Uterine cervical cancer often disseminates via lymphatic spread. It is generally accepted that pelvic lymphadenectomy be conducted systematically at the time of radical hysterectomy, as it provides valuable prognostic information.1–3 Currently, cervical cancer is clinically staged according to International Federation of Gynecology and Obstetrics (FIGO) recommendations. Therefore, in the pretreatment evaluation, the detection of lymph node (LN) involvement and distant metastasis determines the prognosis and the course of treatment.4–6 Although computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and PET/CT are widely used pretreatments to detect metastatic LNs in patients with cervical cancer, recent studies have shown that PET and PET/CT have higher diagnostic significance than does CT or MRI.5,7–10
Positron emission tomography, which uses the glucose analog 2-deoxy-2-F18-fluoro-D-glucose (FDG), is a noninvasive functional imaging modality used to detect primary tumors, plan and monitor therapy, and detect metastasis and recurrence.11,12 The combination of PET with CT increases its specificity and helps localize hot spots through software imaging fusion. However, FDG uptake is not specific for malignancy and is less accurate in detecting lesions smaller than 1.5 cm.4,13
In cancer, higher FDG uptake compared to that of noncancerous cells has been attributed to increased metabolic turnover and increased glycolytic activity with up-regulation of glycolysis-associated genes.14 At the molecular level, expression of the facilitative glucose transporter (GLUT) proteins, especially GLUT-1, has been shown to be a good predictor of FDG uptake in various cancers, such as colorectal, lung, ovarian, and cervical cancers15–18; whereas GLUT-1 is undetectable in most normal epithelial tissues and benign epithelial tumors.19 Glucose phosphorylation enzyme type II (hexokinase II [HK-II]) is also known to be the most important subtype for glucose metabolism in cancer cells.20,21
In this study, our objectives were to determine whether GLUT-1 or HK-II expression is related to FDG uptake in lesions identified as false positive or false negative on PET/CT, in addition to the factors affecting these relationships. To pursue these objectives, we explored the association of FDG uptake with the expressions of GLUT-1 and HK-II in metastatic cervical cancer LNs, as well as LNs pathologically confirmed as negative.
MATERIALS AND METHODS
This prospective study involved 20 women with histopathologically confirmed FIGO stage IB to stage IIA invasive cervical cancer, as determined by a conventional workup that included pelvic and rectovaginal examination, routine laboratory testing, chest radiography, cystoscopy, and sigmoidoscopy. All patients underwent a preoperative workup, including both MRI and PET/CT scans. The patients were recruited between June 2007 and May 2009, and they ranged in age from 32 to 68 years (mean, 49 years). The patients had no contraindications to the surgical procedure, had no evidence of distant metastases, and had a Gynecological Oncology Group (GOG) performance status of 0 to 1. We excluded those women who did not want to undergo preoperative PET/CT (n = 4). The institutional review board of our institute approved the study protocol, and informed consent was obtained from all patients participating in the study.
FDG PET/CT Study
Positron emission tomography/CT was performed on a dual-slice Philips Gemini System (Philips, Eindhoven, The Netherlands). After the patients had fasted for at least 6 hours, 4.0-MBq/kg body weight of fluorine-18-fluorodeoxyglucose (18F-FDG) was administered at a normal blood glucose level. A low-dose CT scan was acquired for attenuation correction at a 120-kV tube potential with 30 mA s current, covering a range from the skull base to the thighs. Positron emission scans were acquired with a 15.5-cm field of view for 3 minutes per table position using a 3-dimensional acquisition mode, and an average of 8 to 10 table positions were required to cover the range. The subsequent diagnostic CT scan was acquired during a single breath-hold of 70 seconds after administration of 120 mL of nonionic contrast material (Ultravist, 623 mg/mL of Iopromide, Bayer Schering, Berlin, Germany) at a tube current of 160 mA s. Positron emission tomography data were reconstructed with attenuation correction, and both CT and PET data were reconstructed in 5-mm slices. Computed tomography and PET data sets were reviewed on a postprocessing workstation in axial, coronal, and sagittal orientations and were fused with a color-coded superimposition of the PET onto the CT image.
The images were preoperatively reviewed by an experienced nuclear medicine physician. The presence of abnormally increased FDG uptake was noted, and its exact anatomic location was indicated on the CT. In the report, the readers indicated the presence or absence of elevated specific uptake values, which increase the suspicion of distant metastases. Positron emission tomography/CT images were interpreted as negative when no areas of abnormal FDG uptake were seen. The presence of abnormal FDG uptake was indicated when accumulation of the tracer was moderately to markedly increased relative to the uptake in comparable normal structures or surrounding tissue, with the exclusion of physiological bowel, vessel, and urinary activity. The classification of LNs on PET/CT images as cancer positive was based on the presence of focally increased FDG uptake on the PET images in a location that corresponded to an LN chain on the CT images. Lymph nodes with increased tracer uptake were considered positive for metastasis, even if they were smaller than 1 cm in the short-axis diameter.
All patients underwent surgery with the same instruments and techniques performed by 2 experienced gynecologic oncologists with knowledge of the PET/CT results. All patients underwent radical hysterectomy with pelvic and para-aortic lymphadenectomy. All harvested LNs were grouped according to the name of the adjacent vessel (abdominal aorta, both common iliac arteries, both external iliac arteries, and both internal iliac and obturator arteries). For nodal matching, each surgical specimen was labeled according to site and side.
Immunohistochemical Staining for GLUT-1 and HK-II
The surgical specimens were fixed in buffered formalin and embedded in paraffin, and sections 3-μm in thickness were stained with hematoxylin and eosin in an autostainer (Bond MAX, Vision BioSystems Ltd, Mount Waverley, Australia). Formalin-fixed, paraffin-embedded tissue sections were immunostained with rabbit anti–GLUT-1 polyclonal antibody (1:50, Neomarkers, Fremont, CA) and goat anti–HK-II antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA).
Evaluation of Stained Sections
Cancer involvement, the sizes of individual LNs (millimeter), and tumor proportions (percent) within LNs were assessed. The proportions (percent) of GLUT-1– and HK-II–positive tumor cells in tumors and normal tissue were also simultaneously evaluated by an experienced pathologist. Staining intensities were graded as follows: zero, negative; 1, weak; 2, medium; and 3, intense. The staining intensity of red blood cells was defined as grade 3.
We performed node-based analyses and calculated sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy using standard statistical formulas. Independent Student t tests were used to compare the sizes of LNs, the tumor proportions within LNs, and the proportions of immunostain-positive cells in tumor and normal tissues. The Mann-Whitney nonparametric 2-sample test was used to assess differences in the staining grades of both GLUT-1 and HK-II. All statistical analyses were performed using SPSS version 12.0 and were considered significant at P < 0.05.
Table 1 summarizes the characteristics of patients in this study. Of the 20 patients, 16 had a diagnosis of squamous cell carcinoma, 3 patients had adenocarcinoma, and the remaining patient had a diagnosis of adenosquamous cell carcinoma. Clinical FIGO stage was IB1 in 6 patients, IB2 in 9 patients, and IIA in 5 patients. On histopathological analysis, 70 of the 333 LNs were positive for metastasis. For LN metastasis, PET/CT was true positive in 16 of the 70 metastatic nodes and true negative in 259 of the 263 nonmetastatic nodes. Thus, the overall sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of PET/CT on node-based analysis were 22.8%, 98.5%, 80%, 82.7%, and 82.6%, respectively (Table 2).
We analyzed the patterns of GLUT-1 and HK-II expression in both the cell membrane and cytoplasm (Fig. 1). In cancer cells, positive staining was present mainly around the necrotic area; the intensity of expression was heterogeneous within the tumor. The mean percentage of GLUT-1–positive cells in pathologically confirmed negative LNs was higher in PET-positive nodes than it was in PET-negative nodes (mean [SD]; 3.75% [2.99%] vs. 1.01% [2.2%]; P = 0.015). Similarly, the mean percentage of HK-II-positive cells in pathologically negative LNs was higher in PET-positive nodes than it was in PET-negative nodes (mean [SD]; 2.25% [1.5%] vs. 0.72% [0.94%], P = 0.001). However, the mean staining intensities of GLUT-1 and HK-II in pathologically negative nodes were not significantly different between PET-positive and PET-negative nodes (Table 3).
In LNs pathologically proven as metastatic, there were statistically significant differences between PET-negative and PET-positive LNs in size, tumor involvement (percent), and percentage of GLUT-1–positive cells (Table 4). The LN diameters of PET-negative and PET-positive nodes were 8.22 and 11.88 mm, respectively (P = 0.043). The proportion of tumor cells was 40.0% and 63.75% in PET-negative and PET-positive nodes, respectively (P = 0.008). The mean percentage of GLUT-1-positive tumor cells was higher in PET-positive nodes than it was in PET-negative nodes (P = 0.042). The mean GLUT-1 staining intensity was greater in PET-positive nodes than it was in PET-negative nodes, although the difference was not statistically significant (P = 0.052). However, no significant difference was observed between FDG uptake and HK-II expression in pathologically confirmed metastatic LNs.
A recent meta-analysis by Choi et al10 reported that PET and PET/CT had higher diagnostic performances than did CT or MRI in detecting metastatic LNs in patients with cervical cancer. However, FDG uptake is not specific to cancer and can be taken up by some benign tumors, infections, inflammatory cells, and normal tissue. Images of the abdomen and female pelvis can be especially difficult to interpret owing to FDG uptake in the ovaries, intestines, and ureters.In addition, false-negative analysis may result from small metastaticLNs or micrometastases.22 Although the image fusion of function and anatomy in a PET/CT scan increases the diagnostic accuracy, pitfalls still exist.23,24
2-Deoxy-2-F18-fluoro-D-glucose uptake in cancer cells is associated with the following: (1) facilitated diffusion through glucose transport proteins, (2) subsequent phosphorylation by HK isoforms that produce FDG-6-phosphate, and (3) decreased dephosphorylation.25 Therefore, the association between FDG uptake and the expressions of GLUT-1 and HK-II has been evaluated in many other cancers, such as lung cancer,25 cholangiocellular carcinoma,26 lymphoma,27 pancreatic cancer,28 ovarian cancer,18 and cervical cancer.17
Our study found that false-positive LNs on PET/CT had higher percentages of GLUT-1– and HK-II–positive cells than did true-negative LNs. However, the expressions of GLUT-1 and HK-II were significantly lower in pathologically negative LNs than those in metastatic LNs. About metastatic LNs, PET/CT-positive nodes had a higher proportion of GLUT-1–positive cells and stronger GLUT-1 staining intensity than did PET/CT-negative nodes, whereas HK-II expression was not correlated with FDG uptake. These results suggest that GLUT-1 plays a role in FDG uptake on PET/CT evaluation in cervical cancer. In addition, the mean tumor size was significantly larger in PET/CT-positive nodes than it was in PET/CT-negative metastatic LNs. Finally, tumor involvement was significantly higher in PET/CT-positive nodes than in PET/CT-negative metastatic LNs (64% vs 40%; P = 0.008). This suggests that tumor involvement is an important factor associated with the PET/CT positivity of metastatic LNs in cervical cancer.
This study has several limitations. First, the surgicopathological localization of PET/CT-positive LNs is difficult. We attempted to minimize this limitation by adopting a mapping system for LN classification.8 Second, our results show a lower sensitivity (22.8%) for PET/CT compared to that in a previously reported meta-analysis (54%).10 It has been reported that PET/CT has low sensitivity in LN evaluation of patients with early-stage cervical cancer (FIGO stage ≤IB1)4 and high false-negative rates with small or early nodal involvement.8,29 Although the sensitivity was not different between stage IB and IIA in this study, compared to previous reports,29,30 our study had smaller negative LNs (mean [SD] size, 6.68 [4.98] mm) and metastatic LNs (mean [SD] size, 9.06 [6.37] mm), which may explain the low sensitivity of PET/CT.
In conclusion, the positivity of PET/CT in metastatic LNs of cervical cancer was related to the node size, tumor involvement, and GLUT-1 expression.
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Keywords:Copyright © 2012 by IGCS and ESGO
Cervical cancer; Lymph node metastasis; PET/CT; Glucose transporter 1; Hexokinase II