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Immunohistochemical expression of ARID1A in penile squamous cell carcinomas: a tissue microarray study of 112 cases
Human Pathology (2015) 46, 761–766
www.elsevier.com/locate/humpath
Original contribution
Immunohistochemical expression of ARID1A in penile squamous cell carcinomas: a tissue microarray study of 112 cases☆ Sheila F. Faraj MD a,1 , Alcides Chaux MD a,b,⁎,1 , Nilda Gonzalez-Roibon MD a , Enrico Munari MD a , Antonio L. Cubilla MD c , Ie-Ming Shih MD a , George J. Netto MD a,d,e a
Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD 31231 Department of Scientific Research, Norte University, Asunción, 1614, Paraguay c Instituto de Patología e Investigación, Asunción 1817, Paraguay d Department of Urology, Johns Hopkins Medical Institutions, Baltimore, MD 31231 e Department of Oncology, Johns Hopkins Medical Institutions, Baltimore, MD 31231 b
Received 2 December 2014; revised 28 January 2015; accepted 30 January 2015
Keywords: ARID1A; BAF250A; SWI/SNF; mTOR pathway; Penile SCC
Summary ARID1A, a member of the chromatin remodeling genes family, has been suggested as a novel tumor suppressor gene in gynecologic malignancies. However, its role in penile cancer has yet to be determined. This study assesses the immunohistochemical expression of ARID1A in penile squamous cell carcinoma (SCC) and its association with pathologic features, human papillomavirus (HPV) status, and previously reported mammalian target of rapamycin pathway markers in the same cohort. Four tissue microarrays were constructed from 112 cases of formalin-fixed, paraffin-embedded penile SCC from Paraguay. Each tumor was sampled 3 to 12 times. ARID1A expression was evaluated by immunohistochemistry using a polyclonal rabbit anti-ARID1A (BAF250A) antibody. An H score was calculated in each spot as the sum of expression intensity (0-3+) by extent (0%-100%). Median H score per case was used for statistical analysis. ARID1A expression was observed in all cases, ranging from 3% to 100% of tumor cells (median, 95%). In 96 cases (86%), ARID1A expression was observed in 90% or more tumor cells. HPV DNA was detected in 20 (38%) of 52 analyzed samples. There was a significant trend of association between ARID1A and histologic grade. ARID1A expression was not associated with histologic subtype (P = .61) or HPV status (P = .18). ARID1A expression decreased with decreasing levels of PTEN expression (P = .01). ARID1A was expressed in penile SCC, in most cases at high levels. A significant trend of association was found between histologic grade and ARID1A expression, with lower ARID1A expression, lower histologic grades, and decreased PTEN expression. © 2015 Elsevier Inc. All rights reserved.
☆ Funding/Support: This study was partially supported by the Johns Hopkins Medicine–Patana Fund for Research. Dr Alcides Chaux was partially supported by an award granted by the CONACYT (National Council of Science and Technology) dependent of the Presidency of the Republic of Paraguay, as an Active Researcher of Level 1 of the PRONII (National Incentive Program for Researchers). ⁎ Corresponding author. Department of Scientific Research, Norte University, Gral. Santos e/25 de Mayo, Asunción, Paraguay. E-mail address: [email protected] (A. Chaux). 1 These authors have contributed equally to this work.
http://dx.doi.org/10.1016/j.humpath.2015.01.018 0046-8177/© 2015 Elsevier Inc. All rights reserved.
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1. Introduction The incidence of penile cancer varies worldwide, with the highest rates seen in developing countries, particularly in Africa and South America, suggesting a relationship between epidemiologic factors and cancer development. Squamous cell carcinoma (SCC) accounts for most of penile cancer, and about half of them are classified as usual SCC [1]. Penile SCC is associated with several established risk factors including phimosis, chronic inflammation, poor hygiene, and smoking. Human papillomavirus (HPV) is an additional risk factor for the development of penile SCC. Its presence has been reported in 20% to 40% of penile cancer. Different subtypes are known to have distinctive clinicopathologic features. HPV-related tumors are more frequently seen in younger patients with basaloid and warty carcinomas [2]. Penile cancer is primarily treated with surgical resection [3]. Locally advanced or metastatic disease often requires a multidisciplinary approach, involving both surgery and systemic therapy [3,4]. Despite improvements in cancer therapy, mortality and morbidity rates remain high for patients with advanced penile cancer [5]. Regional lymph node metastasis is followed by distant metastasis and is the most important prognostic factor for survival. Detection of patients with higher risk for lymph node disease is crucial for appropriate clinical management [6,7]. Reliable prognostic factors for the development of lymph node metastasis are being sought. Histologic features such as pathologic tumor stage, grade, and vascular invasion have been suggested to predict lymph node metastasis [8]. In addition, several biomarkers have been investigated for their potential prediction of lymph node metastasis. The association between p53 expression and lymph node metastasis has been observed [9]. In addition to p53 expression, FerrandizPulido et al [10] have recently suggested a role for the mammalian target of rapamycin (mTOR) pathway in the development of lymph node metastasis. Recently, inactivating mutations in AT-rich interactive domain 1A (ARID1A) have been recognized in several tumors [11,12], suggesting that it is a tumor suppressor gene in many different cell types, including ovarian and endometrial epithelium [13,14]. However, its role in penile cancer has yet to be determined. The aim of this study is to evaluate the ARID1A immunohistochemical expression in a cohort of penile SCC. Furthermore, the association with pathologic features, HPV status and mTOR pathway markers is assessed.
S. F. Faraj et al.
2.1. Case selection, tissue microarray construction, morphologic evaluation, and HPV detection The present study includes tissue samples from 112 patients with invasive SCCs of the penis diagnosed at the Instituto de Patología e Investigación (Asunción, Paraguay) between 2000 and 2011. Cases were selected based on availability of formalin-fixed, paraffin-embedded tissue blocks. From each case, 1 to 4 blocks were selected. Four tissue microarrays (TMAs) were built at the Johns Hopkins TMA Lab Core (Baltimore, MD) using a previously described procedure [15]. Three tissue cores of 1 mm each were obtained per block, giving a representation of 3 to 12 TMA spots per case. Histologic subtyping was performed in whole-tissue sections using previously published morphologic criteria [16]. Histologic grading was performed spot by spot using previously published and validated criteria [17]. For statistical analysis, the highest grade at the TMA spots was assigned as the histologic grade of the case. In 52 cases, HPV detection was done by SPF 10 polymerase chain reaction and DEIA, a DNA enzymeimmunoassay for general HPV detection, as previously described [18]. Tissue samples (5% of the total) in which no HPV presence was expected were used as controls.
2.2. ARID1A expression and scoring system ARID1A expression was evaluated by immunohistochemistry on 5-μm TMA sections using a polyclonal rabbit (BAF250A) anti-ARID1A antibody (HPA005456; Sigma-Aldrich, St Louis, MO), whose specificity has been confirmed by Western blot [19]. Antigen retrieval was performed by submerging the tissue sections in citrate buffer (pH 6.0) and then in a steamer for 10 minutes. The sections were then incubated with the rabbit antibody at a dilution of 1:200 at 4°C overnight. A positive reaction was detected by the EnVision+ System (Dako, Carpinteria, CA). Only nuclear staining was scored, and tumor stromal cells served as positive internal controls. An H score was assigned in each TMA spot as the sum of the products of the intensity (0 for negative, 1 for weakly positive, 2 for moderately positive, and 3 for strongly positive) multiplied by the extent of immunoexpression (0%-100%), obtaining a value ranging from 0 to 300. For each case, the pooled median of the TMA H scores was used for statistical analyses.
2.3. mTOR pathway markers
2. Materials and methods The current study was approved by the institutional review board at the Johns Hopkins School of Medicine (Baltimore, MD).
The mTOR pathway biomarkers (PTEN, phospho-AKT, phospho-mTOR, and phospho-S6) were previously reported on this cohort [20]. Immunohistochemistry was performed using the PowerVision Poly-HRP IHC Detection System (Leica Microsystems, Bannockburn, IL). Sections were deparaffinized, rehydrated, and subjected to heat-induced
ARID1A immunoexpression in penile SCC
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Fig. 1 Immunohistochemical expression of ARID1A in penile carcinomas. Only nuclear positivity was considered as indicative of ARID1A expression. A, Negative ARID1A expression. B, Weak ARID1A expression. C, Moderate ARID1A expression. D, Strong ARID1A expression (×200 magnification).
antigen retrieval with a buffer solution using a steamer. Sections were then incubated with the appropriate primary antibody at 4°C overnight. Appropriate cell lines were used as
external controls, and internal controls were checked for negative and positive expression. From the 4 TMAs, 732 TMA spots were scanned using the Aperio System (Aperio
Fig. 2 ARID1A expression in penile carcinomas by histologic subtypes, histologic grades, and HPV status. The y-axis corresponds to median ARID1A H scores. Values in brackets on top or bottom of the boxplots correspond to [median, interquartile range]. The dashed line represents the median H score of the sample. A, ARID1A H scores by histologic subtypes; H scores were not significantly different among histologic subtypes. The category “Other Subtypes” includes verrucous and sarcomatoid carcinomas. B, ARID1A H scores by histologic grade; higher ARID1A H scores were observed at increasing histologic grades, with a significant trend. C, ARID1A H scores by HPV status; H scores were not significantly different depending on the presence or absence of HPV DNA.
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S. F. Faraj et al.
Fig. 3
Association between PTEN and ARID1A expression.
Technologies, Vista, CA) and uploaded to TMAJ (available at http://tmaj.pathology.jhmi.edu). PTEN expression was evaluated according to a previously described approach [21]. Each spot was classified as (i) retained PTEN expression, (ii) decreased PTEN expression, or (iii) loss of PTEN expression. If all spots showed retained expression, the case was classified as such. If all spots showed loss of PTEN expression, the case was classified as such. In all other instances, the case was classified as showing decreased PTEN expression.
2.4. Statistical analysis ARID1A H scores were compared against histologic subtype, histologic grade, and HPV status using the Kruskal-Wallis equality-of-populations rank test. The Cuzick test for trend across ordered groups was used as an adjunct to the Kruskal-Wallis test when appropriate. A 2-tailed P b .05 was required for statistical significance. Data were analyzed using Stata/SE 12.0 (StataCorp LP, College Station, TX). Pairwise comparisons between biomarkers were performed using the Pearson product-moment correlation test. Correlation matrices were plotted using the Performance Analytics package in R version 3.1.1 “Sock it to Me” (R Foundation for Statistical Computing, Vienna, Austria).
Table P values for correlation coefficients between phospho-AKT, phospho-mTOR, and phospho-S6 with ARID1a
ARID1a ARID1a Phos-AKT ARID1a Phos-AKT Phos-mTOR
Phos-AKT Phos-mTOR Phos-mTOR Phos-S6 Phos-S6 Phos-S6
Correlation coefficient
P
0.1291 0.1208 −0.0349 0.1856 −0.0558 0.1077
.1749 .2045 .7147 .0501 .5589 .2585
3. Results Distribution of subtypes was as follows: usual SCC, 48 cases; warty-basaloid carcinoma, 24 cases; warty carcinoma, 17 cases; basaloid carcinoma, 11 cases; papillary carcinoma, 9 cases; verrucous carcinoma, 2 cases; and sarcomatoid carcinoma, 1 case. Distribution of histologic grades was as follows: grade 1, 5 cases; grade 2, 30 cases; and grade 3, 77 cases. HPV DNA was detected in 20 (38%) of 52 analyzed samples. ARID1A expression was observed in all cases, ranging from 3% to 100% of tumor cells (median, 95%). In 96 cases (86%), ARID1A expression was observed in 90% or more of tumor cells. Fig. 1 shows the immunohistochemical patterns of ARID1A expression. ARID1A expression was not associated with histologic subtype (P = .61) or HPV status (P = .18). However, the Kruskal-Wallis test yielded a P = .05 for the association between ARID1A and histologic grade. The Cuzick test showed that this trend was significant (P = .03). Fig. 2 summarizes all these results. ARID1A expression decreased with decreasing levels of PTEN expression (P = .01), as shown in Fig. 3. The Table and Fig. 4 show the correlation between biomarkers. A weak positive (correlation coefficient = 0.19; P = .05) correlation was observed between ARID1A expression and phospho-S6. No association was observed between ARID1A and phospho-AKT and phospho-mTOR.
4. Discussion ARID1A has been extensively assessed in gynecologic malignancies [13,14]. To our knowledge, this is the first study evaluating ARID1A in penile carcinoma. ARID1A is located on chromosome 1p and encodes BAF250a, a large nuclear protein that participates in the formation of a switch/ sucrose nonfermentable (SWI/SNF) chromatin remodeling complex involved in the regulation of cell differentiation, proliferation, DNA repair, and tumor suppression [13].
ARID1A immunoexpression in penile SCC
Fig. 4
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Correlation matrices between biomarkers.
Katagiri et al [22] found low ARID1A expression in 6.5% of cervical SCCs. Wang et al [23] found ARID1A to be down-regulated in cervical cancer leading to inhibition of miR31 that suppresses growth, enhances apoptosis, and decreases invasion of cancer cells. We found 86% penile SCCs to express ARID1A in 90% or more of tumor cells. Cho et al [24] found an association of lower ARID1A expression and high-grade cervical cancer. Previous studies have shown somatic inactivating mutations of ARID1A to be more frequently seen in low-grade uterine and ovarian tumors [14,19]. In the present study, lower ARID1A expression was seen at lower histologic grades. Recent studies have indicated an association of low ARID1A expression with unfavorable prognosis and chemoresistance in gynecologic malignancies [24–26]. Taking into consideration the association between histologic grade and prognosis, future studies should focus on the predictive value of ARID1A in penile SCC. Recent studies have demonstrated an implication of PTEN/PI3K/AKT pathway in penile carcinogenesis [20,27,28]. PTEN/PI3K/AKT pathway has been suggested to interact with ARID1A in gastric adenocarcinoma and ovarian carcinoma [29–32]. We found that lower ARID1A
expression was associated with decreasing PTEN expression. Samartzis et al [32] have shown that PI3K and AKT inhibitors lead to apoptosis in tumor cells with low ARID1A expression, reflecting a significant increase of the sensitivity of cancer cells to small molecule inhibitors of the PI3K/AKT pathway. The involvement of ARID1A in the oncogenesis of penile SCC merits further investigation. TMA technique offers the advantage that it allows the evaluation of a large number of cases under the same immunohistochemistry conditions. Given the known heterogeneity of biomarkers expression within same tumor, the use of TMAs instead of whole-tissue sections could be seen as a limitation in our current study. However, several studies have supported the value of the TMA usage and the appropriate representation of the overall expression levels using multiple TMA spots [33]. The lack of follow-up data is another weakness in the study given the nature of the cohort as part of consultation cases for which no additional data were obtained. In summary, ARID1A was expressed in penile SCC, in most cases at high levels. ARID1A expression was not associated with histologic subtype or HPV presence. A significant trend was found between histologic grade and
766 ARID1A expression, with lower ARID1A expression at lower histologic grades. Lower ARID1A expression was associated with decreasing of PTEN expression.
Acknowledgment We acknowledge the outstanding work of Helen Fedor and Marcella Southerland, from the TMA Lab Core at the Johns Hopkins University, in building the TMAs for the study.
References [1] Chaux A, Cubilla AL. Advances in the pathology of penile carcinomas. HUM PATHOL 2012;43:771-89. [2] Chaux A, Velazquez EF, Algaba F, Ayala G, Cubilla AL. Developments in the pathology of penile squamous cell carcinomas. Urology 2010;76:S7-S14. [3] Pizzocaro G, Algaba F, Horenblas S, et al. European Association of Urology Guidelines Group on Penile C. EAU penile cancer guidelines 2009. Eur Urol 2010;57:1002-12. [4] Bermejo C, Busby JE, Spiess PE, Heller L, Pagliaro LC, Pettaway CA. Neoadjuvant chemotherapy followed by aggressive surgical consolidation for metastatic penile squamous cell carcinoma. J Urol 2007;177: 1335-8. [5] Theodore C, Skoneczna I, Bodrogi I, et al. A phase II multicentre study of irinotecan (CPT 11) in combination with cisplatin (CDDP) in metastatic or locally advanced penile carcinoma (EORTC PROTOCOL 30992). Ann Oncol 2008;19:1304-7. [6] Chaux A, Cubilla AL. Penile cancer: optimal management of T1G2 penile cancer remains unclear. Nat Rev Urol 2013;10:9-11. [7] Mosconi AM, Roila F, Gatta G, Theodore C. Cancer of the penis. Crit Rev Oncol Hematol 2005;53:165-77. [8] Zhu Y, Zhang HL, Yao XD, et al. Development and evaluation of a nomogram to predict inguinal lymph node metastasis in patients with penile cancer and clinically negative lymph nodes. J Urol 2010;184: 539-45. [9] Lopes A, Bezerra AL, Pinto CA, Serrano SV, de MellO CA, Villa LL. p53 as a new prognostic factor for lymph node metastasis in penile carcinoma: analysis of 82 patients treated with amputation and bilateral lymphadenectomy. J Urol 2002;168:81-6. [10] Ferrandiz-Pulido C, Masferrer E, Toll A, et al. mTOR signaling pathway in penile squamous cell carcinoma: pmTOR and peIF4E over expression correlate with aggressive tumor behavior. J Urol 2013;190: 2288-95. [11] Jones S, Li M, Parsons DW, et al. Somatic mutations in the chromatin remodeling gene ARID1A occur in several tumor types. Hum Mutat 2012;33:100-3. [12] Wu JN, Roberts CW. ARID1A mutations in cancer: another epigenetic tumor suppressor? Cancer Discov 2013;3:35-43. [13] Guan B, Wang TL, Shih Ie M. ARID1A, a factor that promotes formation of SWI/SNF-mediated chromatin remodeling, is a tumor suppressor in gynecologic cancers. Cancer Res 2011;71:6718-27. [14] Kurman RJ, Shih Ie M. Molecular pathogenesis and extraovarian origin of epithelial ovarian cancer—shifting the paradigm. HUM PATHOL 2011;42:918-31. [15] Fedor HL, De Marzo AM. Practical methods for tissue microarray construction. Methods Mol Med 2005;103:89-101.
S. F. Faraj et al. [16] Epstein JH, Cubilla AL, Humphrey PA. Tumors of the prostate gland, seminal vesicles, penis, and scrotum. Washington, DC: American Registry of Pathology in collaboration with the Armed Forces Institute of Pathology; 2011. p. 405-612. [17] Chaux A, Torres J, Pfannl R, et al. Histologic grade in penile squamous cell carcinoma: visual estimation versus digital measurement of proportions of grades, adverse prognosis with any proportion of grade 3 and correlation of a Gleason-like system with nodal metastasis. Am J Surg Pathol 2009;33:1042-8. [18] Cubilla AL, Lloveras B, Alejo M, et al. The basaloid cell is the best tissue marker for human papillomavirus in invasive penile squamous cell carcinoma: a study of 202 cases from Paraguay. Am J Surg Pathol 2010;34:104-14. [19] Guan B, Mao TL, Panuganti PK, et al. Mutation and loss of expression of ARID1A in uterine low-grade endometrioid carcinoma. Am J Surg Pathol 2011;35:625-32. [20] Chaux A, Munari E, Cubilla AL, et al. Immunohistochemical expression of the mammalian target of rapamycin pathway in penile squamous cell carcinomas: a tissue microarray study of 112 cases. Histopathology 2014;64:863-71. [21] Schultz L, Chaux A, Albadine R, et al. Immunoexpression status and prognostic value of mTOR and hypoxia-induced pathway members in primary and metastatic clear cell renal cell carcinomas. Am J Surg Pathol 2011;35:1549-56. [22] Katagiri A, Nakayama K, Rahman MT, et al. Frequent loss of tumor suppressor ARID1A protein expression in adenocarcinomas/adenosquamous carcinomas of the uterine cervix. Int J Gynecol Cancer 2012; 22:208-12. [23] Wang N, Zhou Y, Zheng L, Li H. MiR-31 is an independent prognostic factor and functions as an oncomir in cervical cancer via targeting ARID1A. Gynecol Oncol 2014;134:129-37. [24] Cho H, Kim JS, Chung H, Perry C, Lee H, Kim JH. Loss of ARID1A/ BAF250a expression is linked to tumor progression and adverse prognosis in cervical cancer. HUM PATHOL 2013;44:1365-74. [25] Katagiri A, Nakayama K, Rahman MT, et al. Loss of ARID1A expression is related to shorter progression-free survival and chemoresistance in ovarian clear cell carcinoma. Mod Pathol 2012;25:282-8. [26] Yokoyama Y, Matsushita Y, Shigeto T, Futagami M, Mizunuma H. Decreased ARID1A expression is correlated with chemoresistance in epithelial ovarian cancer. J Gynecol Oncol 2014;25:58-63. [27] Stankiewicz E, Prowse DM, Ng M, et al. Alternative HER/PTEN/Akt pathway activation in HPV positive and negative penile carcinomas. PLoS One 2011;6:e17517. [28] Guan B, Rahmanto YS, Wu RC, et al. Roles of deletion of Arid1a, a tumor suppressor, in mouse ovarian tumorigenesis. J Natl Cancer Inst 2014;106:dju146. [29] Wiegand KC, Hennessy BT, Leung S, et al. A functional proteogenomic analysis of endometrioid and clear cell carcinomas using reverse phase protein array and mutation analysis: protein expression is histotype-specific and loss of ARID1A/BAF250a is associated with AKT phosphorylation. BMC Cancer 2014;14:120. [30] Yamamoto S, Tsuda H, Takano M, Tamai S, Matsubara O. Loss of ARID1A protein expression occurs as an early event in ovarian clearcell carcinoma development and frequently coexists with PIK3CA mutations. Mod Pathol 2012;25:615-24. [31] Zang ZJ, Cutcutache I, Poon SL, et al. Exome sequencing of gastric adenocarcinoma identifies recurrent somatic mutations in cell adhesion and chromatin remodeling genes. Nat Genet 2012;44:570-4. [32] Samartzis EP, Gutsche K, Dedes KJ, Fink D, Stucki M, Imesch P. Loss of ARID1A expression sensitizes cancer cells to PI3K- and AKTinhibition. Oncotarget 2014;5:5295-303. [33] Camp RL, Neumeister V, Rimm DL. A decade of tissue microarrays: progress in the discovery and validation of cancer biomarkers. J Clin Oncol 2008;26:5630-7.
www.elsevier.com/locate/humpath
Original contribution
Immunohistochemical expression of ARID1A in penile squamous cell carcinomas: a tissue microarray study of 112 cases☆ Sheila F. Faraj MD a,1 , Alcides Chaux MD a,b,⁎,1 , Nilda Gonzalez-Roibon MD a , Enrico Munari MD a , Antonio L. Cubilla MD c , Ie-Ming Shih MD a , George J. Netto MD a,d,e a
Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD 31231 Department of Scientific Research, Norte University, Asunción, 1614, Paraguay c Instituto de Patología e Investigación, Asunción 1817, Paraguay d Department of Urology, Johns Hopkins Medical Institutions, Baltimore, MD 31231 e Department of Oncology, Johns Hopkins Medical Institutions, Baltimore, MD 31231 b
Received 2 December 2014; revised 28 January 2015; accepted 30 January 2015
Keywords: ARID1A; BAF250A; SWI/SNF; mTOR pathway; Penile SCC
Summary ARID1A, a member of the chromatin remodeling genes family, has been suggested as a novel tumor suppressor gene in gynecologic malignancies. However, its role in penile cancer has yet to be determined. This study assesses the immunohistochemical expression of ARID1A in penile squamous cell carcinoma (SCC) and its association with pathologic features, human papillomavirus (HPV) status, and previously reported mammalian target of rapamycin pathway markers in the same cohort. Four tissue microarrays were constructed from 112 cases of formalin-fixed, paraffin-embedded penile SCC from Paraguay. Each tumor was sampled 3 to 12 times. ARID1A expression was evaluated by immunohistochemistry using a polyclonal rabbit anti-ARID1A (BAF250A) antibody. An H score was calculated in each spot as the sum of expression intensity (0-3+) by extent (0%-100%). Median H score per case was used for statistical analysis. ARID1A expression was observed in all cases, ranging from 3% to 100% of tumor cells (median, 95%). In 96 cases (86%), ARID1A expression was observed in 90% or more tumor cells. HPV DNA was detected in 20 (38%) of 52 analyzed samples. There was a significant trend of association between ARID1A and histologic grade. ARID1A expression was not associated with histologic subtype (P = .61) or HPV status (P = .18). ARID1A expression decreased with decreasing levels of PTEN expression (P = .01). ARID1A was expressed in penile SCC, in most cases at high levels. A significant trend of association was found between histologic grade and ARID1A expression, with lower ARID1A expression, lower histologic grades, and decreased PTEN expression. © 2015 Elsevier Inc. All rights reserved.
☆ Funding/Support: This study was partially supported by the Johns Hopkins Medicine–Patana Fund for Research. Dr Alcides Chaux was partially supported by an award granted by the CONACYT (National Council of Science and Technology) dependent of the Presidency of the Republic of Paraguay, as an Active Researcher of Level 1 of the PRONII (National Incentive Program for Researchers). ⁎ Corresponding author. Department of Scientific Research, Norte University, Gral. Santos e/25 de Mayo, Asunción, Paraguay. E-mail address: [email protected] (A. Chaux). 1 These authors have contributed equally to this work.
http://dx.doi.org/10.1016/j.humpath.2015.01.018 0046-8177/© 2015 Elsevier Inc. All rights reserved.
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1. Introduction The incidence of penile cancer varies worldwide, with the highest rates seen in developing countries, particularly in Africa and South America, suggesting a relationship between epidemiologic factors and cancer development. Squamous cell carcinoma (SCC) accounts for most of penile cancer, and about half of them are classified as usual SCC [1]. Penile SCC is associated with several established risk factors including phimosis, chronic inflammation, poor hygiene, and smoking. Human papillomavirus (HPV) is an additional risk factor for the development of penile SCC. Its presence has been reported in 20% to 40% of penile cancer. Different subtypes are known to have distinctive clinicopathologic features. HPV-related tumors are more frequently seen in younger patients with basaloid and warty carcinomas [2]. Penile cancer is primarily treated with surgical resection [3]. Locally advanced or metastatic disease often requires a multidisciplinary approach, involving both surgery and systemic therapy [3,4]. Despite improvements in cancer therapy, mortality and morbidity rates remain high for patients with advanced penile cancer [5]. Regional lymph node metastasis is followed by distant metastasis and is the most important prognostic factor for survival. Detection of patients with higher risk for lymph node disease is crucial for appropriate clinical management [6,7]. Reliable prognostic factors for the development of lymph node metastasis are being sought. Histologic features such as pathologic tumor stage, grade, and vascular invasion have been suggested to predict lymph node metastasis [8]. In addition, several biomarkers have been investigated for their potential prediction of lymph node metastasis. The association between p53 expression and lymph node metastasis has been observed [9]. In addition to p53 expression, FerrandizPulido et al [10] have recently suggested a role for the mammalian target of rapamycin (mTOR) pathway in the development of lymph node metastasis. Recently, inactivating mutations in AT-rich interactive domain 1A (ARID1A) have been recognized in several tumors [11,12], suggesting that it is a tumor suppressor gene in many different cell types, including ovarian and endometrial epithelium [13,14]. However, its role in penile cancer has yet to be determined. The aim of this study is to evaluate the ARID1A immunohistochemical expression in a cohort of penile SCC. Furthermore, the association with pathologic features, HPV status and mTOR pathway markers is assessed.
S. F. Faraj et al.
2.1. Case selection, tissue microarray construction, morphologic evaluation, and HPV detection The present study includes tissue samples from 112 patients with invasive SCCs of the penis diagnosed at the Instituto de Patología e Investigación (Asunción, Paraguay) between 2000 and 2011. Cases were selected based on availability of formalin-fixed, paraffin-embedded tissue blocks. From each case, 1 to 4 blocks were selected. Four tissue microarrays (TMAs) were built at the Johns Hopkins TMA Lab Core (Baltimore, MD) using a previously described procedure [15]. Three tissue cores of 1 mm each were obtained per block, giving a representation of 3 to 12 TMA spots per case. Histologic subtyping was performed in whole-tissue sections using previously published morphologic criteria [16]. Histologic grading was performed spot by spot using previously published and validated criteria [17]. For statistical analysis, the highest grade at the TMA spots was assigned as the histologic grade of the case. In 52 cases, HPV detection was done by SPF 10 polymerase chain reaction and DEIA, a DNA enzymeimmunoassay for general HPV detection, as previously described [18]. Tissue samples (5% of the total) in which no HPV presence was expected were used as controls.
2.2. ARID1A expression and scoring system ARID1A expression was evaluated by immunohistochemistry on 5-μm TMA sections using a polyclonal rabbit (BAF250A) anti-ARID1A antibody (HPA005456; Sigma-Aldrich, St Louis, MO), whose specificity has been confirmed by Western blot [19]. Antigen retrieval was performed by submerging the tissue sections in citrate buffer (pH 6.0) and then in a steamer for 10 minutes. The sections were then incubated with the rabbit antibody at a dilution of 1:200 at 4°C overnight. A positive reaction was detected by the EnVision+ System (Dako, Carpinteria, CA). Only nuclear staining was scored, and tumor stromal cells served as positive internal controls. An H score was assigned in each TMA spot as the sum of the products of the intensity (0 for negative, 1 for weakly positive, 2 for moderately positive, and 3 for strongly positive) multiplied by the extent of immunoexpression (0%-100%), obtaining a value ranging from 0 to 300. For each case, the pooled median of the TMA H scores was used for statistical analyses.
2.3. mTOR pathway markers
2. Materials and methods The current study was approved by the institutional review board at the Johns Hopkins School of Medicine (Baltimore, MD).
The mTOR pathway biomarkers (PTEN, phospho-AKT, phospho-mTOR, and phospho-S6) were previously reported on this cohort [20]. Immunohistochemistry was performed using the PowerVision Poly-HRP IHC Detection System (Leica Microsystems, Bannockburn, IL). Sections were deparaffinized, rehydrated, and subjected to heat-induced
ARID1A immunoexpression in penile SCC
763
Fig. 1 Immunohistochemical expression of ARID1A in penile carcinomas. Only nuclear positivity was considered as indicative of ARID1A expression. A, Negative ARID1A expression. B, Weak ARID1A expression. C, Moderate ARID1A expression. D, Strong ARID1A expression (×200 magnification).
antigen retrieval with a buffer solution using a steamer. Sections were then incubated with the appropriate primary antibody at 4°C overnight. Appropriate cell lines were used as
external controls, and internal controls were checked for negative and positive expression. From the 4 TMAs, 732 TMA spots were scanned using the Aperio System (Aperio
Fig. 2 ARID1A expression in penile carcinomas by histologic subtypes, histologic grades, and HPV status. The y-axis corresponds to median ARID1A H scores. Values in brackets on top or bottom of the boxplots correspond to [median, interquartile range]. The dashed line represents the median H score of the sample. A, ARID1A H scores by histologic subtypes; H scores were not significantly different among histologic subtypes. The category “Other Subtypes” includes verrucous and sarcomatoid carcinomas. B, ARID1A H scores by histologic grade; higher ARID1A H scores were observed at increasing histologic grades, with a significant trend. C, ARID1A H scores by HPV status; H scores were not significantly different depending on the presence or absence of HPV DNA.
764
S. F. Faraj et al.
Fig. 3
Association between PTEN and ARID1A expression.
Technologies, Vista, CA) and uploaded to TMAJ (available at http://tmaj.pathology.jhmi.edu). PTEN expression was evaluated according to a previously described approach [21]. Each spot was classified as (i) retained PTEN expression, (ii) decreased PTEN expression, or (iii) loss of PTEN expression. If all spots showed retained expression, the case was classified as such. If all spots showed loss of PTEN expression, the case was classified as such. In all other instances, the case was classified as showing decreased PTEN expression.
2.4. Statistical analysis ARID1A H scores were compared against histologic subtype, histologic grade, and HPV status using the Kruskal-Wallis equality-of-populations rank test. The Cuzick test for trend across ordered groups was used as an adjunct to the Kruskal-Wallis test when appropriate. A 2-tailed P b .05 was required for statistical significance. Data were analyzed using Stata/SE 12.0 (StataCorp LP, College Station, TX). Pairwise comparisons between biomarkers were performed using the Pearson product-moment correlation test. Correlation matrices were plotted using the Performance Analytics package in R version 3.1.1 “Sock it to Me” (R Foundation for Statistical Computing, Vienna, Austria).
Table P values for correlation coefficients between phospho-AKT, phospho-mTOR, and phospho-S6 with ARID1a
ARID1a ARID1a Phos-AKT ARID1a Phos-AKT Phos-mTOR
Phos-AKT Phos-mTOR Phos-mTOR Phos-S6 Phos-S6 Phos-S6
Correlation coefficient
P
0.1291 0.1208 −0.0349 0.1856 −0.0558 0.1077
.1749 .2045 .7147 .0501 .5589 .2585
3. Results Distribution of subtypes was as follows: usual SCC, 48 cases; warty-basaloid carcinoma, 24 cases; warty carcinoma, 17 cases; basaloid carcinoma, 11 cases; papillary carcinoma, 9 cases; verrucous carcinoma, 2 cases; and sarcomatoid carcinoma, 1 case. Distribution of histologic grades was as follows: grade 1, 5 cases; grade 2, 30 cases; and grade 3, 77 cases. HPV DNA was detected in 20 (38%) of 52 analyzed samples. ARID1A expression was observed in all cases, ranging from 3% to 100% of tumor cells (median, 95%). In 96 cases (86%), ARID1A expression was observed in 90% or more of tumor cells. Fig. 1 shows the immunohistochemical patterns of ARID1A expression. ARID1A expression was not associated with histologic subtype (P = .61) or HPV status (P = .18). However, the Kruskal-Wallis test yielded a P = .05 for the association between ARID1A and histologic grade. The Cuzick test showed that this trend was significant (P = .03). Fig. 2 summarizes all these results. ARID1A expression decreased with decreasing levels of PTEN expression (P = .01), as shown in Fig. 3. The Table and Fig. 4 show the correlation between biomarkers. A weak positive (correlation coefficient = 0.19; P = .05) correlation was observed between ARID1A expression and phospho-S6. No association was observed between ARID1A and phospho-AKT and phospho-mTOR.
4. Discussion ARID1A has been extensively assessed in gynecologic malignancies [13,14]. To our knowledge, this is the first study evaluating ARID1A in penile carcinoma. ARID1A is located on chromosome 1p and encodes BAF250a, a large nuclear protein that participates in the formation of a switch/ sucrose nonfermentable (SWI/SNF) chromatin remodeling complex involved in the regulation of cell differentiation, proliferation, DNA repair, and tumor suppression [13].
ARID1A immunoexpression in penile SCC
Fig. 4
765
Correlation matrices between biomarkers.
Katagiri et al [22] found low ARID1A expression in 6.5% of cervical SCCs. Wang et al [23] found ARID1A to be down-regulated in cervical cancer leading to inhibition of miR31 that suppresses growth, enhances apoptosis, and decreases invasion of cancer cells. We found 86% penile SCCs to express ARID1A in 90% or more of tumor cells. Cho et al [24] found an association of lower ARID1A expression and high-grade cervical cancer. Previous studies have shown somatic inactivating mutations of ARID1A to be more frequently seen in low-grade uterine and ovarian tumors [14,19]. In the present study, lower ARID1A expression was seen at lower histologic grades. Recent studies have indicated an association of low ARID1A expression with unfavorable prognosis and chemoresistance in gynecologic malignancies [24–26]. Taking into consideration the association between histologic grade and prognosis, future studies should focus on the predictive value of ARID1A in penile SCC. Recent studies have demonstrated an implication of PTEN/PI3K/AKT pathway in penile carcinogenesis [20,27,28]. PTEN/PI3K/AKT pathway has been suggested to interact with ARID1A in gastric adenocarcinoma and ovarian carcinoma [29–32]. We found that lower ARID1A
expression was associated with decreasing PTEN expression. Samartzis et al [32] have shown that PI3K and AKT inhibitors lead to apoptosis in tumor cells with low ARID1A expression, reflecting a significant increase of the sensitivity of cancer cells to small molecule inhibitors of the PI3K/AKT pathway. The involvement of ARID1A in the oncogenesis of penile SCC merits further investigation. TMA technique offers the advantage that it allows the evaluation of a large number of cases under the same immunohistochemistry conditions. Given the known heterogeneity of biomarkers expression within same tumor, the use of TMAs instead of whole-tissue sections could be seen as a limitation in our current study. However, several studies have supported the value of the TMA usage and the appropriate representation of the overall expression levels using multiple TMA spots [33]. The lack of follow-up data is another weakness in the study given the nature of the cohort as part of consultation cases for which no additional data were obtained. In summary, ARID1A was expressed in penile SCC, in most cases at high levels. ARID1A expression was not associated with histologic subtype or HPV presence. A significant trend was found between histologic grade and
766 ARID1A expression, with lower ARID1A expression at lower histologic grades. Lower ARID1A expression was associated with decreasing of PTEN expression.
Acknowledgment We acknowledge the outstanding work of Helen Fedor and Marcella Southerland, from the TMA Lab Core at the Johns Hopkins University, in building the TMAs for the study.
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