RT-PCR data for SAA in EEC versus NEC as controls is shown in Figure 1. Significant expression differences between EEC and NEC were readily apparent (Figure 1). Relative copy numbers in NEC control samples had a mean ± SEM of 1.9 ± 0.6 and ranged from 1.0 to 3.1. By contrast, relative copies in EEC samples had a mean ± SEM of 182 ± 69 and ranged from 1.7 to 1090. The fold change in mean relative copy numbers was 95.8 (Figure 1; P=0.01).

Figure 1
SAA mRNA copy number by quantitative RT-PCR in 3 normal endometrial control cell samples (NEC) and 18 EEC snap frozen biopsies. The vertical axis represents the relative number of copies compared to the lowest-expressing NEC (value of 1).

Intracellular EEC expression in EEC cell lines by Flow cytometry

To determine whether the high expression of SAA gene detected by q-RT-PCR assays in flash frozen EEC also results in high expression of the SAA protein, we performed intracellular flow-cytometry analysis of SAA protein expression in 3 primary EEC established as short-term cultures in vitro in our laboratory. As shown in Table 2, all 3 primary EEC-culture cell lines were found positive for intracellular SAA expression by flow cytometry (i.e., 100% positive cells; mean fluorescence intensity [MFI] range from 26 to 65) (Table 2). In contrast, significantly lower expression of SAA was detected in Epstein-Barr transformed B cells (LCL) and cervical cancer cell lines (CVX) used as controls (i.e., 65–71% positive cells; MFI range from 11 to 20 and 69–84% positive cells; MFI range from 9 to 15, respectively) by flow cytometry (P=0.03 for both EEC vs LCL and EEC vs CVX, Table 2).

Table 2
Intracellular SAA expression in EEC and control cell lines by flow cytometry

SAA Expression by Immunohistochemistry in EEC

To evaluate whether the high SAA expression detected by flow cytometry on primary EEC cell lines was comparable to the expression of SAA of the EEC specimens from which EEC primary tumor cell lines were derived and/or whether in vitro expansion conditions may have modified protein expression, we evaluated SAA by immunohistochemical staining on formalin fixed tumor tissue. As representatively shown in Figure 2 for EEC-ARK-1 as well as another unrelated G3 EEC, cytoplasmic positivity for SAA was detected by IHC in all tissues derived from poorly differentiated (G3) EEC. In contrast, no SAA positivity was detected in well differentiated (G1) and moderately differentiated (G2) EEC (Figure 2). The intensity of staining for SAA in G3 EEC was significantly higher when compared to normal endometrial controls and G1 and G2 EEC (P<.001). Normal liver tissue (i.e., positive control) was found strongly positive for SAA expression (Figure 2).

Figure 2
Representative immunohistochemical staining for SAA on NEC paraffin-embedded specimen (A), G1 EEC (B), G2 EEC (C), G3 EEC (D) and G3 EEC-ARK-1 (E), and a liver biopsy (F). NEC 1, G1 EEC and G2 EEC, showed negative staining for SAA while cytoplasmic staining ...

SAA Secretion by Primary EEC Cell Cultures

Primary short-term tumor cultures, minimizing the risk of a selection bias inherent in any long-term in vitro growth, may provide an opportunity to study differential SAA secretion between highly enriched populations of tumor-derived epithelial cells. Cell-free supernatants from freshly isolated gynecologic malignancies including 3 EEC and 3 squamous cervical carcinoma cell lines, as well as cultures of normal human fibroblasts and EBV cell lines, were collected and analyzed for SAA expression levels by a sensitive bead-based immunoassay (Millipore, Corp. Danvers, MA). Because prolonged passages in vitro are known to alter the physiology and phenotype of primary tumor cells, we performed all our experiments with highly purified tumor cells and normal cells (i.e., fibroblasts and EBV-transformed B cells) grown for less than 20 passages in vitro. Growth control medium was always analyzed at the same time. In this regard, KSFM and RPMI 1640 media containing 10% fetal bovine serum had no detectable endogenous levels of SAA immunoreactivity (data not shown). All three primary EEC tumor cell lines tested secreted SAA (mean = 2.2 ng/ml, range: 0,3 to 5.5 ng/mL/105 cells/72 hr). In contrast, undetectable to low secretion was identified in the supernatant of normal human fibroblasts (mean = 0.12 ng/ml), EBV-transformed B cells (mean = 0.16 ng/ml) or in those of 3 primary cervical carcinoma cell lines (i.e., not detectable) run in parallel (P<.001).

Serum SAA Concentration in EEC and Noncancer Patients

To investigate whether SAA is detectable in the serum of patients harboring EEC, samples from 102 EEC including 49 G1, 38 G2 and 15 G3 patients, 50 healthy female controls and 42 women harboring benign gynecologic diseases were evaluated by a sensitive bead-based immunoassay (Millipore, Corp. Danvers, MA). SAA serum levels (μg/ml) from 50 healthy female controls had a median of 6.0 and ranged from 1.6 to 169, while 42 patients with benign gynecologic diseases had a median of 6.0 and ranged from 1.1 to 189; their distributions were not statistically significantly different (P=0.92) (Table 3 and Figure 3A). In contrast, serum SAA values from 102 EEC patients had a median of 23.7 and ranged from 0.9 to 2228; these values were statistically significantly higher than those in the healthy group (P=0.0001) and benign group (P=0.0001, Table 3 and Figure 3A). Importantly, when the levels of SAA in EEC patients harboring tumors with different degrees of differentiation (i.e., G1, G2 and G3) were evaluated, we found G3 EEC patients to have higher circulating levels of SAA when compared to G1 and G2 tumors (Table 3). Indeed, as representatively shown in Figure 3B, G3 EEC patients had a mean SAA ± SEM of 252.6 ± 148.9 (range from 0.9 to 2228.2), versus a mean ± SEM of 47.9 ± 9.5 (range from 1.8 to 226.5) in G2 patients and a mean ± SEM of 39.7 ± 8.0 (range from 1.4 to 273.2) in G1 EEC patients.

Figure 3Figure 3
A) Serum SAA levels in 50 healthy subjects, 42 benign-disease subjects, and 102 EEC patients. B) Serum SAA levels in EEC with different degrees of differentiation (i.e., 49 G1, 38 G2 and 15 G3 EEC patients). Data are presented as mean ± SEM.
Table 3
Serum SAA levels in non cancer (healthy), benign disease and EEC patients

DISCUSSION

Because of the early declaration of the disease by vaginal bleeding and the fact that most of EC patients are diagnosed at an early stage with Type I EEC, endometrial cancer is generally considered a neoplasia with good prognosis. Nevertheless, up to 35% of EC patients may be diagnosed with biologically aggressive poorly differentiated endometrioid endometrial cancer (G3-ECC) (2,3). For several of these patients the prognosis remains poor, regardless of their treatment with gold standard therapies including surgery, adjuvant radiation and/or chemotherapy. The identification of biomarkers that can be used for early diagnosis, monitoring, and prediction of response to treatment in EEC might greatly contribute to the improvement of clinical management and outcomes of these patients. Unfortunately, no accepted and/or specific serum tumor markers have as yet been identified for this disease. Furthermore, few EC markers are currently available to monitor the effects of adjuvant therapy or to predict early tumor recurrence. In this regard, although CA125 is commonly used in the clinic for these purposes, it is endowed with low sensitivity and specificity (1518). This report represents the first evaluation of SAA as a novel biomarker in endometrial carcinoma.

In this study, we have quantified SAA expression by RT-PCR in snap frozen EEC specimens. In addition, we have studied SAA protein expression and secretion in multiple primary gynecologic malignancies including EEC and cervical squamous carcinoma cultures. We have confirmed the purity of the tumor cells in our short-term cultures by differential counts of Giemsa-stained cytospin slides as well as by cytokeratin expression using immunohistochemical techniques (data not shown). Our fresh tumor samples contained over 99% tumor cells. Finally, we have studied SAA levels in 194 serum samples derived from healthy donors, patients harboring benign gynecologic tumors and EEC patients.

We report for the first time a high level of expression of the SAA gene in EEC. Indeed, SAA gene expression was significantly higher in EEC when compared to NEC by RT-PCR. The average copy number of SAA-gene mRNA was 95 times higher in EEC compared to NEC cells. Consistent with these findings, highly purified primary EEC cultures were found positive for intracellular expression of SAA by flow cytometry as well as IHC and, importantly, were able to secrete high levels of SAA in vitro as detected by a sensitive bead-based immunoassay. In contrast, SAA was not detected in any of the 3 cervical carcinoma cell lines tested and negligible SAA levels were found in the culture supernatants of normal human fibroblasts cultures used as controls. Thus, taken together, our data highlight for the first time a major tumor expression and secretion of SAA directly by EEC. More importantly, these results support our hypothesis that, in EEC patients, SAA is not only a liver-secreted protein but is also an EEC cell product.

Importantly, when SAA levels were quantified in the serum of EEC patients, we found elevated levels in EEC patients when compared to the levels found in healthy women. Furthermore, in the limited number of EEC patients where sequential serum samples were available (i.e., 3 patients), a decrease in SAA levels was observed post-operatively (data not shown). These in vivo data accord with our in vitro results from highly purified EEC primary cultures, and suggest that SAA is actively secreted by biologically aggressive EEC cells in vitro, and potentially in vivo. Importantly, we found no significant elevation of SAA in the serum of patients harboring benign gynecologic disease when compared to healthy females. We conclude that SAA may be a promising biomarker for early detection of recurrent disease and for monitoring EEC response to adjuvant therapy.

It is worth noting that in our series of EEC the 15 patients harboring poorly differentiated (G3) tumors were found to have higher serum SAA when compared to G1 and G2 tumors. These data are in agreement with our IHC results showing higher levels of positivity for SAA in poorly differentiated EEC when compared to G1 and G2 tumors. Of interest, Gutfeld et al., (14) have recently reported on SAA expression in normal, dysplastic, and neoplastic colonic mucosa. Using in situ hybridization and IHC, they demonstrated local and differential expression of SAA in human colon cancer tissues when compared to normal colonic mucosa. Furthermore they showed progressively higher SAA positivity through the different stages of dysplasia to overt carcinoma (14). Although the biological importance of SAA in cancer patients is not well understood, these findings in human colon carcinoma combined with our results in EEC seem to suggest a novel role for SAA autocrine production in colonic and endometrial tumorigenesis, particularly in G3 EEC. Larger studies that include more EC patients harboring poorly differentiated EEC will be necessary to confirm this hypothesis

Recently, our group has used large-scale gene expression profiling analysis to evaluate the genetic fingerprint of EEC. Using the Affymetrics platform several potentially useful serologic markers, including human trefoil factor 3 (TFF3) and Mammaglobin B, have been recently reported to be highly differentially expressed and potentially secreted by EEC (19, 20). It is thus possible that, in analogy to what has recently been shown in ovarian cancer (21), the simultaneous evaluation of multiple markers such as SAA, TFF3 and Mammaglobin B by a multiplex, bead-based immunoassay system may ultimately allow the development of a test endowed with high specificity and sensitivity for the detection of EEC. This possibility is currently being investigated in our laboratory.

In conclusion, we report here the first evidence that SAA is highly expressed in EEC, is actively secreted in vitro, and that high concentrations of SAA are present in the serum of EEC patients. Moreover, we have shown that higher serum SAA levels are present in EEC patients harboring poorly differentiated EEC. Thus, although our results are preliminary and exploratory and will need further validation in future studies, they support the hypothesis that SAA may be used as a biomarker for this aggressive variant of endometrial cancer to risk-stratify patients and, potentially, to follow disease status in high risk patients. The current availability of a highly sensitive and specific assay for measuring SAA protein concentration in serum, either alone or in combination with multiple additional biomarkers, will facilitate further studies to validate the clinical usefulness of the circulating levels of SAA for the management of patients with EEC.

Acknowledgments

The Authors wish to thank Dr. Gil Mor and Irene Visintin for technical help and the use of the Luminex100 instrument and Amos A. Brooks for his help and expertise in IHC staining.

The Authors wish to thank Dr. Gil Mor and Irene Visintin for technical help and the use of the Luminex100 instrument and Amos A. Brooks for his help and expertise in IHC staining. Supported in part by grants from the Angelo Nocivelli, the Berlucchi and the Camillo Golgi Foundation, Brescia, Italy, NIH R01 CA122728-01A2 to AS, and grants 501/A3/3 and 0027557 from the Italian Institute of Health (ISS) to AS. This investigation was also supported by NIH Research Grant CA-16359 from the National Cancer Institute.

Abbreviations

EEC
Endometrial Endometrioid carcinoma
SAA
Serum Amyloid A
SEM
standard error of the mean
NEC
normal endometrial controls
FBS
fetal bovine serum
q-RT-PCR
quantitative Real Time-PCR
CVX
cervical cancer
LCL
Epstein-Barr transformed B cells

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