Relationship of Nonstaging Pathological Risk Factors to Lymph Node Metastasis and Recurrence in Clinical Stage I Endometrial Carcinoma

GYNECOLOGIC ONCOLOGY ARTICLE NO.

66, 388–392 (1997)

GO974788

Relationship of Nonstaging Pathological Risk Factors to Lymph Node Metastasis and Recurrence in Clinical Stage I Endometrial Carcinoma Jeffrey G. Bell, M.D., Angela Minnick, Gary C. Reid, M.D., Jeffrey Judis, M.D., and Mark Brownell, M.D. Department of Obstetrics and Gynecology and Department of Pathology, Riverside Methodist Hospitals, Columbus, Ohio 43214 Received November 18, 1997

Objective. To determine if DNA ploidy, hormone receptors, vascular space invasion (VSI), perivascular lymphocytes (PVL), and the oncogenes HER-2/neu, p53, and bcl-2 are independent prognostic indicators for lymph node metastasis and cancer recurrence in clinical stage I endometrial carcinoma. Methods. Among 349 patients with clinical stage I endometrial cancer 31 patients either had lymph node metastases when surgically staged or developed recurrent cancer. Using a case–control matched-pair technique, controls were selected for each of 24 cases by matching for age, histological grade, depth of myometrial invasion, performance of node dissection, and use of adjuvant radiation therapy. In a blinded fashion a pathologist reviewed all histopathology, and all molecular tests were performed on paraffin-embedded tissue samples. Statistical analysis was performed by x2 and McNemar’s tests. Results. VSI was the only histopathological factor significantly related to positive lymph nodes and cancer recurrence (P Å 0.01), independent of grade and myometrial invasion. Aneuploidy, oncogene expression (p53, HER-2/neu, bcl-2), and hormone receptors were not significantly related to lymph node metastasis and cancer recurrence. Conclusions. The presence of vascular space invasion is a pathological factor independently associated with a risk of nodal metastasis and cancer recurrence in clinical stage I endometrial cancer. DNA ploidy, oncogene expression, and hormone receptor status do not have more predictive value than standard staging pathological criteria. q 1997 Academic Press

Well-recognized histopathological risk factors for lymph node metastasis or recurrent cancer include histologic grade and depth of myometrial invasion. However, oncologists are familiar with experiences whereby patients with low-risk histologic grade and myoinvasion have nodal metastasis or develop recurrent cancer, and vice versa. In addition, patients completely staged surgically with normal nodes may suffer recurrence and death. A number of studies have investigated the prognostic value of histopathologic and molecular parameters not included in FIGO staging. The most commonly reported factors have been vascular space invasion (VSI), hormone receptors, DNA ploidy, and oncogenes including p53 and HER-2/neu. One goal of such studies would be the identification of features that can better determine the prognosis of endometrial cancer with a given histologic grade and myometrial invasion. The data from previously published studies of nonstaging histopathologic prognostic factors are quite conflicting. Possible reasons for the contradictory data include small sample sizes due to the overall good survival for this cancer, study design or statistical methodology, and differences in the selection of controlled variables. The purpose of this study was to determine if nonstaging histopathologic and molecular features are independent prognostic factors for lymph node metastasis or cancer recurrence by controlling for grade and myoinvasion. In addition, we studied bcl-2, an oncogene previously unreported in endometrial cancer literature.

INTRODUCTION

METHODS

In 1988, FIGO (International Federation of Gynecologists and Obstetricians) introduced surgical staging for endometrial cancer that enables clinicians to better define criteria for estimating the chance of cancer recurrence and patient survival. Unfortunately, many patients are unable to undergo complete surgical staging that includes lymph node dissection because of obesity, age, or other medical risks. In these situations, clinicians frequently use uterine histopathological features to assess the need for adjuvant therapy for patients with disease grossly confined to the uterus.

Charts of 349 patients with clinical stage I endometrial carcinoma surgically treated between 1984 and 1992 were reviewed to identify cases who had either lymph node metastasis when surgically staged or cancer recurrence. We excluded patients with disease outside the corpus (adnexa, positive peritoneal cytology, cervical extension) or on the serosa, and patients with papillary serous or clear cell histology. The resulting 31 cases included 7 with lymph node metastasis but no recurrence, 2 with nodal metastasis and recurrence, and 22 with cancer recurrence without

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0090-8258/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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known nodal metastasis. We were able to identify 24 paired controls for 24 of these cases by matching for age less than or greater than 55, histologic grade, depth of myometrial invasion, use of postoperative adjuvant radiation, and performance of node dissection. No patients received preoperative radiotherapy. All controls had to be free of disease for at least 3 years after surgery. In a blinded fashion one pathologist reviewed all cases and controls for grade, myoinvasion, vascular space invasion, and perivascular lymphocytes, and he interpreted DNA ploidy, hormone receptor status, and oncogene expression (p53, HER-2/neu, and bcl-2). All molecular tests were performed on paraffin-embedded specimens. DNA ploidy. Fifty-micrometer sections were cut from paraffin blocks and placed in a centrifuge tube, deparaffinized with xylene, and rehydrated. The tissue was then minced with scalpel and suspended in 0.9% Na Cl containing 0.5% pepsin and 3% polyethylene glycol at pH 1.5 for 1 hr at 377C. The incubation was repeated for another hour with fresh pepsin solution. Following two washes in Hanks’ balanced salt solution containing 3% polyethylene glycol and 0.02% sodium azide, the suspension was incubated for 3 min in 0.1% Triton X-100 in Dulbecco’s phosphate-buffered saline (D-PBS). The suspension was incubated with RNase 180 units/ml in D-PBS for 20 min at 377C and then incubated with propidium iodide 50 mg/ml in D-PBS for 60 min. DNA content was measured on a Becton Dickinson FACScan flow cytometer. Immunohistochemistry. The expression of estrogen receptors (ER), progesterone receptors (PR), and the oncogenes p53, HER-2/neu, and bcl-2 were analyzed by immunohistochemistry using commercially available monoclonal antibodies: ER, Dako 1 D5 clone; PR, Novo castra 1 A6 clone; p53, Biogenex DO7 clone; HER-2/neu, Biogenex CB11 clone; bcl-2, Dako 124 clone. Immunostaining was performed using ABC kit reagents after microwaving in 4 M Tris, pH 10. Breast cancer was used as the control for both p53 and HER-2/neu. Lymphoma was used as the control for the bcl-2 oncogene. A pathologist blinded to the cases and controls graded the slides based on intensity from 0 to 4/ with 4/ demonstrating the greatest overexpression of the oncogene. Statistics. McNemar’s x2 test was used for the statistical analysis of the matched pairs. Continuity correction was used to compensate for the small sample size (n Å 24). P õ 0.05 was considered significant. When variables were graded from 0 to 4/, 0 and 1/ were considered negative results, while 2/ to 4/ were considered positive results.

nodes and subsequent cancer recurrence, and 18 patients who experienced recurrent cancer with either negative nodes at staging laparotomy (n Å 10) or no node dissection performed (n Å 8). Of the 6 patients with positive nodes, 2 had both aortic and pelvic nodes positive, 1 had only positive aortic nodes, and 3 had only positive pelvic nodes. Three other patients had positive nodes, but these were excluded due to lack of suitable controls. The 9 patients with positive nodes represent 4% of aortic node dissections performed and 6% of pelvic node dissections. Table 1 presents the histologic grade, depth of myoinvasion, age, and number of patients who underwent node dissections and adjuvant postoperative radiotherapy. In only two matched pairs did the control have a node dissection performed, whereas the case did not. Of the 24 cases in our study with recurrent disease and/ or positive lymph nodes, 13 (54%) patients died of disease after a mean survival of 25 months. For those patients still alive the mean follow-up was 44 months. The site of recurrent cancer was pelvic in 3 patients, distant in 14 patients, and both pelvic and distant in 3. For the control group there were 5 intercurrent deaths during a mean surveillance of 94 months, ranging from 39 to 145 months. Vascular space invasion (VSI) was the only risk factor that showed a significant (P õ 0.05) association with the cases; it was present in the cases and absent in the control for 10 matched pairs, and yet only one pair showed VSI in the control while it was absent in the case. The presence of all other risk factors—perivascular lymphocytes (PVL), hormone receptors, HER-2/neu, bcl-2, p53, DNA ploidy— was not significantly different between cases and matched controls. However, the absence of estrogen receptors and the presence of bcl-2 in the cases approached significance, P õ 0.1. Table 2 summarizes the relationship between cases and their matched-pair controls for the various nonstaging pathological and molecular risk factors. Although the presence of perivascular lymphocytes was not significantly related to the case group, it was significantly associated with the presence of vascular space invasion. VSI was present in 17 of 48 study patients, and 11 of these 17 (65%) were also positive for PVL; PVL was present in 8 of 31 subjects (26%) without VSI (P Å 0.009, x2). DNA ploidy was evaluable in 20 of the 24 pairs. Ten of 20 cases (50%) were aneuploid, 3 (15%) were tetraploid, and 1 (5%) was hypertetraploid. For analysis, aneuploid, tetraploid, hypertetraploid, and multiploid were grouped as nondiploid. Cases were nondiploid while their matched controls were diploid in 8 of 20 pairs; controls were nondiploid and cases diploid in 2 matched pairs (P Å N.S.).

RESULTS

DISCUSSION

Our case group of 24 patients consisted of 4 patients who had positive lymph nodes from surgical staging but did not experience cancer recurrence, 2 patients who had positive

The prognostic value of histologic grade and myometrial invasion within stage I endometrial cancers is no doubt beyond dispute. Nevertheless, the fact that some grade 1, mini-

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TABLE 1 Controlled Variables for Cases and Matched Control Patients: Age, Histologic Grade, Myoinvasion, Node Dissection, and Adjuvant Pelvic Radiotherapy

Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls

n

Mean age

Grade

Myoinvasion

Node dissection

Radiation

3 3 3 3 3 3 4 4 2 2 3 3 6 6

72 64 71 69 63 56 69 75 72 69 74 66 72 67

1 1 2 2 2 2 2 2 3 3 3 3 3 3

Inner half Inner half None None Inner half Inner half Outer half Outer half None None Inner half Inner half Outer half Outer half

1 1 1 0 3 2 4 3 1 2 2 3 4 4

0 0 0 0 0 0 2 2 0 0 1 1 5 5

mally invasive cancers recur while other grade 3, deeply invasive cancers do not recur challenges the gynecologic oncologist to define appropriate adjuvant therapy. Numerous studies have attempted to identify histopathologic and molecular factors, other than grade and myoinvasion, that would further define the risk of recurrent cancer. Data from studies of prognostic factors are difficult to analyze due to small sample sizes. With the incidence of nodal metastasis being 7 – 15% and the rate of recurrent cancer being 10 – 20% in clinical stage I disease, previous publications reporting on fewer than 100 patients have sample sizes that may not be suitable for multivariate analysis. In addition, rarely do other publications mention either postoperative therapy or node dissection as controlled variables. Our study design employed the matched-pair case – control method to control for the factors known or suspected to influence prognosis: grade, myoinvasion, age, postoperative radiation, and node dissection. The matchedpair case– control design may provide the best means of

investigating a very specific hypothesis in a comparatively small sample [1]. VSI was the only pathologic criterion that was present significantly more frequently in the case group, the patients with either nodal metastasis or recurrent cancer. The implication is that the presence of vascular space invasion increases the chance of nodal metastasis or cancer recurrence for a given histologic grade and depth of invasion. VSI is a prognostic factor independent of grade, myoinvasion, age, postoperative radiotherapy, and performance of node dissection. Other investigators have reached similar conclusions about VSI being an independent prognostic factor [2–7]. In fact, our feeling is that the evidence for vascular space invasion affecting prognosis is sufficient for FIGO staging to include VSI as a criterion or category. The significant relationship between the presence of perivascular lymphocytes and vascular space invasion should suggest to pathologists to thoroughly search for VSI when perivascular lymphocytes are present in the specimen. Others

TABLE 2 Number of Matched Pairs with Risk Factor Present (Positive) or Absent (Negative) in Tissue Matched pairs

VSIa

PVLb

ERc

PRd

HER-2/neu

bcl-2

p53

Case positive/control negative Case negative/control positive Both case and control positive Both case and control negative P value

10 1 3 10 0.01

6 5 4 9 N.Se

3 9 7 5 N.S.

6 6 9 3 N.S.

3 7 4 10 N.S.

7 2 4 11 N.S.

8 5 5 6 N.S.

Note. Results of McNemar’s x2 with continuity correction. a VSI, vascular space invasion. b PVL, perivascular lymphocytes. c ER, estrogen receptor. d PR, progesterone receptor. e N.S., not significant.

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have also reported a significant relationship between PVL and VSI [8]. The fact that our data showed no correlation between PVL and nodal metastasis or cancer recurrence supports conclusions previously published [9]. Two of the other studied pathological/molecular factors may have reached significant association with nodal metastasis and cancer recurrence if the number of matched pairs had been greater: lack of estrogen receptors and bcl-2 oncogene. The P value for these factors was ú0.05 but less than 0.1 when McNemar’s x2 was applied without a continuity correction. The literature on the prognostic importance of estrogen receptors is conflicting. Creasman et al. [10] reported that hormone receptor status was an independent prognostic factor for a group of clinical stage I patients analyzed by a stepwise proportional hazards regression method. Horvath et al. [11] found that low amounts of estrogen receptor were significantly related to higher cancer relapse rates in a small group of 65 patients with stage I and II disease. On the other hand, Morris et al. [12] noted that hormone receptor content did not add independent prognostic information concerning lymph node metastasis, and others have found no correlation between estrogen receptors and survival [13]. bcl-2 is a proto-oncogene responsible for a protein that inhibits apoptosis, a regulated form of cell death. Its study has mainly involved lymphomas and leukemias [14–16], and to our knowledge, our study is the first to report the relationship of bcl-2 and the prognosis of endometrial cancer. Although not reaching statistical significance, the presence of this oncogene in our case group seems relevant and suggests that it may be prognostic for nodal metastasis or cancer recurrence. The nearly significant association warrants further study. Both HER-2/neu and p53 are oncogenes reported to influence the prognosis of endometrial cancer; yet again, the literature is contradictory. Boyd and Risinger [17] found no evidence of HER-2/neu overexpression in any of 11 endometrial cancer cell lines, while Sato et al. [18] detected the expression in 29% of 30 cancer specimens. Although univariate statistical analysis has shown a relationship between HER-2/neu and survival or recurrent cancer, multivariate analysis has not proven a significance for this relationship [7, 19]. Similar inconsistent data exist for p53. While Pisani et al. [7] found p53 to be the strongest prognosticator of survival in a multivariate analysis, the multivariate method of Lukes et al. [19] found no significant association between p53 and recurrent cancer. Our matched-pair case–control analysis also revealed no relationship between the oncogenes, p53 and HER-2/neu, and our patients who had nodal metastases or cancer recurrence. Nondiploid DNA content also was not significantly related to our patients who had nodal metastasis or recurrent cancer. Others also have provided data that indicate lack of prognostic significance for DNA ploidy. Sorbe et al. [20] concluded

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from 106 cases that DNA ploidy did not add significant prognostic information for tumor-related survival. Thornton et al. [21] found that ploidy status was related to grade and survival, but it was not an independent predictor of survival in 257 endometrial cancers. On the other hand, other investigators support the concept of DNA ploidy being prognostic of survival in endometrial cancer, Pisani et al. [7] by univariate analysis, and Lukes et al. [19], Britton et al. [22], and Iversen et al. [23] by multivariate analysis. However, the close association between ploidy and both histologic grade and hormone receptors [24–26] emphasizes the importance of proper control of variables and statistical analysis. Our study and a review of the literature have raised significant questions about the search for histopathological and molecular factors that might have prognostic significance for endometrial carcinoma. As mentioned earlier, possible explanations for lack of consistent conclusions are small sample sizes due to the overall good prognosis of the disease, and study design using various statistical methods. Surgical technique (node dissection) and postoperative treatment are variables that may influence outcome, and thereby require controlling in statistical analysis. In addition, technique of staining for molecular factors may be important. We observed that the grading of immunohistochemical staining for molecular factors is subjective, and thus the decision for what degree of staining constitutes a significant presence of oncogene or hormone receptor might bias the results. Vascular space invasion is the only pathological factor that has consistently shown a significant correlation with nodal metastasis, cancer recurrence, and survival. FIGO staging should incorporate VSI as it does grade and myoinvasion. Solving the search for other histopathological and molecular prognostic factors may require a multi-institutional study using carefully controlled variables. REFERENCES 1. Schlesselman JJ: Case–Control Studies: Design, Conduct, Analysis, in Monographs in Epidemiology and Biostatistics, Oxford, Oxford Univ Press, 1982, pp 120–123 2. Aalders JG, Syde R, Poppema S, Szabo BG, Janssens J: Prognostic factors and changing trends in the treatment of stage I endometrial cancer: a clinical and histopathological study of 182 patients. Int J Radiat Oncol Biol Phys 10(11):2083–2088, 1984 3. De Gois NM, Martins NV, Abrao FS, De Lima GR, Alves AC: Peritumorous lymph–vascular invasion, grade of histologic differentiation, and myometrial infiltration as prognostic factors of endometrial carcinoma (review). Rev Paulista Med 111(3):385–390, 1993 4. Kadar N, Malfetano JH, Homesley HD: Determinants of survival of surgically staged patients with endometrial carcinoma histologically confined to the uterus: implications for therapy. Obstet Gynecol 80(4):655–659, 1992 5. Kaku T, Tsuruchi N, Tsukamoto N, Hirakawa T, Kamura T, Nakano H: Reassessment of myometrial invasion in endometrial carcinoma. Obstet Gynecol 84(6):979–982, 1994 6. Lampe B, Kurzl R, Hantschmann P: Prognostic factors that predict

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