Objective
We sought to investigate the potential predictive value of SLIT/ROBO1 immunoreactivity in recurrent and nonrecurrent endometrial cancer (EC), and the relationship between SLIT/Roundabout (ROBO1) immunoreactivity and microvessel density (MVD) in EC.
Study Design
From a total of 815 consecutive patients histologically diagnosed with EC who had undergone surgery we retrieved 45 patients who had confirmed recurrence and randomly selected 110 patients without recurrence. Their paraffin-embedded tissue blocks were also retrieved and subjected to immunohistochemistry for pan-SLIT and ROBO1. MVD counts were evaluated by CD34 immunohistochemistry. Univariate and multivariate analyses were performed to evaluate the effect of SLIT/ROBO1 on recurrence risk with adjustment for other known risk factors.
Results
Immunoreactivity to pan-SLIT and ROBO1 was higher in recurrence patients than that in nonrecurrence patients. Both SLIT and ROBO1 immunoreactivities were positively correlated with MVD. Cox regression analysis identified SLIT, along with age and International Federation of Gynecology and Obstetrics stage, as risk factors for recurrence. The resultant discrimination model yielded estimated and cross-validated sensitivity and specificity of 79% and 85%, respectively.
Conclusion
Increased immunoreactivity to SLIT is an important factor for recurrence of EC, likely through attracting endothelial cells and promoting neovascularization. Thus, the SLIT immunoreactivity is likely a promising biomarker for recurrence and the SLIT/ROBO1 system may be a potential target for reducing the recurrence risk in EC.
Endometrial cancer (EC) is a common malignancy in women in Western nations, trailing only breast, colon, and lung cancers in overall prevalence. In China, where dramatic social and economic changes have taken place along with living standard (and thus lifestyle) changes in the last 30 years, the EC incidence has increased alarmingly rapidly along with breast, kidney, and colon cancers, and it is now the most prevalent cancer in the female reproductive tract, in conjunction with cervical cancer.
Currently, the treatment of choice for EC is surgery, including complete hysterectomy, and removal of remaining adnexal structures. The overall 5-year survival is approximately 80% for all stages of EC, and the major cause for mortality is recurrence, which mostly occurs within 3 years after surgery. Even though the overall recurrence rate is not very high, the salvage rate is reported to be as low as 10%. Hence, the identification of patients with a high risk of recurrence may accord appropriate intervention that ultimately may increase the survival.
There is a substantial prognostic difference between different histologic types of ECs. The most important prognostic variables in EC are surgical International Federation of Gynecology and Obstetrics (FIGO) stage, myometrial invasion, histologic type, and differentiation grade. As with risk factors in any disease epidemiology, although these prognostic variables may be accurate on the population level, they may not be accurate on the individual level. In addition, these variables have so far revealed little, if any, about possible mechanisms on why and how EC recurs. Consequently, targeted therapy is not available. Although some immunohistochemical prognostic factors such as estrogen receptors (ERs) and progesterone receptors (PRs) are reported to be predictive, more recent studies failed to validate their prognostic value. Therefore, biomarkers with high sensitivity and specificity in identifying patients with a high recurrence risk are sorely needed.
It has been recognized that the prognosis of patients with EC can be improved by suppression of the growth of secondarily spreading and initial recurrent lesions. Although chemotherapy and radiation can serve the purpose, the severe collateral damage that they cause to the bone marrow and renal cells often mitigate or even negate their intended effects. Because tumor growth and metastasis depend critically on blood vessels, it is argued that antiangiogenic therapy could be an excellent strategy to serve the purpose of improving prognosis.
Indeed, angiogenesis is of paramount importance in tumor development and an absolute requirement for tumors to become clinically relevant, and tumor angiogenesis is a complex and highly regulated process under the influence of the host microenvironment and various mediators. In EC, vascular endothelial cell growth factors (VEGF), fibroblast growth factors, cyclooxygenase-2, interleukin-8, and thymidine phosphorylase have, among others, been identified to be angiogenic molecules. Because tumor angiogenesis may involve several pathways and, when one is blocked, other pathways may be activated, the identification of all possible angiogenic molecules and pathways in EC would help devise strategies to suppress tumor angiogenesis, thus cutting off the nutritional supply to the tumor, and ultimately starving the tumor.
SLIT is a family of secretory glycoproteins consisting of 3 members, SLIT1, SLIT, and SLIT3, and was originally found to be secreted repellents in axon guidance and neuronal migration. It has been shown to be an endogenously available inhibitor of leukocyte chemotaxis. The receptor for SLIT is the transmembrane protein Roundabout (ROBO), which currently consists of 4 members: ROBO1-4.
Wang et al demonstrated that SLIT is secreted by various cancer cells and ROBO1 is expressed in vascular endothelial cells, where SLIT can attract vascular endothelial cells in vitro and promote tumor-induced angiogenesis in a xenograft model of human malignant melanoma cells. Consistent with this finding, SLIT–ROBO4 is reported to function as a chemoattractant to recruit vascular endothelial cells to sites for vasculogenesis. More recently, it was reported that, in a chemically induced squamous cell carcinoma model in the hamster buccal pouch, increased SLIT expression was associated with higher tumor angiogenesis as reflected by increased VEGF expression and microvessel density (MVD). More remarkably, treatment with a monoclonal antibody against ROBO1 that interrupts the SLIT–ROBO interaction inhibited tumor angiogenesis and growth, indicating that SLIT-mediated tumor angiogenesis is a critical process in the development of chemical-induced squamous cell carcinoma and perhaps in some human cancers as well.
We hypothesized that SLIT/ROBO1 may be involved in tumor angiogenesis in EC, and their expression levels may thus be indicative of the propensity for metastasis and recurrence. In this study, we sought to investigate the expression and localization of SLIT and ROBO1, and CD34, which is considered to be a marker for MVD and thus a measure of angiogenesis in normal and pathological endometrium, in women with recurrence and nonrecurrence EC and healthy women. We also sought to correlate SLIT/ROBO1 immunoreactivity with some known prognostic variables. Finally, we sought to determine the prognostic value, if any, of SLIT, ROBO1, and MVD, along with other known prognostic variables, in predicting recurrence of EC.
Materials and Methods
Patients and specimens
From 1999 through 2005, 815 consecutive patients were histologically diagnosed to have primary EC at Shanghai Obstetrics and Gynecology Hospital, Fudan University. For this study, patients who previously had received chemotherapy, hormonal therapy, or radiotherapy were excluded. All included patients thus received primary surgery in the hospital, and FIGO staging was assigned following pathological findings before and after surgery. Patients in stage I received extrafascial total hysterectomy with bilateral salpingo-oophorectomy (BSO). Complete lymphadenectomy was reserved for patients deemed to have at least 1 of the following high-risk features: (1) tumor grade 3 (poorly differentiated); (2) deep (≥50%) myometrial invasion; (3) tumor involving >50% of uterine cavity or extended to isthmus uteri; and (4) tumor of either clear cell type, undifferentiated, or squamous cell type. Patients with serous papillary EC received the same surgical treatment as those with ovarian cancer, including an exploratory laparotomy, total hysterectomy, BSO, omentectomy, appendectomy, and biopsy of any suspected lesions. Patients with stage II EC received a radical hysterectomy with BSO and lymphadenectomy, whereas those with stage III/IV EC received a complete staging laparotomy and cytoreductive surgery whenever surgically feasible. All cytoreductive surgeries were satisfactory with no apparent macroscopic residual lesion visualized postoperatively. Depending on the patient’s histologic subtype, grade, depth of myometrial invasion, lymphatic-vascular space invasion, cervical involvement, extrauterine spread, and other known prognostic variables, postoperative adjuvant therapy, including radiotherapy, hormonal therapy, and chemotherapy, was provided individually when indicated.
All patients were followed up every 3 months for the first year after surgery, then every 6 months for the next 2 years, and then annually. At each follow-up visit, gynecologic examination, chest radiographs, vaginal ultrasound, and serum CA125 test were performed, with vaginal smears or biopsy samples, and computed tomography (CT)/magnetic resonance imaging (MRI) taken every 6 or 12 months. Among the 815 patients, 751 (92.1%) were successfully followed up and constituted the patient pool for this study. The length of follow-up ranged from 36-110 months, with a mean (SD) of 60.2 (20.8) and a median of 53 months, respectively.
The recurrence, either local (vaginal or pelvic) or distant (abdominal, chest, and other locations), of EC was defined as follows: (1) imaging, via radiographs, vaginal ultrasonography, CT, or MRI, of persistent (≥3 months) de novo lesion(s) characteristic of tumors, with at least 1 dimension being ≥20 mm by ultrasound/radiographs or ≥10 mm by CT/MRI in conjunction with <30% decrease in the sum of the longest diameters of all de novo lesions between 2 consecutive examinations ; or (2) pathologically diagnosed as EC via biopsy or a secondary surgery, with the histologic subtype being the same as that found in the primary one. Among 751 patients successfully followed up, 45 (6.0%) were identified to have confirmed recurrence. Among them, 19 (42.2%) had recurrence in the pelvic/abdominal cavity, 6 (13.3%) had vaginal stump, and the remaining 20 (44.4%) had distant recurrence. The recurrence was confirmed histologically in 8 patients (17.8%) and by imaging evidence in the remaining 37 (82.2%). The mean time to recurrence was 25.9 ± 19.3 months.
Initially, we attempted to select, from the patient pool, patients without any clinical or imaging evidence for recurrence for at least 36 months after surgery for this study to match with the recurrence cases on age and histologic type, and we then randomly selected nonrecurrence patients for this study after realizing that it was very difficult to do so. Thus, this study consisted of 45 patients who had confirmed recurrence and 110 patients who did not. For these patients, their tissue blocks, which were harvested at the time of surgery and subsequently formalin fixed, paraffin embedded, and archived in the pathology department of the hospital, were retrieved, along with their medical charts. For comparison purposes, we also collected endometrial tissue samples from 20 normally cycling women with tubal infertility or surgically diagnosed benign ovarian cysts after informed consent.
This study was reviewed and approved by the ethics committee of Shanghai Obstetrics and Gynecology Hospital.
Immunohistochemistry
Antibodies to ROBO1 and pan-SLIT were prepared and characterized as reported previously. Mouse monoclonal anti-CD34 was purchased from Beijing Zhongshan Goldenbridge Biotechnology Co Ltd (ZM-0046; Beijing, China). Formalin-fixed, paraffin-embedded sections (3-4 μm) were dried at 65°C for 2 hours, dewaxed in xylene twice for 20 minutes before rehydration through graded alcohols (100%, 95%, 80%, and 70%) and then water. Antigen retrieval was performed by placing the slides in a bath of Tris-EDTA (pH = 9) and boiling for 15 minutes using an 800-W microwave oven. The volume of fluid was topped up, and the slides then were left to cool down for 30 minutes at room temperature before being washed well in phosphate-buffered saline (PBS) (10 mmol/L, pH 7.4). Peroxidase was blocked with methanol and 3% H 2 O 2 for 10 minutes and washed in PBS. All incubations were performed at 37°C. Sections were incubated with the primary antibody of interest for 1 hour (SLIT 5 μg/mL, ROBO1 5 μg/mL, CD34 1:100, diluted in antibody diluent [S2023; Dako, Glostrup, Denmark]). After washing with PBS, sections were incubated for 30 minutes with a secondary antibody (K5007; Dako) and again washed in PBS. Slides then were treated for 3 minutes in diaminobenzidine (K3468; Dako), 30-second counterstained in hematoxylin, and washed with tap water.
Each staining run incorporated a positive control slide from a breast cancer tissue sample. A negative control was also incorporated using PBS instead of the antibody. Immunoreactivity staining was characterized quantitatively by digital image analysis using the Image Pro-Plus 6.0 (Media Cybernetics Inc, Silver Spring, MD) as reported by Wang-Tilz et al without prior knowledge of the recurrence status of the patient being evaluated. Briefly, images were obtained with the microscope (BX51; Olympus, Tokyo, Japan) fitted with a digital camera (DP70; Olympus). A series of 10 random images on several sections were taken for each immunostained parameter to obtain a mean value. Staining was defined via color intensity, and a color mask was made. The mask was then applied equally to all images, and measurements were obtained. Immunohistochemical parameters assessed in the area detected included: (1) integrated optical density; (2) total stained area; and (3) mean optical density, which is defined as mean optical density = integrated optical density/total stained area, equivalent to the intensity of stain in the positive cells.
Quantification of angiogenesis (MVD)
MVD was assessed on CD34-stained slides by light microscopy in the areas having the highest numbers of capillaries and small venules (neovascular hot spots). Then microvessel counting followed on 10 chosen ×200 fields of the “hot plot” by the same investigator without knowledge of the recurrence status of the patient being evaluated. Endothelial cells or cell cluster clearly separated from adjacent microvessels, tumor cells, and other connective tissue elements were taken into account for microvessel counting. Vessel lumens were not necessary for a structure to be defined as a microvessel, and red cells were not used to define a vessel lumen. The MVD was defined to be the mean of the vessel counts obtained in these fields, as reported by Mai et al.
Statistical analysis
For descriptive statistics, we used box plot to graphically depict groups of immunoreactivity data, in which the bottom and top of the box represent the lower and upper quartiles, respectively; the band near the middle of the box represents the median; and the ends of the whiskers represent the smallest and the largest nonoutlier observations. The comparison of distributions of continuous variables between 2 or among ≥3 groups was made using the Wilcoxon test and Kruskal-Wallis test, respectively. Pearson correlation coefficient was used when evaluating correlations between 2 variables when both variables were continuous. When at least 1 variable was ordinal, Spearman rank correlation coefficient was used instead.
To evaluate which factors are associated with the risk of recurrence, the Cox proportional hazard regression model was used in conjunction with a stepwise regression. To facilitate computation, patients’ age was subtracted from their mean, and the histologic type was dichotomized into either endometrioid adenocarcinoma or nonendometrioid adenocarcinoma (which includes clear cell, squamous cell, and papillary serous EC).
To evaluate the effect of SLIT immunoreactivity level and other factors identified to be predictive by the Cox regression on the risk of recurrence, a logistic regression model was used to differentiate recurrence and nonrecurrence cases and to estimate sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and the overall correct classification rate. Because estimates of sensitivity and specificity based on the same data used for model fitting are usually too optimistic, leaving-one-out cross-validation was used to estimate sensitivity, specificity, PPV, NPV, and the overall correct classification rate.
P values of < .05 were considered statistically significant. All computations were made with R 2.8.0 ( www.r-project.org ).
Results
Group-specific and clinicopathological data
Among the 45 patients with recurrence, 33 (73.3%) had recurrence within 3 years after surgery whereas the remaining 12 (26.7%) had recurrence after, consistent with the finding from a recent metaanalysis. The time to recurrence ranged from 6-72 months. In the nonrecurrence group, the length of follow-up ranged from 36-87 months with a mean (SD) and median of 64.0 (16.4) and 66 months, respectively. The lower quartile of follow-up length in the nonrecurrence group (46 months) was longer than the upper quartile of that in the recurrent group (38 months). The group-specific and clinicopathological characteristics in women with and without recurrence are listed in Table 1 . It can be seen that the patients in the recurrent group were considerably older than those in the nonrecurrence group ( P < .05, Wilcoxon rank sum test). The mean (SD) age in the control group was 43.8 (6.1) years, ranging from 33-55 years, which was significantly younger than either the recurrence or nonrecurrence group (both P values < .0001).
| Variable | Recurrence group (n = 45) | Nonrecurrence group (n = 110) | Statistical significance of difference ( P value) |
|---|---|---|---|
| Age, y |
|
| 2.86 × 10 –7 |
| Age at menarche, y |
|
| .785 |
| Menopausal | |||
| No | 10 (22.2%) | 50 (45.5%) | .010 |
| Yes | 35 (77.8%) | 60 (54.5%) | |
| Parity | |||
| 0 | 5 (11.1%) | 9 (8.2%) | .55 |
| ≥1 | 40 (88.9%) | 101 (91.8) | |
| Grade of differentiation | |||
| High | 25 (55.6%) | 84 (76.4%) | .0050 |
| Medium | 8 (17.8%) | 18 (16.4%) | |
| Low | 12 (26.7%) | 8 (7.3%) | |
| Myometrial invasion | |||
| None | 2 (4%) | 17 (14.5%) | |
| Minimal | 23 (51.1%) | 67 (60.9%) | .017 |
| Deep | 20 (44.4%) | 26 (23.6%) | |
| Vascular-space invasion | |||
| No | 35 (77.8%) | 91 (82.7%) | .500 |
| Yes | 10 (22.2%) | 19 (17.3%) | |
| Lymph node involvement | |||
| No | 33 (73.3%) | 105 (95.5%) | .0002 |
| Yes | 12 (26.7%) | 5 (4.5%) | |
| FIGO stage | |||
| I | 16 (35.6%) | 74 (67.3%) | |
| II | 7 (15.6%) | 26 (23.6%) | 6.50 × 10 –7 |
| III | 12 (26.7%) | 8 (7.3%) | |
| IV | 10 (22.2%) | 2 (1.8%) | |
| Histologic type | |||
| Endometrioid adenocarcinoma | 23 (51.1%) | 96 (87.3%) | 1.22 × 10 –5 |
| Papillary serous | 13 (28.9%) | 10 (9.1%) | |
| Clear cell | 6 (13.3%) | 2 (1.8%) | |
| Squamous cell | 3 (6.7%) | 2 (1.8) | |
| Postoperative chemotherapy | |||
| No | 23 (51.1%) | 81 (73.6%) | .009 |
| Yes | 22 (48.9%) | 29 (26.4%) | |
| Postoperative radiotherapy | |||
| No | 39 (86.7%) | 94 (85.5%) | 1.0 |
| Yes | 6 (13.3%) | 16 (14.5%) | |
| ER immunoreactivity | |||
| – | 22 (71.0%) | 40 (41.2%) | |
| +/– | 0 (0.0%) | 2 (2.1%) | .012 |
| + | 9 (29.0%) | 55 (56.7%) | |
| Missing | 14 | 13 | |
| PR immunoreactivity | |||
| – | 23 (69.7%) | 23 (23.2%) | |
| +/– | 2 (6.1%) | 3 (3.0%) | 1.74 × 10 –7 |
| + | 8 (24.2%) | 73 (73.7%) | |
| Missing | 12 | 11 | |
| p53 Immunoreactivity | |||
| – | 76 (80.0%) | 20 (66.7%) | |
| +/– | 2 (2.1%) | 0 (0.0%) | .174 |
| + | 17 (17.9%) | 10 (33.3%) | |
| Missing | 15 | 15 | |
| Mode of surgery | |||
| Hysterectomy + BSO | 7 (15.6%) | 16 (14.5%) | |
| Hysterectomy + BSO + lymphadenectomy | 5 (11.1%) | 21 (19.1%) | .349 |
| Radical hysterectomy + BSO + lymphadenectomy | 26 (57.8%) | 72 (65.5%) | |
| Staging laparotomy and cytoreduction | 7 (15.6%) | 1 (0.9%) |
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
