|Year : 2017 | Volume
| Issue : 3 | Page : 141-147
Immunohistochemical study of the expression of Oct-4 in bladder urothelial carcinoma
Amal Asar1, Samia Gabal2, Noha Helmy1, Sara E Khalifa MD 2
1 Department of Histopathology, Medical Division, National Research Center, Cairo, Egypt
2 Department of Pathology, Faculty of Medicine, Cairo University, Cairo, Egypt
|Date of Submission||25-Jul-2017|
|Date of Acceptance||19-Nov-2017|
|Date of Web Publication||19-Feb-2018|
Sara E Khalifa
Department of Pathology, Faculty of Medicine, Cairo University, Kasr El Ainy Street, Cairo 11728
Source of Support: None, Conflict of Interest: None
A cancer stem cell model was proposed for bladder carcinoma, as there is a subpopulation of tumor cells capable of resisting conventional therapies and surviving treatment to facilitate recurrence and metastasis. Oct-4 is a pluripotency marker of stem cells, which has been found to be associated with worse prognosis in multiple somatic tumors.
We aimed to explore the expression of Oct-4 protein in urothelial carcinoma and some of its variants and also determine whether Oct-4 expression and level of expression are associated with the clinicopathological parameters such as age, sex, grade, stage, morphology, and tumor progression.
Patients and methods
We tested 84 urothelial tumor specimens including all grades, stages, and some variants of urothelial carcinoma for the expression of Oct-4. Two sections were prepared per case for histologic evaluation and immunohistochemical staining by Oct-4 monoclonal antibody. The immunostaining was evaluated semiquantitavely through obtaining an H-score for each case.
All cases took up the Oct-4 stain except for low-grade tumors and carcinoma in-situ cases. The highest percentage of positive cases was seen in invasive urothelial carcinoma with variant morphology other than conventional (19.1%). Presence of Oct-4 expression was significantly associated with grade (P=0.03), histopathologic type of urothelial tumor (P<0.001), category (P<0.001), and tumor progression (P=0.001). Conversely, the level of Oct-4 expression among positive cases was not found to be associated with any of the studied parameters.
Our study calls for considering Oct-4 as a novel marker of prognostic significance that could be implemented in target therapies for urothelial carcinoma.
Keywords: bladder urothelial carcinoma, cancer stem cells, Oct-4, tumor progression, urothelial carcinoma variants
|How to cite this article:|
Asar A, Gabal S, Helmy N, Khalifa SE. Immunohistochemical study of the expression of Oct-4 in bladder urothelial carcinoma. Kasr Al Ainy Med J 2017;23:141-7
|How to cite this URL:|
Asar A, Gabal S, Helmy N, Khalifa SE. Immunohistochemical study of the expression of Oct-4 in bladder urothelial carcinoma. Kasr Al Ainy Med J [serial online] 2017 [cited 2018 Jul 17];23:141-7. Available from: http://www.kamj.eg.net/text.asp?2017/23/3/141/225775
| Introduction|| |
Continuous cancer research shows accumulating evidence supporting the idea that tumors, similar to normal adult tissues, arise from a specific stem-cell-like population, the cancer stem cells (CSCs), which are responsible for tumor growth, the ability to metastasize, and resistance to conventional antitumor therapy .
Stem cells are ‘unspecialized cells that undergo unlimited self-renewal and multilineage differentiation to become specialized cells’. They usually constitute a small percentage of the total cell population in any tissue and are usually indolent, dividing very slowly . It is generally accepted that CSCs may be derived from transformed stem cells or progenitor cells that have regained self-renewal activity, owing to the autonomous cell-cycle control, interference of cellular stress-control mechanisms, and signaling pathways, because of the accumulated genetic and epigenetic changes .
Urothelial carcinoma has been found to originate through two different carcinogenic pathways (so-called dual-track carcinogenesis): carcinoma in-situ (CIS) track, which has a greater metastatic potential, with a poor prognosis, and the papillary tumor track, which generally has a better prognosis .
Castello-Martin et al.  reported that the molecular changes peculiar to the papillary neoplasms are usually gain-of-function mutations affecting H-RAS, FGFR3, and PI3K, and deletions of the long arm of chromosome 9 (9q); however, CIS is characterized by loss-of-function mutations affecting tumor suppressor genes such as p53, RB, and PTEN. Thus, each of these patterns of urothlial carcinoma must be arising through different oncogenic pathways . This explains a lot about their different behavior and phenotype. It was suggested that these profound differences between papillary and invasive cancers to consist in the particular initiated cell. Thus, papillary carcinoma may originate from a transit amplifying cell, as they predominantly differentiate into the intermediate cell phenotype, which has restricted replicative potential and does not usually participate in epithelial–stromal interactions that could facilitate invasion, whereas invasive tumors might originate from an initiated urothelial stem cell, as such tumors usually show heterogeneous phenotypes which could be attributed to the pseudodifferentiation process of the CSC pool. However, the exact cell of origin of urothelial cancer − in either subtype − is still undetermined ,.
The presence and quantity of urothelial carcinoma stem cells in each case may as such carry important prognostic information that might allow a rationale for stratification of patients with urothelial cancer and probably pave the way for CSC targeted therapy .
In cancer biology, the relationship between embryogenesis and oncogenesis has long been a prevailing theme. Cancer cells are similar to very early embryonic cells, which are immortal, undifferentiated, and invasive. It is important to study genes associated with embryogenesis and discover their potential for tumorigenesis. One of these genes is the one coding the POU (Pit-Oct-Unc) family of transcription factors, of which Oct-4 (POU5F1) is a member. This family acts as a key regulator of pluripotency in early stages of mammalian development, acting within a very narrow biological limit. As such, a critical amount of Oct-4 is required to sustain self-renewal of embryonic stem cells and any upregulation or downregulation induces divergent cell fates .
Here, we examined the expression of Oct-4 protein in urothelial carcinoma and some of its variants, aiming at determining whether Oct-4 expression and level of expression are associated with any clinicopathological prognostic parameters.
| Patients and methods|| |
Study design and case selection
This study included 84 archived paraffin blocks of urothelial carcinoma cases collected from January 2013 till February 2016. All procedures performed were in accordance with the ethical standards of the research committee of both Kasr Alainy and National Research Centre. The specimens included the various grades and stages of urothelial carcinoma and some of its variants; which were selected in accordance with the modified 2004 WHO/ISUP classification  and the American Joint Committee on Cancer (7th edition) staging . Forty-three tumor specimens were obtained from archived paraffin blocks of TURT specimens, which were used to collect stages Ta, Tis, and T1. The remaining 41 cases were obtained through selected sections from radical cystectomy specimens. Sections were usually selected from the edge of tumor nodules at the maximum depth of infiltration. Staging employed stratification according to the T category (tumor category) rather than the entire pathological stage.
Serial sections of 5 µm thick were prepared from the formalin-fixed paraffin blocks, deparaffinized in xylene, cleared in alcohol, then mounted on glass slides, and stained by hematoxylin and eosin for histological re-evaluation.
Preparation of glass slides
Serial sections were additionally prepared from the paraffin blocks and mounted on charged slides for immunostaining. These were left overnight incubated at 37°C to allow for the proper adherence of the tissue sections.
Immunostaining was performed with ready-to-use anti-Oct-4 antibody (mouse monoclonal antibody, 309M-18) (MRQ-10; Cell Marque Corp., Rocklin, California, USA) in Benchmark XT IHC/ISH staining module (Ventana, Medical Systems, Roche Group, California, USA). Sections from seminoma specimens were used as positive control in each run of immunostaining to validate the results ([Figure 1]).
|Figure 1 Immunohistochemical staining pattern for Oct-4, positive control (seminoma) (low power).|
Click here to view
Evaluation of immunostaining
Oct-4 nuclear staining was considered positive when nuclei showed the chromogenic reaction, and considered negative when no staining was detected at all or when no nuclei took the chromogen. The intensity of staining was classified and given scores as follows: 0=negative, 1=weak, 2=moderate, and 3=strong. Moreover, each case was given an intensity score according to the most prevalent staining intensity. Weak staining intensity (score 1) ([Figure 2]) was defined by staining of nuclear membrane only, moderate (score 2) ([Figure 3]) was characterized by stippled or faint intranuclear staining, and strong (score 3) ([Figure 4]) staining intensity was defined as homogenous intense staining of the nucleus. The stained cells within the tumor population were then counted in 10 random high-power field (×400), and a mean percentage was given to each case.
|Figure 2 Intensity score 1 expression of Oct 4 in urothelial carcinoma exhibiting nuclear membrane staining (high power).|
Click here to view
|Figure 3 Intensity score 2 expression of Oct 4 in a case of urothelial carcinoma showing moderate intra nuclear staining (high power).|
Click here to view
|Figure 4 Intensity score 3 expression of Oct 4 in urotelial carcinoma with homogenous intense nuclear staining (high power).|
Click here to view
A histochemical score (H-score) was then calculated per case as a final score by multiplying the average intensity score and the average percentage of expression per slide, where a score was designated within the range of 0 (minimum possible score) and 300 (maximum possible score).
The cases were stratified into cases with positive expression (H-score>0) and those with negative expression of Oct-4 (H-score=0), and the results were analyzed accordingly. Moreover, the mean H-score of positive cases only was used as a cutoff to stratify Oct-4-expressing tumors into high expression (H-score of tumor≥mean H-score) and low expression (H-score of tumor<mean H-score) of Oct-4 and used for further analysis of the results where applicable.
Qualitative data were presented as frequencies (numbers and percentages) whereas quantitative data were displayed as arithmetic means and SD. Significance of associations in cross-tables of qualitative data was tested by χ2 or Fischer’s exact tests as appropriate. In this study, P value of less than 0.05 was considered significant.
| Results|| |
Characteristics of studied cases
This study included 76 (90.5%) male cases and eight (9.5%) female cases, with a ratio of males-to-females of 10 : 1. Age ranged between 42 and 82 years, with a mean age of 61.2±9.043 years. The most frequently presented age group being the 50–59 years old group, which including 35 (41.7%) cases ([Table 1]).
|Table 1 Age and sex characteristics of studied cases and association with Oct-4 expression|
Click here to view
Low-grade urothelial tumors represented 14.3% (n=12) of all specimens, whereas high-grade tumors constituted 85.7% (n=72). Noninvasive urothelial tumors constituted 25 cases, which included 17 (20.2%) cases of papillary urothelial carcinoma, both high and low grade, and eight (9.5%) cases of CIS. Invasive tumors constituted most cases and were grouped as conventional urothelial carcinoma and urothelial carcinoma with variants. The aggregate number of urothelial tumors with variant morphology was 25 (29.8%) cases. Conventional urothelial carcinoma represented 34 (40.5%) cases, being the most frequent of all cases. The most prevalent categories were T3 (n=21, 25%), Ta (n=17, 20.2%), and T2 (n=17, 20.2%), whereas the least frequent were the Tis and T4, each representing 9.5% (n=8) ([Table 2]).
|Table 2 Grade, categories, morphologies, and tumor progression of studied cases and associations with Oct-4|
Click here to view
The cases were also stratified on the basis of whether they are muscle invasive or non-muscle invasive. Most of the non-muscle-invasive cases (n=37, 44%) were conventional urothelial carcinoma; however, only one (1.2%) case showed glandular differentiation (stage T1). Muscle-invasive cases showed 22 (26.2%) cases with conventional morphology and 24 (28.6%) with variant morphology ([Table 2]).
Expression of Oct-4 and its clinicopathologic associations
Of all the tumor specimens, 30 (35.8%) cases showed tumor positivity whereas 54 (64.2%) cases were negative for Oct-4 staining, with an H-score ranging between 0 and 33.6. The mean H-score of all cases was 4.57±8.227. The mean H-score of the positive cases only was 12.79±9.214, which was used as a cutoff value to stratify the positive tumors into high expression (≥mean) and low expression (<mean) ([Table 2]).
There was a significant association between Oct-4 expression and tumor grade (P=0.003), tumor morphology (P<0.001), tumor stage (P<0.001) and progression of tumor from non-muscle-invasive to invasive urothelial carcinoma (P=0.001). Further investigation of the association of Oct-4 expression with the specific expression of variant morphology, however, proved insignificant (P=0.400). Similarly, association of all relevant studied parameters with the level of Oct-4 expression proved insignificant ([Table 2]).
Another striking observation was that in tumors with desmoplastic reaction, the fibroblasts of the desmoplasia always showed strong staining, even when the surrounding tumor population was negative.
| Discussion|| |
Stem cell research has become a hot topic, and in cancer biology, CSCs seem to provide a promising ground for the eradication of the limitations we face in cancer treatment. CSCs have been isolated from many parenchymal tumors such as breast, brain, skin, thyroid, and organs of the gastrointestinal tract ,,,.
All the mounting evidence for the presence of CSCs directed toward the idea that a CSC model is similarly applicable on bladder urothelial carcinoma owing to the following: first, it tends to recur after treatment, sometimes displaying a higher grade or stage than the original tumor , suggesting the presence of cancer initiating population that is resistant to conventional cancer remedies. Second, it displays a wide range of grades and morphologies, sometimes with heterogeneous morphologies coexisting in the same tumor , suggesting the presence of tumor cells capable of differentiating along multiple lineages.
Oct-4 was chosen as a marker for CSC to test its expression in various grades, stages, and morphologies of bladder urothelial carcinoma and to assess whether it was associated with worse outcome. It was suggested that because Oct-4 is a marker of undifferentiated phenotype that it would probably be associated with worse outcome. This theory has been put to the test in breast, liver, pancreatic, and kidney cancers, together with prostatic cancer and cervical cancers ,,,, all of which proved significant association of Oct-4 with one or more of parameters of poor prognosis.
In the current study, the histopathological type of urothelial carcinoma was given special attention. It was an objective to evade categorization based solely on whether the tumor is flat or papillary, and therefore, another defining factor was added for the sake of elaboration, which is the presence/absence of variant morphology. Variant morphology is defined by the presence of subpopulation within the invasive urothelial tumor that shows divergent differentiation, or more simply, it entails presence of nonurothelial malignant population together with the urothelial malignant elements, and could be associated with worse outcome. To our knowledge, none of the previous studies concerned with Oct-4 expression in urothelial carcinoma explored potential association of Oct-4 with variant morphology.
In our study, 35.7% of all 84 patients showed positive expression of Oct-4, which is in contrast to the highest reported result of 93.6%  and lowest reported result of 68% . As per this study, only high-grade tumors exhibited positive Oct-4 expression, whereas all low-grade tumors were negative. There is a consensus, however, in all published literature that low-grade tumors do significantly express Oct-4. The only reasonable explanation for this discrepancy in our results was probably the minimum representation of low-grade specimens in this study (14.3%) as compared with other studies by Sedaghat et al. , Huang et al. , Xu et al. , and Zhao et al. . Our work shows that grade was significantly associated with Oct-4 expression (P=0.03), as was stage and tumor progression, where correlation of Oct-4 expression with the different stages and with progression from non-muscle-invasive to muscle-invasive tumor was highly significant (P<0.001 and P=0.001, respectively).
Per the current study, all morphologies expressed Oct-4 except for the CIS category, which is in contrast to the work by Sedaghat et al. , reporting positive expression in CIS. It was found in this study that Oct-4 was significantly associated with the histopathologic type, whether the tumor is flat or papillary (P<0.001), yet not specifically associated with differentiation along variant lineages (P=0.400). In contrast to these results, Chang et al.  concluded that Oct-4 expression was not significantly associated with the histopathologic type (P=0.44).
A final parameter that was tested is whether the level of expressed Oct-4 would have an effect on whether tumor expresses a certain grade, stage, or morphology, which was explored by dividing cases into high-expression versus low-expression cases. This technique was used by Sedaghat et al.  and proved no significant association with grade or stage. In our work, we tried to elaborate further this point and employed a different parameter in setting the cutoff H-score based on the number of positively expressing cases only, and we correlated Oct-4 expression with grade, stage and morphology as an added parameter. Our results, however, showed no significant association between Oct-4 level and any of the studied parameters.
In our work, it was notable that in certain tumor specimens, where the desmoplastic reaction was prominent, the desmoplastic stroma took up the Oct-4 stain with strong intensity, even when the tumor tissue itself proved negative for Oct-4 expression. This observation was seen in multiple immunostaining runs, which excludes the possibility of faulty run procedure. This raises questions regarding the tumor microenvironment, and whether the microenvironment too can express markers of stemness that would allow survival of tumor tissues.
It is worth noting that this study’s rationale was to detect level of Oct-4 protein with disregard to the different Oct-4 splice variants or Oct-4 pseudogene protein products. Atlas More Detailsi et al.  conducted a research to characterize the expression of Oct-4 splice variants and its pseudogenes in various pluripotent (embryonal carcinoma) and differentiated cell lines (urothelial, colonic, cervical carcinomas, and others), and they concluded that it is important to specify the exact variant of Oct-4 when addressing the issue of Oct-4 expression in different human cells, which could be the answer to resolving discrepancies in the results of authors working in this domain.
| Conclusion|| |
Oct-4 is expressed in urothelial carcinoma and its variants, and it is probably related to tumor prognosis and outcome, but further research would still be needed to delineate the specific isoform of Oct-4 implicated with prognosis. Further identification of the specific cause-effect relationship between Oct-4 expression and tumor behavior would also require investigation. This could be achieved by knock-in/ knock-out studies of cancer cell lines for Oct-4 expression and proceeding either by observing the changes of presence/absence of Oct-4 expression on the characteristics of cell culture (in vitro) or xenograft implantation of tumors in laboratory animals (in vivo), and follow-up of their behavior.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Dimov I, Visnjic M, Stefanovic V. Urothelial cancer stem cells. ScientificWorldJournal 2010; 10:1400–1415.
Stricker TP, Kumar V. Neoplasia. In: Kumar V, Abbas AK, Fausto N, Aster JC, editors. Robbins and Cotran pathologic basis of disease. 8th ed. Philadelphia, PA: Saunders; 2010. pp. 267–268.
Hatina J, Schulz WA. Stem cells in the biology of normal urothelium and urothelial carcinoma. Neoplasma 2012; 59:728–736.
Castello-Martin M, Domingo-Domenech J, Karni-Schmidt O, Matos T, Cordon-Cardo C. Molecular pathways of urothelial development and bladder tumorigenesis. Urol Oncol 2010; 28:401–408.
Brandt WD, Matsui W, Rosenberg GE, He X, Ling S, Schaeffer EM, Berman DM. Urothelial carcinoma: stem cells on the edge. Cancer Metastasis Rev 2009; 28:291–304.
Chang CC, Shieh GS, Wu P, Lin CC, Shiau AL, Wu CL. Oct-3/4 expression reflects tumour progression and regulates motility of bladder cancer cells. J Cancer Res 2008; 68:6281–6291.
Eble JN, Sauter G, Epstein JI, Sesterhenn IA. Chapter 2: tumours of the urinary system. In: Eble JN, Sauter G, Epstein JI, Sesterhenn IA, editors. Pathology and genetics of tumours of the urinary system and male genital organs. Lyon, France: IARC Press; 2004. p. 90.
Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A. Urinary bladder. In: Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A, editors. AJCC Cancer Staging Manual. 7th ed. New York, NY: Springer; 2010. pp. 497–505.
Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 2003; 100:3983–3988.
Ignatova TN, Kukekov VG, Laywell ED, Suslov ON, Vrionis FD, Steindler DA. Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro. Glia 2002; 39:193–206.
Meng M, Zhao X, Ning Q, Hou L, Xin G, Liu L. Tumor stem cells: a new approach for tumor therapy (review). Oncol Lett 2012; 4:187–193.
Mimeault M, Hauke R, Mehta PP, Batra SK. Recent advances in cancer stem/progenitor cellresearch: therapeutic implications for overcoming resistance to the most aggressive cancers. J Cell Mol Med. 2007; 11:981–1011.
Chamie K, Litwin MS, Bassett JC, Daskivich TJ, Lai J, Hanley JM et al.
Recurrence of high risk bladder cancer: a population-based analysis. Cancer 2013; 119:3219–3227.
Lopez-Beltran A, Sauter G, Gasser T, Hartmann A, Schmitz-Drager BJ, Helpap B et al.
Infiltrating urothelial carcinoma [chapter 2]. In: Eble JN, Sauter G, Epstein JI, Sesterhenn IA, editors. Pathology and genetics of tumours of the urinary system and male genital organs. Lyon, France: IARC Press; 2004. pp. 97–103.
Kim RJ, Nam JS. Oct-4 expression enhances features of cancer stem cells in a mouse model of breast cancer. Lab Anim Res 2011; 27:147–152.
Tai MH, Chang CC, Kiupel M, Webster JD, Olson LK, Trosko JE. Oct4 expression in adult human stem cells: evidence in support of the stem cell theory of carcinogenesis. Carcinogenesis 2005; 26:495–502.
Kosaka T, Mikami S, Yoshimine S, Miyazaki Y, Daimon T, Kikuchi E et al.
The prognostic significance of Oct-4 expression in patients with prostate cancer. Hum Pathol 2016; 51:1–8.
Kim BW, Cho H, Choi CH, Ylaya K, Chung JY, Kim JH, Hewitt SM. Clinical significance of Oct-4 and SOX2 protein expression in cervical cancer. BMC Cancer 2015; 15:1015.
Sedaghat S, Gheytanchi E, Asgari M, Roudi R, Keymoosi H, Madjd Z. Expression of cancer stem cell markers Oct-4 and CD133 in transitional cell carcinomas. Appl Immunohistochem Mol Morphol 2017; 25:196–202. doi: 10.1097/PAI.0000000000000291
Huang P, Chen J, Wang L, Na Y, Kaku H, Ueki H et al.
Implications of transcriptional factor, Oct-4, in human bladder malignancy and tumour recurrence. Med Oncol 2012; 29:829–834.
Xu K, Zhu Z, Zeng F. Expression and significance of Oct-4 in bladder cancer. J Huazhong Univ Sci Technolog Med Sci 2007; 27:675–677.
Zhao P, Liu C, Xu K, Zheng S, Li H, Xu Y et al.
Expression of Oct-4 protein in bladder cancer and its clinicopathological implications. Nan Fang Yi Ke Da Xue Xue Bao 2012; 32:643–646.
Atlasi Y, Mowla SJ, Ziaee SA, Gokhale PJ, Andrews PW. Oct-4 spliced variants are differentially expressed in human pluripotent and nonpluripotent cells. Stem Cells 2008; 26:3068–3074.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]