Material and Method: The expression of IMP3 in 91 patients with benign urothelium (20 cases), low grade invasive (17 cases) / noninvasive (20 cases) urothelial carcinoma and high grade invasive (20 cases) / non-invasive (14 cases) urothelial carcinoma was evaluated by immunohistochemistry in this study.

Results: IMP3 was not expressed in benign urothelium, low-grade non-invasive urothelial carcinoma and high grade non-invasive urothelial carcinoma. Expression of IMP3 was found in 11.76% of low-grade invasive urothelial carcinomas and 55% of high grade invasive urothelial carcinomas. Statistical analysis including χ2 tests showed that IMP3 expression of invasive urothelial carcinomas was statistically significant (p<0.000).

Conclusion: The detection of IMP3 only in invasive carcinomas although some of them were low grade showed that the expression of IMP3 may be related to aggressive behavior of urothelial carcinomas.

Our aim in this study was to determine the IMP3 expression profile in benign urothelium and urothelial carcinomas of the bladder and evaluate its relationship with the histopathological stage and grade of the tumor.

The clinical information of the patients such as age, gender, number of tumors, tumor diameter, and type of biopsy were obtained from the pathology reports, cystoscopy reports and patient files.

All sections stained with H&E were assessed for prognosisrelated parameters such as tumor histopathological stage and grade and lymphovascular invasion and re-evaluated according to the World Health Organization/International Society of Urologic Pathology 2004 Classification¹.

The sections that best reflected tumor tissue were determined for all cases and consecutive serial sections 4-5 μm thick were obtained from their relevant paraffin blocks for immunohistochemical IMP3 evaluation.

Section 4-5 μm thick were obtained onto electrostaticcharged slides (X-traTM, Surgipath Medical Industries, Richmond, Illinois, USA) from tissues that had been fixed in formalin and embedded in paraffin, and these were then dried at 60°C for at least two hours. The whole staining procedure including the deparaffinization and antigen exposure steps was performed on the Ventana, BenchMark XT fully automatic immunohistochemistry staining device. Counterstaining was completed on the device with Hematoxylin and blue-dying solution. The immunohistochemistry staining protocol was completed with dehydration, clarification with xylene and closure with a coverslip of the sections. The primary antibody used was the IMP3 monoclonal antibody (dilution: 1/100, Clone: 69.1, Code: L523S, Dako SA, Glostrup, Denmark). The cytoplasmic staining observed in neuroendocrine lung carcinoma tumor cells for IMP3 in was used as positive control.

The tumor areas with the densest positive staining and thinnest section were chosen by screening the whole section with the 10x magnification of the microscope (Eclipse E200, Nikon, Japan) for each case when evaluating the immunohistochemical staining for IMP3 antibody. All cytoplasmic staining areas found in tumor cells on the sections were considered positive whether mild, moderate or severe.

All analyses were performed with the SPSS software (version 16.0, SPSS Inc., Chicago, IL, USA) and the χ² test. A p value <0.05 for a result was accepted as significant

We found no IMP3 staining in 20 cases diagnosed with lowgrade non-invasive urothelial carcinoma (Figure 1) and 14 cases with high-grade non-invasive urothelial carcinoma (Figure 2) along with 20 cases with benign urothelium. Staining was positive for IMP3 (Figure 3) in 2 (12.5%) of the 16 pT1 tumor cases in the low-grade invasive urothelial carcinoma group while there was no IMP3 staining in one pT2 tumor case. These two IMP3-positive low-grade invasive urothelial carcinoma, pT1 cases had both superficial and deep moderate staining. In the high-grade invasive urothelial carcinoma group, 9 of the 12 pT2 tumor cases (75%) were IMP3 positive (Figure 4) while 2 of 9 pT1 tumor cases (25%) were also IMP3 positive (Table I). The staining was deep and strong in 6 of the 9 pT2 cases in the IMP3-positive high-grade invasive urothelial carcinoma group while it was superficial and weak in 2 and superficial and strong in 1. The IMP3 staining of the 2pT1 cases in the same group was deep and strong in one and superficial and weak in the other.

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Figure 1: Low-grade non-invasive urothelial carcinoma (H&E; x200).

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Figure 2: High-grade non-invasive urothelial carcinoma (H&E; x200).

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Figure 3: Low-grade invasive urothelial carcinoma (H&E; x400).

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Figure 4: High-grade invasive urothelial carcinoma (H&E; x200).

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Table I: Distribution of cases by IMP3 expression

Evaluation according to these data showed that 11.76% of low-grade invasive urothelial carcinoma cases and 55% of high-grade invasive urothelial carcinoma cases had IMP3 expression while there was no staining in low/high grade non-invasive tumors and benign urothelium. The statistical analysis with the χ² test according to this distribution showed a statistically significant relationship between invasive tumors and IMP3 staining (p<0.000).

Invasive and non-invasive urothelial bladder cancers are known to develop through two separate pathways⁶. A partial or total loss of chromosome 9q is a frequently encountered genetic event in low- or high-grade bladder tumors^1,6. Loss of heterozygosity in chromosome 11p is seen in approximately 40% of some pT1 tumors including pTa tumors while it is more common in tumors with high histopathological stage and grade⁶.

Low-grade pTa tumors are genetically stable. Low-grade (pTa) tumors are known to develop following events that activate the Mitogen-activated Protein Kinase pathway such as H-ras mutations and Fibroblast growth factor receptor 3 mutations⁶. The cyclin-dependent kinase inhibitor p16/CDKN2 (INK4a), an important cell cycle regulator and tumor suppressor gene, also plays an important role in the multistep carcinogenesis of papillary tumors³.

Invasive tumors are genetically unstable tumors⁶. A disturbance in the cell cycle control mechanism following DNA methyl transferase I upregulation and defects in the p53 and Rb pathways are accused of playing a role in the development of invasive tumors^3,6. Missense mutations found in the p53 tumor suppressor gene have been detected in approximately 50% of bladder tumors with an unfavorable prognosis^1,4. Overexpression of epiregulin that acts as a ligand for the epidermal growth factor receptor (EGFR) has also been shown to cause invasive and metastatic tumor development⁷.

IMP3 is an oncofetal protein of the insulin-like growth factor (IGF)-II m-RNA binding protein family, just like IMP1 and IMP2⁸. Members of the IMP family are proteins that play an important role in RNA movement and stabilization, cell growth and cell migration in the early stages of embryogenesis⁹. IMP3 is equivalent to the K homolog domain containing protein overexpressed in cancer (KOC) protein cloned from pancreatic tumors¹⁰. IMP3, is secreted from the developing epithelium, muscle and placenta during human and mouse embryogenesis. It has been reported to be secreted at undetectably low levels from adult tissues^8,9.

Expression of the IMP3 gene has first been defined by Gress et al. in pancreas cancers in 1996¹¹. IMP3 is secreted in many other malignant tumors such as lung, gastric and colon cancers, renal cell carcinomas and soft tissue sarcomas while it is not expressed in benign tissues neighboring the tumor (Table II). Many studies have shown that IMP3 is associated with advanced stage and aggressive tumor behavior in the tumors it is secreted from^3,4,8-10. These results indicate that the oncofetal protein IMP3 has a critical role in regulation of cell proliferation and tumor invasion. The role played by IMP3 in carcinogenesis is through stimulation of thyrosine phosphorylation by the IGF-I receptor and regulation of IGF-II gene expression through binding to the 5’-3’ mRNA region of IGF-II⁴. The thyrosine-phosphorylated IGF-I receptor sends mitogenic signals to the cell and stimulates cell proliferation and tumorigenesis³.

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Table II: IMP expression in various tissues

We did not find IMP3 expression in benign urothelium or low- and high-grade non-invasive tumors in our study. IMP3 expression was present in 25% of the pT1 urothelial carcinomas and 75% of the pT2 urothelial carcinomas in the high-grade invasive group. IMP3 expression was found in 12.5% of the pT1 urothelial carcinomas in the low-grade invasive group while it was not present in only one pT2 urothelial carcinoma case in this group. This may seem to contradict the expression observed in the pT1 and pT2 urothelial carcinomas in the high-grade invasive group but the one negative IMP3 case in pT2 urothelial carcinomas may be due to tissue processing. The higher expression rate of IMP3 in deep invasive (pT2) urothelial carcinomas and the lack of expression in low- and high-grade noninvasive urothelial carcinomas and benign urothelium were statistically significant (p<0.000). Our results are consistent with those reported in the literature and indicate that it may be associated with aggressive tumor behavior in urothelial carcinomas.

The 2007 study by Li et al. did not detect IMP3 expression in 99% of cases with a diagnosis of non-neoplastic urothelium or low-grade urothelial tumor while there was strong diffuse cytoplasmic staining with IMP3 in 54% of the cases that had been diagnosed as high-grade urothelial tumor. They found a statistically significant difference regarding IMP3 expression between high-grade tumors and non-neoplastic urothelium and high-grade tumors and low-grade tumors similar to these findings. They also found a positive IMP expression rate of 49% in superficial (pTa and pT1) tumors and 78% in deep invasive tumors (pT2 or higher tumors). However, this difference was not statistically significant. Li et al. state in light of these findings that IMP3 may play a role in tumor progression more than in tumor initiation³.

A study by Sitnikova et al. in 2008 investigated the biopsy material of 214 patients who received a diagnosis of urothelial carcinoma of the bladder regarding the IMP3 expression profile. They found no IMP3 expression in benign urothelial tissues adjacent to the urothelial carcinoma. IMP3 expression was found in 16% of Ta urothelial carcinomas, 35% of T1 urothelial carcinomas and 36% of T1 urothelial carcinoma in situ cases among the superficial urothelial carcinoma group while it was present in 93% of metastatic urothelial carcinomas. IMP3 expression was found to correlate between the markers known to be unfavorable prognostic factors (age, gender, tumor size, tumor multicentricity, tumor histopathological stage and grade, response to intravesical treatment) in superficial urothelial carcinomas. Superficial urothelial carcinomas that expressed IMP3 were found to have 6 times the risk of progressing to deep invasive cancer or metastasize compared to those that did not express IMP3. Metastatic disease was found during follow-up in 60% of IMP3 positive cases that received a diagnosis of T1 urothelial carcinoma while metastasis was not found in any IMP3 negative case. Sitnikova et al. state in light of these findings that IMP3 may play a direct role in the progression and metastasis of urothelial carcinomas⁴.

The 5-year survival among high-risk superficial urothelial carcinomas that undergo early cystectomy is about 90%^23,24. However, the quality of life decreases markedly in patients who undergo cystectomy and the patients can also die because of cystectomy-related complications^2,25,26. It is therefore important to determine in the early stages the patients with superficial urothelial carcinoma who have high risk and whose tumors will have an unfavorable prognosis. The assessment of IMP3 expression can be a helpful marker to determine the disease course beforehand and decide on the best therapeutic option when it is difficult to choose between intravesical treatment and cystectomy options in superficial urothelial carcinomas and especially when the biopsy sample is small and superficial and does not contain muscularis propria. IMP3 immunohistochemical staining is also an inexpensive and reliable method that is easy to use in routine practice. The material obtained from TUR used in the treatment of superficial urothelial carcinomas is also an appropriate material for routine immunohistochemical analysis. The use of standard staining protocols and fully automatic staining devices can minimize any conflicts between staining results.

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