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Prospective Multicenter Validation Confirms the Prognostic Superiority of the Endometrial Carcinoma Prognostic Index in International Federation of Gynecology and Obstetrics Stage 1 and 2 Endometrial Carcinoma

Jan P.A. Baak, Wim Snijders, Bianca van Diermen, Paul J. van Diest, Fred W. Diepenhorst, Jantine Benraadt

From the Department of Pathology, Rogaland Central Hospital, Stavanger, Norway; Vrije Universiteit; Vrije Universiteit Medical Center; Comprehensive Cancer Center Amsterdam, Amsterdam; and Department of Gynecology, Medisch Centrum Alkmaar, Alkmaar, The Netherlands.

Address reprint requests to Jan P.A. Baak, MD, PhD, Department of Pathology, Rogaland Central Hospital, PO Box 8100, 4068 Stavanger, Norway; e-mail: baja{at}sir.no.

	ABSTRACT

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Purpose: To validate the prognostic value of the endometrial carcinoma prognostic index (ECPI; combined myometrium invasion, flow cytometric DNA ploidy, and morphometric mean shortest nuclear axis [MSNA]) versus classic prognosticators.

Patients and Methods: Prospective multicenter ECPI analysis was conducted in 463 endometrial carcinomas with a median of 6.5 years (range, 1 to 10 years) follow-up, review of pathology features, and univariate (Kaplan-Meier) and multivariate (Cox) analyses.

Results: Initial routine and review diagnoses varied considerably (invasion depth, 11%; type, 20%; grade, 34%; vessel invasion, 72%); the review diagnoses were stronger prognostically. In International Federation of Gynecology and Obstetrics stage 1 (after histopathologic examination; pFIGO-1; n = 372; 38 deaths occurred as a result of disease [10.2%]), DNA ploidy was prognostic in hysterectomies (P < .00001) but not in curettages (P = .06). ECPI was a stronger prognostic indicator than other features. ECPI, MSNA, and DNA ploidy were also prognostic in pFIGO-1B and -1C subgroups. Multivariate analysis in pFIGO-1 showed that uterine MSNA versus > 7.93 µm (hazard ratio [HR], 3.4) and grade (as 1 + 2 v 3; HR, 2.6) added to the ECPI (HR, 32), but only in patients with an unfavorable ECPI of > 0.87. Adjuvant radiotherapy was not an independent prognostic factor in any of the subgroups. In pFIGO-2 (n = 46), ECPI, DNA-ploidy, and age ( 64, > 64 years) were significant. In FIGO-3 (n = 31) and FIGO-4 (n = 14), none of the classic or other features analyzed was of prognostic value, which explains why in previous studies using different mixtures of FIGO stages, DNA ploidy prognostic results varied.

Conclusion: In endometrial carcinoma, DNA-ploidy is prognostic in hysterectomy and not in curettage samples. The ECPI is prognostically much stronger than the classic features widely used for therapy triage in pFIGO-1 and -2.

	INTRODUCTION

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ENDOMETRIAL CARCINOMA is the most common gynecologic cancer and International Federation of Gynecology and Obstetrics stage 1 (after histopathologic examination; pFIGO-1) has good cure rates of 90% and higher. However, the disease-related death rate in FIGO-2 to -4 is high (25% to 80%), and is 5% to 20% even for the early-stage FIGO-1 patients. Moreover, for decades, the disease-related death rate for patients with pFIGO-1 has been stable.¹ This motivates the development of other prognostic determinants for more accurate triage of patients through various treatment modalities and to provide better insight into the cell biology of the disease. FIGO stage is prognostically strong but most patients are FIGO-1.² In these early-stage cancers, age, histologic type, grade, and myometrium invasion depth (MI) predict outcome; grade and MI are often used to determine individual therapy, but reproducibility of grade is far from optimal.^1,3

Many alternative factors are promising prognostic indicators, such as neovascularization,⁴ p53,⁵ proliferation factors,^1,6,7 steroid receptors,⁸ p16,⁹ plasminogen activator inhibitor,¹⁰ tumor-associated macrophages,¹¹ high stromal macrophage thymidine phosphorylase expression,¹² vascular endothelial growth factor,¹³ and others. However, most of these studies exhibit methodologic shortcomings (the methods have mixtures of different stages, small numbers, no independent prognostic validation, or poor reproducibility, or are not formalized with fixed decision thresholds). DNA ploidy and certain nuclear morphometric features repeatedly have been proven to be well reproducible and strongly prognostic in independent analyses.^1,14–17 In one prospective FIGO-1 cancer study, a multivariate combination of MI, DNA ploidy, and the morphometric mean shortest nuclear axis (MSNA; the combination forms the endometrial carcinoma prognostic index [ECPI]) prognostically overshadowed grade, type, and estrogen receptor status.¹ This was confirmed in a subsequent study.¹⁷ In these studies, the prognostic difference between FIGO-1 patients with an ECPI 0.87 (favorable) and more than 0.87 (unfavorable) was impressive (99% v 55% 8-year survival). Others confirmed that combined DNA ploidy and MSNA was prognostically strong.¹⁶ This could have therapeutic consequences because the technology is simple and highly cost effective.¹⁸ However, good laboratory practice requires independent prospective multicenter validation of a laboratory test with therapeutic implications.¹⁹ Therefore, we evaluated prospectively the ECPI, MI, DNA ploidy, and MSNA in comparison with other prognosticators in consecutive endometrial carcinoma patients.

	PATIENTS AND METHODS

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All 601 patients that presented clinically with primary endometrial malignancies diagnosed from 1986 to 1990 in 22 hospitals of the Comprehensive Cancer Center Amsterdam and Stedendriehoek Twente (together covering 12% of the Netherlands) have been enrolled prospectively. In accordance with the routine procedures in the hospitals, surgery was performed shortly after the initial diagnosis was made by curetting unless the patient’s condition or stage of disease did not allow surgery (which happened in 16 patients). This left 585 patients: 478 clinically FIGO-1 (80%), 39 FIGO-2 (6%), 60 FIGO-3 (10%), and eight FIGO-4 (1%). Diagnostic and therapeutic procedures were applied according to local practice at the various institutions. Patients considered to have FIGO-1 or -2 cancers did not receive preoperative radiotherapy (RT); surgical treatment was total abdominal hysterectomy with bilateral salpingo-oophorectomy but no lymphadenectomy or extensive staging. In FIGO-3 to -4 patients, RT was usually given as primary therapy. In the pFIGO-1 to -2 patients, adjuvant postoperative RT depended on stage and grade of the pathology report. The pathologic material was fixed in 4% buffered formaldehyde, dehydrated, embedded in paraffin, cut into 4-µm sections, and stained with hematoxylin and eosin (HE). Follow-up data were collected annually. Fifty-one of the 585 malignancies histopathologically were nonendometrial malignancies and were therefore excluded. In 30 other patients, the amount or quality of the material (poor fixation or severe autolysis) was inadequate for analyses other than confirmation of carcinoma. In 42 patients (usually those > 80 years old), the primary or follow-up data were inadequate (< 10 months; in most, < 6 months), leaving 462 patients. The median age of the patients was 66 years (range, 26 to 91 years); median follow-up was 79 months (range, 10 to 117 months).

Cervical invasion, MI, and histologic grade and type were routinely assessed in each center and re-evaluated by two independent gynecopathologists (J.B., P.D.). In agreement with other studies,^20,21 assessment of vessel invasion was poorly reproducible and was discontinued. Discrepancies between the initial routine and review diagnoses were considerable (for MI, 11%; histologic type, 20%; grade, 34%); the review diagnoses were stronger prognostically and these were used for additional study. At review (FIGO 1988²), there were 372 pFIGO-1 (80%), 46 pFIGO-2 (10%), 31 pFIGO-3 (7%), and 14 pFIGO-4 cancers (3%). For typing and grading, the worst differentiated well-preserved tumor area per patient was selected (from both the curetting and the hysterectomy).

The following criteria were applied for grading: less than 5% poorly differentiated tissue was designated as well differentiated, grade 1; 5% to 50% poorly differentiated tissue was designated as grade 2; and more than 50% poorly differentiated tissue was designated as grade 3. For assessment of the MI, all sections were investigated to ensure analysis of deepest invasion and were classified as more (pFIGO-1C) or less (pFIGO-1B) than one half of the myometrium thickness or no infiltration at all (pFIGO-1A; the latter only after a repeated search by the two reviewing pathologists, and under the condition that there were unambiguous cytologically atypical malignant glands with characteristic architecture).²² The depth of myometrial invasion was the ratio of the thickness of the invasive part divided by the total wall thickness, and this criterion was applied. However, if the cancer infiltrates over a broad front, the original myometrial wall thickness cannot always be determined with certainty. In such cases, the approximation of the deepest vascular plexus can provide diagnostic support and result in better agreement between pathologists. Deep invasion was confirmed with the following criterion: approximation of cancer cells to the serosa or to the deep myometrial vascular plexus. This additional diagnostic criterion was used to classify a patient case as FIGO-1C. The FIGO-2 category was assigned if invasion in the cervix was observed.

Quantitative Image Analysis and DNA Cytometry
Nuclear morphometric analysis of the representative HE sections used for revision grading was performed with the motorized QPRODIT 6.1 image analysis system (Leica, Cambridge, UK) as described.^17,23 Because this investigation was a validation study, we measured the MSNA only, with the straight-line-length module of QPRODIT at 1,800x screen magnification,²⁴ using rigid point-weighted systematic random sampling.²⁵ This guarantees unbiased high reproducibility and stronger prognostic value.²³ At least 50 and at most 225 (median, 79) nuclei were measured per patient case (one nucleus per field of vision) and the MSNA was calculated. The coefficient of error was continuously calculated: if it was less than 5%, the measurement was terminated but only if at least 50 nuclei were measured. Intra- and interobserver reproducibility of this method previously was proven to be high.²⁶ Figure 1 illustrates assessment of the MSNA.

View larger version (54K): [in this window] [in a new window]   Fig 1. Highly reproducible mean shortest nuclear axis assessment takes approximately 5 minutes. (A) Grid-guided nuclei selection diminishes bias; (B) shortest nuclear axis measurement (numbers refer to panel A): 1 = 7.7, 2 = 6.0, 3 = 6.7, and 4 = 9.3 µm. (C) Measurements are terminated if the coefficient of error of the running mean is less than 5%.

ECPI
ECPI was calculated as follows:¹ =+ 0.6494 x (mean shortest nuclear axis, in micrometers, with one decimal) + 0.6939 x (DNA ploidy coded as 1 = diploid, 2 = tetraploid, 3 = aneuploid) + 0.2398 x (MI coded as 1 = < 0.5 and 2 0.5) - 5.7283

where ECPI 0.87 means a good prognosis and ECPI more than 0.87 means a poor prognosis.

Statistics
SPSS 11 for Windows (SPSS Inc, Chicago, IL) was used. Event-free survival was taken as the primary (curetting) diagnosis date until death or recurrence. The date of death or last follow-up was the end point and the cause of death was recorded. Patients who died as a result of nonendometrial cancer–related causes or were no longer observed without signs of recurrent disease were censored at the last follow-up date. Variables analyzed were pFIGO stage, age, revised grade and type, MI, postoperative RT, DNA ploidy, MSNA, and the ECPI. Histologic types were combined as 15 favorable (adenoacanthoma, n = 10; mucinous, n = 3; and secretory adenocarcinoma, n = 2), 294 intermediate (endometrioid, n = 293), and 63 unfavorable (adenosquamous, n = 33; serous papillary, n = 12; undifferentiated, n = 1; clear cell, n = 15; glassy cell, n = 1; or combined clear cell and glassy cell, n = 1). Continuous variables were divided into two, three, four, or five groups of approximately the same size using thresholds of the median, tertiles, quartiles, or pentiles, or thresholds from previous studies (Table 1). When neighboring subgroups had the same survival they were grouped as one. Survival analysis was performed (Kaplan-Meier). The hazard ratios (HRs) and the 95% CIs were calculated (HRs in subgroups without events could not be calculated). Multivariate analysis (Cox model) was performed to identify the best prognostic feature combination. If features with equal characteristics were identified, well-reproducible features were given priority.

	RESULTS

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View larger version (19K): [in this window] [in a new window]   Fig 2. Kaplan-Meier survival curves by International Federation of Gynecology and Obstetrics staging system (after histopathologic examination; pFIGO).

View larger version (33K): [in this window] [in a new window]   Fig 3. Kaplan-Meier curves in International Federation of Gynecology and Obstetrics staging system, stage 1 (after histopathologic examination; pFIGO-1) for (A) grade, (B) myometrial invasion (Myom. Inv.), (C) mean shortest nuclear axis (MSNA), (D) ploidy, and (E) the endometrial carcinoma prognostic index (ECPI). HR, hazard ratio.

View larger version (35K): [in this window] [in a new window]   Fig 4. Prognostic decision tree using endometrial carcinoma prognostic index (ECPI), mean shortest nuclear axis (MSNA), and grade. FIGO-1, International Federation of Gynecology and Obstetrics Staging System, Stage 1.

	DISCUSSION

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The results confirm that the ECPI, MSNA, DNA ploidy, and MI have exceedingly strong prognostic value in pFIGO-1 endometrial cancers, and ECPI and DNA ploidy are important prognosticators in FIGO-2 patients as well. Grade was also prognostic but not as strong as the ECPI. Moreover, reproducibility of grade is inadequate, whereas the quantitative methods are highly reproducible. Histologic type was not selected in multivariate analysis; the prognostic significance of the ECPI and its constituent features clearly overshadow the prognostic value of histologic type.

Prognostic DNA ploidy analyses have produced conflicting results in previous studies. The results of this study make clear the importance of using large unselected material before final conclusions can be drawn. Smaller patient groups carry a serious risk of selection bias and incorrect conclusions. A typical example is a recent study, in which the material consisted of a mixture of pFIGO-1, -2, -3, and -4 cancers but was biased because many pFIGO-1B grade 3, FIGO-1C, and FIGO-2 cancers in the intake period had been referred for RT and were not included. We found in the unselected material in this prognostic multicenter study that in FIGO-3 and -4 samples, DNA ploidy was not prognostic. In contrast, in the FIGO-1B, -1C, and -2 samples, the prognostic value of DNA ploidy was strong. Clearly, analysis of mixtures of different FIGO stages could either detect whether DNA ploidy and other features have prognostic value, depending on the type of patient cases selected and mixed.

It is important that the prognostic threshold of the ECPI (0.87) is the same as that in earlier studies. The thresholds for the MSNA are slightly higher than in one of the previous studies,¹ probably because of the application of newer measurement technologies used, which are based on (more highly reproducible) volume-weighted sampling with consequently higher mean values of size features. A new finding is that the measurements in the hysterectomy specimens are more prognostic than those in the curetting specimens. The estrogen receptor concentrations differ in samples from curettages and hysterectomies.¹ Perhaps the superficial cancer cells in curetting specimens are more highly differentiated than the samples taken from hysterectomies and thus provide a less representative sample from a larger tumor with less prognostic value. An alternative explanation is that the active invasive front of the cancer is in the deep infiltrating parts, which obviously can be better represented in the hysterectomy samples than in the superficial curettages.

In a general surgical pathology practice, application of the morphometric and cytometric technology in endometrial cancer could best be done with the ECPI, rather than grade-dependent selection of either the ECPI, MSNA, or DNA ploidy. The lack of reproducibility of grade is so high that grade cannot be used as an up-front criterion to select the best prognostic method. It certainly is an advantage that both the morphometric MSNA and DNA ploidy are easy to assess. MSNA is especially simple; it can be measured in standard histologic HE sections. DNA ploidy takes somewhat more time, especially the preparation of single-cell suspensions, but an advantage is that routine paraffin blocks can be used. Highly automated and well-reproducible measurement systems with high resolution also facilitate the use of quantitative methods.²⁹

One could argue that serous papillary and clear-cell cancers are nearly always aneuploid, but cell type was not an independent factor in this study. If a laboratory is not equipped for ECPI assessment, paraffin blocks can be sent to reference laboratories for analysis. Careful quality control of DNA ploidy analysis is important. Likewise, for MSNA, unbiased motorized sampling and accurate assessment of MI are essential for accurate ECPI value determinations.

It is interesting that DNA ploidy was a strong prognosticator in higher-grade cancers but not in well-differentiated carcinomas that grow superficially, in which MSNA was prognostic. In endometrial hyperplasias, DNA ploidy also was not a significant predictor of outcome,³⁰ but morphometric nuclear and glandular architectural features are strong predictors of cancer progression.¹⁸ Morphometric features in these lesions are strongly correlated to molecular-genetic monoclonality, a consistent marker of neoplastic growth.²⁶ Expert gynecopathologists using conventional light microscopy failed to distinguish mono- and polyclonal growth.²⁶ It thus seems that morphometric features are highly sensitive markers to detect subvisual underlying molecular changes long before usual subjective analysis or whole genome DNA ploidy analysis can detect these subcellular changes.

The fact that the ECPI (threshold, 0.87) and the MSNA (threshold, 7.93 µm) both are selected with multiple regression shows that they have independent additional prognostic value. Indeed, the MSNA is one of the three features of the ECPI but as a continuous variable, and the correlation coefficient between the ECPI and MSNA was only 0.48. Further analysis shows that the additional value of the MSNA at a threshold of 7.93 µm occurs only in the patients with an ECPI of more than 0.87. Clearly, this subgroup with MSNA above 7.93 µm yields information that is not contained in the ECPI formula. It may be argued that a new prognostic formula should be developed that describes the prognostic information of the MSNA in a more appropriate manner, but we then would have to pass all the different validation phases of a new laboratory test¹⁹ and the net effect would be similar to the results of the ECPI plus MSNA value at a threshold of 7.93 µm.

It is tempting to interpret the findings against a cell and molecular biologic background. What are the switches that trigger transition from highly favorable (ECPI 0.87) to unfavorable high-risk cancer (ECPI > 0.87)? Genetically mutated cells have a slight growth advantage compared with normal cells. It has been suggested that inactivation of the PTEN suppressor gene plays an essential role in endometrial carcinogenesis.³¹ As a result, neoplastically changed cells will show clonal expansion. In low-grade endometrioid adenocarcinoma, the proliferation markers mitotic and MIB-1 indices were statistically significant independent prognostic indicators strongly correlated with p53 expression in one study.⁷ Patients with endometrial cancers who have p53 overexpression had a much higher risk than those without overexpression; patients with overexpression had a seven-fold higher risk of dying as a result of disease.⁵ The ECPI also yields high HRs of approximately 30, and is composed of markers of invasion, differentiation (MSNA), and genomic instability (DNA ploidy). Lundgren et al¹⁴ found that the degree of differentiation, MIB-1, and p53 lost their prognostic capability when DNA ploidy was included, which was the strongest predictor of outcome. Because DNA ploidy is such an important prognostic factor, the prognostic value of the ECPI probably exceeds that of p53 and MIB-1. Aneuploidy as detected by flow cytometric methods is a genetic change characteristic for high mortality, irrespective of tumor presentation as pFIGO-1 or as advanced disease. Interleukin-8 is in itself not prognostic but might act as an angiogenic switch in myometrial invasion in stage I uterine endometrial cancers.³²

In conclusion, the ECPI multivariate combination of MI, DNA ploidy, and MSNA has an exceedingly strong prognostic value in pFIGO-1 and -2, and could therefore be used to select high-risk patients for adjuvant therapy. Alternatively, the ECPI could be used as a prognostically superior and highly reproducible intermediate end point biomarker; one could question if grade and MI still should be regarded as the only and best gold standard to evaluate therapeutic intervention studies. Given that RT did not improve prognosis in any of the important subgroups analyzed in this prospective study, it would be important to evaluate prospectively the prognostic effect of RT in a specially designed prospective intervention trial using the ECPI 0.87 versus more than 0.87 as the entry criterion.

	APPENDIX

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The following hospitals have participated in this study: Medisch Centrum Alkmaar, Alkmaar; Twenteborg ziekenhuis, Almelo; Academisch Medisch Centrum; Boven IJ ziekenhuis; Lucas Andreas ziekenhuis; NKI/Antonie van Leeuwenhoek ziekenhuis; Onze Lieve Vrouwe Gasthuis; Vrije Universiteit Medisch Centrum, Amsterdam; Ziekenhuis Amstelveen, Amstelveen; Gelreziekenhuizen loc Juliana en Lucas, Apeldoorn; Gemini ziekenhuis, Den Helder; Deventer ziekenhuizen, Deventer; Medisch Spectrum Twente, Enschede; Kennemergasthuis, Haarlem; Spaarne ziekenhuis, Haarlem/Heemstede; West Fries Gasthuis, Hoorn; Ijsselmeer ziekenhuis, loc Lelystad, Lelystad; Waterland ziekenhuis, Purmerend; Streekziekenhuis Koningin Beatrix, Winterswijk; and De Heel-Zaans Medisch Centrum, Zaandam, The Netherlands

	AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicated no potential conflicts of interest.

	ACKNOWLEDGMENTS

 
We thank Emily Armée, Leonard Schuurmans, Erna Matze-Cok, and Marc Broeckaert, and the many gynecologists, pathologists, technicians, and secretaries in the different hospitals who have contributed to this study.

	NOTES

 
Supported by grant 28-1203 of the National Health Research Council of the Netherlands (ZonMw), a grant of the Comprehensive Cancer Center Amsterdam, and grant 97-98 of the Stichting Bevordering Diagnostische Morfometrie.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION APPENDIX AUTHORS’ DISCLOSURES OF... REFERENCES

 
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Submitted February 19, 2003; accepted July 1, 2003.

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