IGF2BP3 (IMP3) Expression Is a Marker of Unfavorable Prognosis in Ovarian Carcinoma of Clear Cell Subtype

Martin Köbel; Haodong Xu; Patricia A. Bourne; Betsy O. Spaulding; Ie-Ming Shih; Tsui-Lien Mao; Robert A. Soslow; Carol A. Ewanowich; Steve E. Kalloger; Erika Mehl; Cheng-Han Lee; David Huntsman; C. Blake Gilks


Mod Pathol. 2009;22(3):469-475. 

In This Article

Materials and Methods

Patients and Tumor Specimens

IGF2BP3 expression was assessed for 475 ovarian carcinomas from a population-based cohort from British Columbia and a validation set of 150 ovarian clear cell carcinomas from three other institutions of North America: these cohorts were described earlier.[14] Briefly, the cohort from British Columbia was obtained from a population of approximately four million people in British Columbia. For the period 1984–2000, 2555 patients with ovarian carcinoma were registered in the Cheryl Brown Ovarian Cancer Outcomes Unit, which records a large majority of cases of ovarian carcinomas in the region. A total of 834 patients without macroscopic residual disease after surgery were selected for further study. Ninety-one patients with excellent prognosis (grade 1, stage 1a or 1b) were excluded from the study; only 3% of women in this group died of disease during the follow-up period. As clear cell carcinomas are by definition grade III, no clear cell carcinomas were excluded. After a full gynecopathological review[11] according to WHO criteria,[13] 541 tissue blocks were available and used for tissue microarray construction. A representative area of each tumor was selected and duplicate 0.6-mm tissue cores were punched to construct a tissue microarray (Beecher Instruments, Silver Springs, MD, USA). A review after tissue microarray construction revealed that an additional 23 cases were not sampled adequately. Also excluded were 18 cases of rare histological type (including seven undifferentiated, six transitional, and one squamous carcinoma) and five histologically unclassified cases. The serous subtype was further subdivided into low and high grade,[15] and all 11 low-grade serous carcinomas were excluded because of insufficient numbers for subtype analysis. This selection resulted in a study population of 489 cases belonging to one of the four major cell types (high-grade serous, clear cell, endometrioid, and mucinous). Table 1 shows the clinicopathological data from 475 patients for whom IGF2BP3 expression data were assessable.

The validation set was composed of 150 cases in which pure clear cell carcinomas were selected after a file search in the period from 1980 to 2006. These cases were obtained from three institutions (Johns Hopkins University, Baltimore, MD, USA, N = 69, University of Alberta, Edmonton, Canada, N = 42, Memorial Sloan-Kettering Cancer Center, New York, NY, USA, N = 39).

To validate immunohistochemistry by analysis of mRNA expression, fresh frozen as well as paraffin-embedded tissue was obtained for 35 ovarian tumors from the Vancouver General Hospital tumor bank from patients who were undergoing surgery during 2004–2005.[16] These cases include the following subtypes: high-grade serous (N = 27), clear cell (N = 3), endometrioid (N = 4), and one serous borderline tumor. Approval for the study was obtained from the Research Ethics Board (H04-60102).

Treatment and Outcome

Minimal staging with inspection and palpation of all peritoneal surfaces and the retroperitoneal area, biopsies of any suspect lesions for metastases, peritoneal washing, and infracolic omentectomy was the standard of care in all the four institutions. The majority of patients were treated during a time period when current comprehensive surgical staging was not the standard of care, and information regarding extent of surgical staging is missing in a significant number of cases. Hence, these cohorts must be considered as incompletely staged, with an approximately 25% risk that residual tumor was left behind after primary surgery.[17] Patients with ovarian carcinoma from the British Columbia cohort received adjuvant therapy according to the provincial treatment guidelines of the British Columbia Cancer Agency that changed over the years[18] and are available at http://www.bccancer.bc.ca/PPI/TypesofCancer/Ovary/default.htm. Similarly, the treatment regimens for ovarian clear cell carcinomas of the other centers were revised over time and are shown in Supplementary Table 1; adjuvant therapy for ovarian clear cell carcinoma was consequently quite heterogeneous. The study endpoint was defined as disease-specific survival. Disease-specific survival was defined as ovarian carcinoma-specific death, in which ovarian cancer was the primary or underlying cause of death. Death from concurrent disease (ie second malignancy) was coded as 'died of other cause'; death resulting from toxicities relating to treatments for ovarian carcinoma was coded as 'died of toxicities' and these patients were censored for disease-specific survival. This information was available for all (100%) patients of the British Columbia cohort (Cheryl Brown Ovarian Cancer Outcomes Unit) and 91.3% of patients from the validation set. Mean follow-up time was 5.9 years (British Columbia cohort) and 4.6 years (validation set).

Immunohistochemistry and Scoring

Serial 4-µm sections were cut for immunohistochemical analysis. After heat-antigen-induced retrieval, staining was carried out on an automated system as per the manufacturer's protocol (DAKO, Carpenteria, CA, USA). The expression of IGF2BP3 in ovarian carcinomas was analyzed by immunohistochemistry using a mouse monoclonal antibody against IGF2BP3 (clone 69.1, dilution 1:100, DAKO, Carpenteria, CA, USA).[19] Normal fallopian tube, ovarian surface epithelium, ovarian stroma, and endometrium were non-immunoreactive and were used as negative controls. A malignant melanoma sample with known IGF2BP3 expression served as a positive control. IGF2BP3 staining on tissue microarrays was scored by a pathologist (MK) blinded to clinical outcome. The cut-off point for positive cases was any convincing cytoplasmic expression in more than 5% of tumor cells. Comparison between the results of tissue microarray assessment of IGF2BP3 staining and full-section immunohistochemical assessment was done in 22 cases (20 that were negative on tissue microarray assessment and two that were positive). Results of assessment of full sections were concordant in 21/22 cases; a single case that was negative on tissue microarray assessment showed focal positivity on full section staining.

Antibody Confirmation

The Human Exonic Evidence Based Oligonucleotide microarray (HEEBO) (Stanford, CA, USA) was used to examine the mRNA expression profiles of 35 ovarian carcinomas. After confirmation of the presence of viable tumor by frozen section, total RNA from the tumor samples was extracted using mirVana™ RNA isolation kit (Ambion, Austin, TX, USA). The total RNA was reverse transcribed into cDNA using a mixture of oligo dT (Operon, HPLC purified) and random hexamer (Amersham, Cat 27-2166-01) primers with incorporation of amino allyl-dUTP (Ambion 8439). Cy3 and Cy5 dyes (Amersham RPN 5661) were used for indirect labeling of the cDNA from reference RNA (Stratagen, Universal Human Reference RNA, Cat 740000) and cDNA from tumor specimens, respectively. Microarray hybridization and washing were carried out using standard procedures.[20,21] Microarrays were scanned on a GenePix 4000 microarray scanner and fluorescence ratios (tumor/reference) were calculated using GenePix software. Only spots with a ratio of signal over background of at least 1.5 in the Cy5 and 1.5 in the Cy3 channel were included. Gene centering was applied to the expression values for this series of tumors. Our current analysis was restricted to the expression level of IGF2BP3 (IMP3) mRNA and was correlated to the status of protein expression by immunohistochemistry.

Statistical Analysis

Univariate disease-specific survival analysis was carried out by generating Kaplan–Meier curves and differences were assessed with the log-rank statistic. Multivariable disease-specific survival analysis was assessed with the Cox proportional hazards regression model. Differential expression of IGF2BP3 across the four histopathological subtypes was assessed with contingency analysis and statistical differences were quantified using the Pearson's chi-square statistic. For all analyses, a P-value < 0.05 was considered statistically significant. Statistical analyses were carried out using SPSS software (version 15.0; SPSS, Chicago, IL, USA).


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