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• W2044256105 abstract "Penile squamous cell carcinoma (PSCC) is a rare tumor associated with high-risk human papillomavirus (HR-HPV) infection in 30% to 60% of cases. Altered expression of miRNAs has been reported in HPV-related cervical and head and neck cancers, but such data have not been available for PSCC. We analyzed a series of 59 PSCCs and 8 condylomata for presence of HPV infection, for p16INK4a, Ki-67, and p53 immunohistochemical expression, and for expression of a panel of cellular miRNAs (let-7c, miR-23b, miR-34a, miR-145, miR-146a, miR-196a, and miR-218) involved in HPV-related cancer. HR-HPV DNA (HPV16 in most cases) was detected in 17/59 (29%) PSCCs; all penile condylomata (8/8) were positive for low-risk HPV6 or HPV11. HR-HPV+ PSCCs overexpressed p16INK4a in 88% cases and p53 in 35% of cases, whereas HR-HPV− PSCCs were positive for p16INK4a and p53 immunostaining in 9% and 44% of cases, respectively. Among the miRNAs investigated, expression of miR-218 was lower in PSCCs with HR-HPV infection and in p53− cancers. Hypermethylation of the promoter of the SLIT2 gene, which contains miR-218-1 in its intronic region, was frequently observed in PSCCs, mainly in those with low miR-218 expression. Epigenetic silencing of miR-218 is a common feature in HR-HPV+ PSCCs and in HR-HPV− PSCCs without immunohistochemical detection of p53. Penile squamous cell carcinoma (PSCC) is a rare tumor associated with high-risk human papillomavirus (HR-HPV) infection in 30% to 60% of cases. Altered expression of miRNAs has been reported in HPV-related cervical and head and neck cancers, but such data have not been available for PSCC. We analyzed a series of 59 PSCCs and 8 condylomata for presence of HPV infection, for p16INK4a, Ki-67, and p53 immunohistochemical expression, and for expression of a panel of cellular miRNAs (let-7c, miR-23b, miR-34a, miR-145, miR-146a, miR-196a, and miR-218) involved in HPV-related cancer. HR-HPV DNA (HPV16 in most cases) was detected in 17/59 (29%) PSCCs; all penile condylomata (8/8) were positive for low-risk HPV6 or HPV11. HR-HPV+ PSCCs overexpressed p16INK4a in 88% cases and p53 in 35% of cases, whereas HR-HPV− PSCCs were positive for p16INK4a and p53 immunostaining in 9% and 44% of cases, respectively. Among the miRNAs investigated, expression of miR-218 was lower in PSCCs with HR-HPV infection and in p53− cancers. Hypermethylation of the promoter of the SLIT2 gene, which contains miR-218-1 in its intronic region, was frequently observed in PSCCs, mainly in those with low miR-218 expression. Epigenetic silencing of miR-218 is a common feature in HR-HPV+ PSCCs and in HR-HPV− PSCCs without immunohistochemical detection of p53. Penile squamous cell carcinoma (PSCC) is a relatively rare cancer that usually originates in the epithelium of the inner prepuce and glans.1Pizzocaro G. Algaba F. Horenblas S. Solsona E. Tana S. Van Der Poel H. Watkin N.A. European Association of Urology (EAU) Guidelines Group on Penile CancerEAU penile cancer guidelines 2009.Eur Urol. 2010; 57: 1002-1012Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar It accounts for less than 0.5% of cancers in men in Western countries and for approximately 10% of cancers in men in high-incidence countries of South America and Africa.1Pizzocaro G. Algaba F. Horenblas S. Solsona E. Tana S. Van Der Poel H. Watkin N.A. European Association of Urology (EAU) Guidelines Group on Penile CancerEAU penile cancer guidelines 2009.Eur Urol. 2010; 57: 1002-1012Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar Mortality, however, is low. HPV DNA is detectable in 30% to 60% of all invasive PSCCs, and prevalence varies across different series.2Miralles-Guri C. Bruni L. Cubilla A.L. Castellsagué X. Bosch F.X. de Sanjosé S. Human papillomavirus prevalence and type distribution in penile carcinoma.J Clin Pathol. 2009; 62: 870-878Crossref PubMed Scopus (254) Google Scholar, 3Backes D.M. Kurman R.J. Pimenta J.M. Smith J.S. Systematic review of human papillomavirus prevalence in invasive penile cancer.Cancer Causes Control. 2009; 20: 449-457Crossref PubMed Scopus (309) Google Scholar, 4Cubilla A.L. Lloveras B. Alejo M. Clavero O. Chaux A. Kasamatsu E. Velazquez E.F. Lezcano C. Monfulleda N. Tous S. Alemany L. Klaustermeier J. Muñoz N. Quint W. de Sanjose S. Bosch F.X. 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: 104-114Crossref PubMed Scopus (89) Google Scholar The presence of HPV infection strongly correlates with either mixed basaloid and verrucous or purely basaloid histotype and is only weakly associated with the usual type of keratinizing SCC.2Miralles-Guri C. Bruni L. Cubilla A.L. Castellsagué X. Bosch F.X. de Sanjosé S. Human papillomavirus prevalence and type distribution in penile carcinoma.J Clin Pathol. 2009; 62: 870-878Crossref PubMed Scopus (254) Google Scholar, 3Backes D.M. Kurman R.J. Pimenta J.M. Smith J.S. Systematic review of human papillomavirus prevalence in invasive penile cancer.Cancer Causes Control. 2009; 20: 449-457Crossref PubMed Scopus (309) Google Scholar, 4Cubilla A.L. Lloveras B. Alejo M. Clavero O. Chaux A. Kasamatsu E. Velazquez E.F. Lezcano C. Monfulleda N. Tous S. Alemany L. Klaustermeier J. Muñoz N. Quint W. de Sanjose S. Bosch F.X. 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: 104-114Crossref PubMed Scopus (89) Google Scholar HPV16 and, to a lesser extent, HPV18 are the most frequent viral types associated with PSCC; HPV31 and HPV33 are rarely detected.2Miralles-Guri C. Bruni L. Cubilla A.L. Castellsagué X. Bosch F.X. de Sanjosé S. Human papillomavirus prevalence and type distribution in penile carcinoma.J Clin Pathol. 2009; 62: 870-878Crossref PubMed Scopus (254) Google Scholar, 3Backes D.M. Kurman R.J. Pimenta J.M. Smith J.S. Systematic review of human papillomavirus prevalence in invasive penile cancer.Cancer Causes Control. 2009; 20: 449-457Crossref PubMed Scopus (309) Google Scholar, 4Cubilla A.L. Lloveras B. Alejo M. Clavero O. Chaux A. Kasamatsu E. Velazquez E.F. Lezcano C. Monfulleda N. Tous S. Alemany L. Klaustermeier J. Muñoz N. Quint W. de Sanjose S. Bosch F.X. 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: 104-114Crossref PubMed Scopus (89) Google Scholar The mechanisms of HPV oncogenesis have been extensively investigated in cervical cancer, but only limited data are available for PSCC. Oncogenic HPV types cause cancer mainly by inducing degradation of the tumor suppressor proteins p53 and pRb (by E6 and E7 viral proteins, respectively), but also important are alterations of additional pathways involved in regulation of cell-cycle progression, telomere maintenance, apoptosis, chromosomal stability, and cell adhesion and migration.5Moody C.A. Laimins L.A. Human papillomavirus oncoproteins: pathways to transformation.Nat Rev Cancer. 2010; 10: 550-560Crossref PubMed Scopus (1160) Google Scholar Recently, high-risk HPV (HR-HPV) infection was reported in association with aberrant expression of oncogenic and tumor-suppressive host miRNAs.6Zheng Z.M. Wang X. Regulation of cellular miRNA expression by human papillomaviruses.Biochim Biophys Acta. 2011; 1809: 668-677Crossref PubMed Scopus (158) Google Scholar Most of these miRNAs are deregulated via the E6–p53 and E7–pRb pathways, including such as miR-34a/b/c, miR-23b, miR-145, and miR-218, which are down-regulated by HR-E6, and miR-146a and the let-7 family of miRNAs, which are down-regulated by c-Myc (which in turn is activated by both HR-E6 and HR-E7).6Zheng Z.M. Wang X. Regulation of cellular miRNA expression by human papillomaviruses.Biochim Biophys Acta. 2011; 1809: 668-677Crossref PubMed Scopus (158) Google Scholar Altered expression of these miRNAs has been reported in cervical and head and neck cancers,6Zheng Z.M. Wang X. Regulation of cellular miRNA expression by human papillomaviruses.Biochim Biophys Acta. 2011; 1809: 668-677Crossref PubMed Scopus (158) Google Scholar but there have been no previous studies on PSCC. We investigated expression of a panel of cellular miRNAs involved in HPV-related cancer in a series of PSCCs and analyzed its relationship with the presence of HPV infection and with p53, Ki-67, and p16INK4a expression. The specimens studied were 59 formalin-fixed, paraffin-embedded PSCC samples from patients who underwent surgical biopsy or resection (median age, 68 years; range, 44 to 87 years) and 8 condyloma samples as control (median age, 42 years; range, 26 to 60 years) retrieved from the archives of the Pathology Unit at the University of Padua, from January 2002 to December 2010. All diagnoses were based on World Health Organization criteria7Eble J.N. Sauter G. Epstein J.I. Sesterhenn I.A. Pathology and Genetics of Tumours of the Urinary System and Male Genital Organs. World Health Organization Classification of Tumours. IARC press, Lyon2004: 279-298Google Scholar and were confirmed jointly by three masked pathologists (A.F., R.C., and M.F.). The study was approved (no. 0037713) by the Institutional Ethical Review Board of the University of Padua and was conducted in accord with the Institute's ethical regulations on research involving human tissues. Automated immunohistochemistry staining (Leica Microsystems BOND-MAX; Menarini, Florence, Italy) was performed on 4-μm formalin-fixed, paraffin-embedded sections with primary antibodies for p53 (clone DO-7, prediluted; Dako, Glostrup, Denmark; Carpinteria, CA), p16INK4a (clone JC8, prediluted; Santa Cruz Biotechnology, Dallas, TX), and Ki-67 (clone SP6, dilution 1:200; Spring Bioscience, Pleasanton, CA), as described previously.8Fassina A. Cappellesso R. Guzzardo V. Dalla Via L. Piccolo S. Ventura L. Fassan M. Epithelial-mesenchymal transition in malignant mesothelioma.Mod Pathol. 2012; 25: 86-99Crossref PubMed Scopus (115) Google Scholar, 9Rosato A. Menin C. Boldrin D. Santa S.D. Bonaldi L. Scaini M.C. Del Bianco P. Zardo D. Fassan M. Cappellesso R. Fassina A. Survivin expression impacts prognostically on NSCLC but not SCLC.Lung Cancer. 2013; 79: 180-186Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar Appropriate positive and negative controls were run concurrently. Three observers (A.F., R.C., and M.F.) independently scored protein expression and a consensus score was reached. p53 staining was defined as positive if ≥75% of the cells exhibited a strong nuclear staining (indicative of p53 accumulation due to mutation). For evaluation of p16INK4a expression, patterns of immunohistochemistry reactions were scored on a four-point scale: 0, complete absence of p16INK4a staining in all neoplastic cells; 1, staining only in isolated and dispersed neoplastic cells; 2, staining in patchy and scattered clusters of neoplastic cells; and 3, dense and continuous cytoplasmic/nuclear staining in all neoplastic cells (except hyperkeratotic or parakeratotic areas).10Cubilla A.L. Lloveras B. Alejo M. Clavero O. Chaux A. Kasamatsu E. Monfulleda N. Tous S. Alemany L. Klaustermeier J. Muñoz N. Quint W. de Sanjose S. Bosch F.X. Value of p16(INK)(4)(a) in the pathology of invasive penile squamous cell carcinomas: a report of 202 cases.Am J Surg Pathol. 2011; 35: 253-261Crossref PubMed Scopus (92) Google Scholar In each tumor, the highest score was assumed to be representative of the whole lesion, and only score 3 was considered indicative of p16INK4a overexpression.10Cubilla A.L. Lloveras B. Alejo M. Clavero O. Chaux A. Kasamatsu E. Monfulleda N. Tous S. Alemany L. Klaustermeier J. Muñoz N. Quint W. de Sanjose S. Bosch F.X. Value of p16(INK)(4)(a) in the pathology of invasive penile squamous cell carcinomas: a report of 202 cases.Am J Surg Pathol. 2011; 35: 253-261Crossref PubMed Scopus (92) Google Scholar Expression of Ki-67 was classified as weak if ≤15% of the neoplastic cells exhibited nuclear staining, intermediate if 15% to 60% of nuclei were positive, and strong if >60% of nuclei were positive.11Protzel C. Knoedel J. Zimmermann U. Woenckhaus C. Poetsch M. Giebel J. Expression of proliferation marker Ki67 correlates to occurrence of metastasis and prognosis, histological subtypes and HPV DNA detection in penile carcinomas.Histol Histopathol. 2007; 22: 1197-1204PubMed Google Scholar From the corresponding significant non-necrotic areas of the slides (hematoxylin and eosin stain), 2-mm cores of tissue were microdissected from the paraffin block and deparaffinized in xylene for 3 minutes at 50°C. Genomic DNA was extracted with a MagNA Pure 96 nucleic acid kit in a MagNA Pure 96 system (Roche Diagnostics, Mannheim, Germany; Indianapolis, IN) and was eluted in 100 μL, according to the manufacturer’s recommendations. For PCR amplification and reverse hybridization, 5 μL of the DNA solution was used with the INNO-LiPA HPV Genotyping Extra line probe assay (Innogenetics, Ghent, Belgium) for HPV detection and typing, as described previously.12Barzon L. Militello V. Pagni S. Palù G. Comparison of INNO-LiPA Genotyping Extra and Hybrid Capture 2 assays for detection of carcinogenic human papillomavirus genotypes.J Clin Virol. 2012; 55: 256-261Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar The presence of HPV types in all samples positive by the line probe assay was also investigated by type-specific quantitative real-time PCR (qPCR), which allowed estimation of the viral genome copy number per cell (ratio to the β-globin gene copy number), as described previously.13Militello V. Trevisan M. Squarzon L. Biasolo M.A. Rugge M. Militello C. Palù G. Barzon L. Investigation on the presence of polyomavirus, herpesvirus, and papillomavirus sequences in colorectal neoplasms and their association with cancer.Int J Cancer. 2009; 124: 2501-2503Crossref PubMed Scopus (33) Google Scholar Total RNA was extracted from tissue cores with an Ambion RecoverAll kit (Life Technologies, Carlsbad, CA), as described previously.8Fassina A. Cappellesso R. Guzzardo V. Dalla Via L. Piccolo S. Ventura L. Fassan M. Epithelial-mesenchymal transition in malignant mesothelioma.Mod Pathol. 2012; 25: 86-99Crossref PubMed Scopus (115) Google Scholar, 14Fassina A. Cappellesso R. Fassan M. Classification of non-small cell lung carcinoma in transthoracic needle specimens using microRNA expression profiling.Chest. 2011; 140: 1305-1311Crossref PubMed Scopus (61) Google Scholar, 15Fassina A. Cappellesso R. Simonato F. Siri M. Ventura L. Tosato F. Busund L.T. Pelizzo M.R. Fassan M. A 4-microRNA signature can discriminate primary lymphomas from anaplastic carcinomas in thyroid cytology smears.Cancer Cytopathol. 2014; 122: 274-281Crossref PubMed Scopus (26) Google Scholar To detect and quantify mature hsa-miR-145, hsa-miR-146a, hsa-miR-23b, hsa-miR-218, hsa-miR-196a, hsa-miR-34a, and hsa-let-7c, an NCode miRNA real-time quantitative PCR with reverse transcription (RT-qPCR) method (Life Technologies) was used on a LightCycler 480 real-time PCR system (Roche Diagnostics); results were normalized to the small nuclear RNA U6B (RNU6b assay; Life Technologies). Primer sequences are reported in Table 1. All reactions were run in triplicate, including no-template controls.Table 1Primers Used for miRNA Real-Time Quantitative PCR with Reverse TranscriptionmiRNAForward primerhsa-let-7c5′-CGCTGAGGTAGTAGGTTGTATGGTT-3′hsa-miR-23b5′-ATCACATTGCCAGGGATTACC-3′hsa-miR-34a5′-GGCAGTGTCTTAGCTGGTTGT-3′hsa-miR-1455′-CAGTTTTCCCAGGAATCCCT-3′hsa-miR-146a5′-TGAGAACTGAATTCCATGGGTT-3′hsa-miR-196a5′-GCTAGGTAGTTTCATGTTGTTGGG-3′hsa-miR-2185′-CGTTGTGCTTGATCTAACCATGT-3′RNU6B5′-ACGCAAATTCGTGAAGCGTT-3′ Open table in a new tab Methylation analysis of the SLIT2 and SLIT3 promoters was performed by treatment of genomic DNA with bisulfite using an EpiTect bisulfite kit (Qiagen, Hilden, Germany; Valencia, CA) followed by PCR amplification with methylation-sensitive primers, as described previously.16Dallol A. Da Silva N.F. Viacava P. Minna J.D. Bieche I. Maher E.R. Latif F. SLIT2, a human homologue of the Drosophila Slit2 gene, has tumor suppressor activity and is frequently inactivated in lung and breast cancers.Cancer Res. 2002; 62: 5874-5880PubMed Google Scholar Sequence analysis of PCR amplicons was performed by cycle sequencing using a BigDye Terminator version 1.1 cycle sequencing kit (Life Technologies). The correlation between continuous variables was computed as Pearson's r. Nonparametric testing (U-test) was used to study differences between miRNA amounts for condyloma and PSCC cases, among PSCC cases positive and negative for HPV16, and among PSCC cases positive and negative for p16INK4a and p53. In addition, levels of within-sample agreement between HPV type and p16INK4a expression were assessed by χ2 test. P < 0.05 was considered statistically significant. All statistical analyses were performed using R software version 2.9 (R Foundation for Statistical Computing, Vienna, Austria). Data are expressed as means ± SD, as median and range, or as percentages for categorical variables. The PSCC specimens exhibited a wide spectrum of microscopic findings (Figure 1). Overall, the 59 specimens (54 invasive and 5 in situ) exhibited a classic squamous appearance; the series covered the full range from well to poorly differentiated (although the majority had a moderate degree of differentiation and keratinization). In two of the poorly differentiated PSCCs, a comedo-like necrosis was observed. Invasion presented as individual cells or as sheets and nests of atypical cells. A verrucous feature was present in 10 PSCCs, with papillomatous growth and hyperparakeratosis with low to moderate grade tumor cells and common koilocytosis. Finally, four PSCCs exhibited a basaloid appearance, with tightly closed tumor cell nests always associated with comedo-type necrosis. These cells were small, with scant cytoplasm, oval or round nuclei, and inconspicuous nucleoli. Condylomata presented complex papillary arrangements of squamous epithelium in association with vacuolization of keratinocytes and enlarged, hyperchromatic, and folded nuclei (koilocytosis), along with stromal lymphocytic infiltration. The presence of HPV infection in penile specimens was investigated by line probe assay and HPV type-specific real-time PCR. Specimens were considered HPV+ if HPV DNA copy number was greater than 1 genome equivalent per cell, consistent with a clonal expansion of HPV+ cancer cells. These stringent criteria were chosen to differentiate pathogenic HPV types from co-infecting or contaminating HPV types, which may be present in cancer tissues. According to these criteria, HPV DNA was detected in two of five (40%) cases of PSCC in situ and in 16/54 (30%) cases of invasive PSCC (Table 2). Overall, 17/59 (29%) PSCCs were positive for HR-HPV (Table 3). All eight penile condylomata were positive for low-risk HPV6 or HPV11. Estimation of HPV DNA copy number in PSCCs by qPCR indicated the presence of approximately 1 to 100 genome equivalents per cell. The presence of HR-HPV infection was detected in 3/4 (75%) basaloid PSCCs, 2/10 (20%) verrucous PSCCs, and 2/6 (33%) grade 3 PSCCs (Table 3).Table 2Results of HPV Detection and Typing and of p16INK4a, p53, and Ki-67 Immunostaining in PSCC and Control SpecimensImmunostainingKi-67+‡Ki-67 positivity is defined as immunoreaction in >60% of cells.11 [no. (%)]HPV+ [no. (%)]HPV type§High risk: HPV16, HPV31, HPV45, and HPV68. Low risk: HPV6, HPV11. (no.)p16∗p16INK4a positivity is defined as pattern 3 staining: dense and continuous cytoplasmic/nuclear staining in all neoplastic cells (except hyperkeratotic or parakeratotic areas).10p53†p53 positivity is defined as p53 nuclear staining in ≥75% of the cells.[no. (%)]Invasive PSCC (n = 54) +−10 (18.5)5 (50.0)9 (90)HPV16+ (9), HPV− (1) ++8 (14.8)4 (50.0)5 (62.5)HPV16+ (4), HPV68+ (1), HPV− (3) −−16 (29.6)9 (56.3)2 (13.3)HPV16+ (1), HPV45+ (1), HPV− (14) −+20 (37.1)13 (65)0 (0)HPV− (20)PSCC in situ (n = 5) ++1 (20.0)0 (0)1 (100)HPV16+ (1) −−4 (80.0)2 (50)1 (25)HPV11+ (1), HPV− (3)Condyloma (n = 8) −+3 (37.5)1 (33.3)3 (100)HPV6+ (2), HPV11+ (1) −−5 (62.5)0 (0)5 (100)HPV6+ (4), HPV6+HPV31+ (1)HPV, human papillomavirus; PSCC, penile squamous cell carcinoma.∗ p16INK4a positivity is defined as pattern 3 staining: dense and continuous cytoplasmic/nuclear staining in all neoplastic cells (except hyperkeratotic or parakeratotic areas).10Cubilla A.L. Lloveras B. Alejo M. Clavero O. Chaux A. Kasamatsu E. Monfulleda N. Tous S. Alemany L. Klaustermeier J. Muñoz N. Quint W. de Sanjose S. Bosch F.X. Value of p16(INK)(4)(a) in the pathology of invasive penile squamous cell carcinomas: a report of 202 cases.Am J Surg Pathol. 2011; 35: 253-261Crossref PubMed Scopus (92) Google Scholar† p53 positivity is defined as p53 nuclear staining in ≥75% of the cells.‡ Ki-67 positivity is defined as immunoreaction in >60% of cells.11Protzel C. Knoedel J. Zimmermann U. Woenckhaus C. Poetsch M. Giebel J. Expression of proliferation marker Ki67 correlates to occurrence of metastasis and prognosis, histological subtypes and HPV DNA detection in penile carcinomas.Histol Histopathol. 2007; 22: 1197-1204PubMed Google Scholar§ High risk: HPV16, HPV31, HPV45, and HPV68. Low risk: HPV6, HPV11. Open table in a new tab Table 3Histopathological Findings in PSCCs According to HR-HPV Detection and Biomarker ExpressionFeaturePSCC [no. (%)]Subtype [no. (%)]Grade [no. (%)]WDBasaloidVerrucous123HR-HPV −42∗Of the 42 cases lacking HR-HPV infection, 1 was positive for HPV11. (71.2)33 (55.9)1 (1.7)8 (13.6)13 (22.0)25 (42.4)4 (6.8) +17 (28.8)12 (20.3)3 (5.1)2 (3.4)9 (15.3)6 (10.2)2 (3.4)p16INK4a pattern†p16INK4a immunostaining pattern: 0, complete absence of p16INK4a staining in all of the neoplastic cells; 1, staining only in isolated and dispersed neoplastic cells; 2, staining in patchy and scattered clusters of neoplastic cells; and 3, dense and continuous cytoplasmic/nuclear staining in all neoplastic cells (except hyperkeratotic or parakeratotic areas).10 018 (30.5)13 (22.0)1 (1.7)4 (6.8)3 (5.1)12 (20.3)3 (5.1) 116 (27.1)13 (22.0)03 (5.1)6 (10.2)10 (16.9)0 26 (10.2)6 (10.2)002 (3.4)3 (5.1)1 (1.7) 319 (32.2)13 (22.0)3 (5.1)3 (5.1)10 (16.9)7 (11.9)2 (3.4)p53‡p53 positivity is defined as p53 nuclear staining in ≥75% of the cells. −29 (49.2)21 (35.6)2 (3.4)6 (10.2)15 (25.4)12 (20.3)2 (3.4) +30 (50.8)24 (40.7)2 (3.4)4 (6.8)8 (13.6)18 (30.5)4 (6.8)Total59 (100)45 (76.3)4 (6.8)10 (16.9)21 (35.6)32 (54.2)6 (10.2)HR-HPV, high-risk human papilloma virus; WD, well differentiated.∗ Of the 42 cases lacking HR-HPV infection, 1 was positive for HPV11.† p16INK4a immunostaining pattern: 0, complete absence of p16INK4a staining in all of the neoplastic cells; 1, staining only in isolated and dispersed neoplastic cells; 2, staining in patchy and scattered clusters of neoplastic cells; and 3, dense and continuous cytoplasmic/nuclear staining in all neoplastic cells (except hyperkeratotic or parakeratotic areas).10Cubilla A.L. Lloveras B. Alejo M. Clavero O. Chaux A. Kasamatsu E. Monfulleda N. Tous S. Alemany L. Klaustermeier J. Muñoz N. Quint W. de Sanjose S. Bosch F.X. Value of p16(INK)(4)(a) in the pathology of invasive penile squamous cell carcinomas: a report of 202 cases.Am J Surg Pathol. 2011; 35: 253-261Crossref PubMed Scopus (92) Google Scholar‡ p53 positivity is defined as p53 nuclear staining in ≥75% of the cells. Open table in a new tab HPV, human papillomavirus; PSCC, penile squamous cell carcinoma. HR-HPV, high-risk human papilloma virus; WD, well differentiated. Overexpression of p16INK4a (Figure 1) (ie, pattern 3, as described above and in Ref. 10Cubilla A.L. Lloveras B. Alejo M. Clavero O. Chaux A. Kasamatsu E. Monfulleda N. Tous S. Alemany L. Klaustermeier J. Muñoz N. Quint W. de Sanjose S. Bosch F.X. Value of p16(INK)(4)(a) in the pathology of invasive penile squamous cell carcinomas: a report of 202 cases.Am J Surg Pathol. 2011; 35: 253-261Crossref PubMed Scopus (92) Google Scholar), which is a considered a surrogate biomarker of HR-HPV infection, was identified in 19/59 (32%) PSCCs (Table 3) and was significantly associated with the presence of HR-HPV infection (P < 0.001, χ2 test), with sensitivity of 88% and specificity of 95%. Interestingly, p16INK4a overexpression seemed to be associated, although not significantly, with the presence of basaloid features and with low histological grading (grades 1 and 2) (Table 3). Because this observation is based on few PSCCs, it should be confirmed by further studies involving an adequate number of cases. p53 immunoreactivity was detected in 30/59 (51%) PSCCs. No significant correlation was observed between p53 expression and HR-HPV infection, p16INK4a immunostaining, and histological features (Figure 1); p53+ immunostaining was more frequently observed in grade 2 and grade 3 PSCCs than in grade 1 PSCCs (Table 3). Immunostaining for the proliferation marker Ki-67 was also investigated in penile specimens. No relationship was observed between the percentage of Ki-67+ cells and histological features, HR-HPV infection, or p16INK4a and p53 immunostaining. Expression of a panel of miRNAs (let-7c, miR-34a, miR-23b, miR-145, miR-146a, miR-196a, and miR-218) was investigated by RT-qPCR in all samples. The panel was selected based on literature indicating an association with HPV16 infection and/or cervical carcinoma.6Zheng Z.M. Wang X. Regulation of cellular miRNA expression by human papillomaviruses.Biochim Biophys Acta. 2011; 1809: 668-677Crossref PubMed Scopus (158) Google Scholar, 17Au Yeung C.L. Tsang T.Y. Yau P.L. Kwok T.T. Human papillomavirus type 16 E6 induces cervical cancer cell migration through the p53/microRNA-23b/urokinase-type plasminogen activator pathway.Oncogene. 2011; 30: 2401-2410Crossref PubMed Scopus (120) Google Scholar, 18Wang X. Wang H.K. McCoy J.P. Banerjee N.S. Rader J.S. Broker T.R. Meyers C. Chow L.T. Zheng Z.M. Oncogenic HPV infection interrupts the expression of tumor-suppressive miR-34a through viral oncoprotein E6.RNA. 2009; 15: 637-647Crossref PubMed Scopus (185) Google Scholar, 19Martinez I. Gardiner A.S. Board K.F. Monzon F.A. Edwards R.P. Khan S.A. Human papillomavirus type 16 reduces the expression of microRNA-218 in cervical carcinoma cells.Oncogene. 2008; 27: 2575-2582Crossref PubMed Scopus (287) Google Scholar, 20Lajer C.B. Garnaes E. Friis-Hansen L. Norrild B. Therkildsen M.H. Glud M. Rossing M. Lajer H. Svane D. Skotte L. Specht L. Buchwald C. Nielsen F.C. The role of miRNAs in human papilloma virus (HPV)-associated cancers: bridging between HPV-related head and neck cancer and cervical cancer.Br J Cancer. 2012; 106: 1526-1534Crossref PubMed Scopus (176) Google Scholar, 21Huang L. Lin J.X. Yu Y.H. Zhang M.Y. Wang H.Y. Zheng M. Downregulation of six microRNAs is associated with advanced stage, lymph node metastasis and poor prognosis in small cell carcinoma of the cervix.PLoS One. 2012; 7: e33762Crossref PubMed Scopus (115) Google Scholar, 22Lee J.W. Choi C.H. Choi J.J. Park Y.A. Kim S.J. Hwang S.Y. Kim W.Y. Kim T.J. Lee J.H. Kim B.G. Bae D.S. Altered microRNA expression in cervical carcinomas.Clin Cancer Res. 2008; 14: 2535-2542Crossref PubMed Scopus (281) Google Scholar Expression of miR-218 showed a downward trend in PSCCs with HR-HPV infection (P = 0.069), and it was down-regulated in p53− PSCCs (P = 0.011). In the subgroup of HR-HPV− PSCCs, expression of miR-218 was significantly lower in p53− than p53+ PSCCs (P = 0.020); no difference in miR-218 expression was observed between p53+ and p53− tumors in the HR-HPV+ PSCC group (Figure 2A). miR-218 expression levels were similar among HR-HPV+ cases and those lacking both p53 immunolabeling and HR-HPV infection. Levels of miR-23b, miR-145, and miR146a were lower in HR-HPV+ (and p16INK4a+) PSCCs than in HR-HPV− PSCCs, although the difference was not statistically significant (Figure 2B). No association was observed between these miRNAs and p53 or Ki-67 expression and histological features, except that miR-196a levels were significantly higher (P < 0.05) in grade 2 and 3 PSCCs than in grade 1 PSCCs (relative expression: 3.0 ± 5.6 versus 1.0 ± 0.6, respectively). Because promoter hypermethylation has been reported to be a common mechanism of miR-218 down-regulation in several cancers, including oral SCCs,23Uesugi A. Kozaki K. Tsuruta T. Furuta M. Morita K. Imoto I. Omura K. Inazawa J. The tumor suppressive microRNA miR-218 targets the mTOR component Rictor and inhibits AKT phosphorylation in oral cancer.Cancer Res. 2011; 71: 5765-5778Crossref PubMed Scopus (220) Google Scholar we performed methylation analysis of the promoter of SLIT2 and SLIT3 (tumor suppressor genes that respectively contain the miR-218-1 and miR-218-2 precursors in the intronic region), using a methylation-specific PCR method that qualitatively assesses the presence of a hypermethylated CpG island in the promoters.16Dallol A. Da Silva N.F. Viacava P. Minna J.D. Bieche I. Maher E.R. Latif F. SLIT2, a human homologue of the Drosophila Slit2 gene, has tumor suppressor activity and is frequently inactivated in lung and breast cancers.Cancer Res. 2002; 62: 5874-5880PubMed Google Scholar For SLIT2, both methylated and unmethylated alleles were detected in tumor samples by PCR, indicating partial methylation. However, in tumors with low miR-218 levels, the degree of promoter methylation was greater than in those with higher miR-218 levels (Figure 3). Sequence analysis of methylation-specific PCR amplicons of the SLIT2 promoter confirmed the presence of methylated CpG sites. For SLIT3, only unmethylated alleles were observed (Figure 3). In this retrospective study, we investigated expression of a group of miRNAs associated with HPV-related cancer on a series of PSCCs and correlated miRNA expression with the presence of HPV infection, with p16INK4a, p53, and Ki-67 immunostaining, and with pathological features. The main finding of the present study is the demonstration that miR-218 was significantly down-regulated in p53− PSCCs. This finding was confirmed among HR-HPV− cancers. Interestingly, miR-218 expression showed a downward trend in HR-HPV+ PSCCs, compared with HR-HPV− PSCCs. Moreover, among HR-HPV+ cases the expression levels of miR-218 were similar to those cases lacking both p53 immunolabeling and HR-HPV infection, irrespective of p53 expression. In addition, by using stringent criteria to define HPV+ tumors, we demonstrated the presence of HR-HPV infection in approximately one third of PSCCs and confirmed the significant association between HR-HPV infection and p16INK4a overexpression. Our results on miR-218 expression in PSCCs are consistent with the literature on other HPV-related cancers. miR-218 has been shown to be significantly down-regulated in cervical carcinoma cell lines and in cervical tissues containing HPV16, compared with the normal cervix,19Martinez I. Gardiner A.S. Board K.F. Monzon F.A. Edwards R.P. Khan S.A. Human papillomavirus type 16 reduces the expression of microRNA-218 in cervical carcinoma cells.Oncogene. 2008; 27: 2575-2582Crossref PubMed Scopus (287) Google Scholar, 24Li Y. Liu J. Yuan C. Cui B. Zou X. Qiao Y. High-risk human papillomavirus reduces the expression of microRNA-218 in women with cervical intraepithelial neoplasia.J Int Med Res. 2010; 38: 1730-1736Crossref PubMed Scopus (38) Google Scholar as well as in HR-HPV+ head and neck SCCs, compared with HR-HPV− cancers.25Wald A.I. Hoskins E.E. Wells S.I. Ferris R.L. Khan S.A. Alteration of microRNA profiles in squamous cell carcinoma of the head and neck cell lines by human papillomavirus.Head Neck. 2011; 33: 504-512Crossref PubMed Scopus (139) Google Scholar The E6 gene of high-risk HPV16 (but not of low-risk HPV6) reduced expression of both miR-218 and of SLIT2, which contains miR-218-1 sequence in its intronic region.19Martinez I. Gardiner A.S. Board K.F. Monzon F.A. Edwards R.P. Khan S.A. Human papillomavirus type 16 reduces the expression of microRNA-218 in cervical carcinoma cells.Oncogene. 2008; 27: 2575-2582Crossref PubMed Scopus (287) Google Scholar miR-218 deregulation by E6 in HPV-related cancers has been suggested to occur via promoter hypermethylation of the SLIT2 gene.26Wu D.W. Cheng Y.W. Wang J. Chen C.Y. Lee H. Paxillin predicts survival and relapse in non-small cell lung cancer by microRNA-218 targeting.Cancer Res. 2010; 70: 10392-10401Crossref PubMed Scopus (118) Google Scholar However, miR-218 has been found to be underexpressed also in HPV− oral SCC, and the mechanism of silencing was again through promoter hypermethylation,23Uesugi A. Kozaki K. Tsuruta T. Furuta M. Morita K. Imoto I. Omura K. Inazawa J. The tumor suppressive microRNA miR-218 targets the mTOR component Rictor and inhibits AKT phosphorylation in oral cancer.Cancer Res. 2011; 71: 5765-5778Crossref PubMed Scopus (220) Google Scholar thus indicating that promoter hypermethylation is a common mechanism of miR-218 silencing in SCCs. In the present study, methylation of the SLIT2 promoter was observed in both HR-HPV+ and HR-HPV− PSCCs, and the degree of promoter methylation correlated with miR-218 silencing. In three cases, however, we observed equal signals for methylated and unmethylated promoter DNA. Such findings suggest the possibility that other mechanisms (eg, chromatin structure in the promoter region) may play an important role in the regulation of miR-218 expression in addition to DNA methylation. Furthermore, our findings identify miR-218 down-regulation as an important event in PSCC oncogenesis, which in p53+ cases could be related to a HR-HPV effect. Hypermethylation of the promoter of the SLIT3 gene, which contains miR-218-2 in its intronic region, has been also reported in different cancers, but less frequently than hypermethylation of the SLIT2 promoter.23Uesugi A. Kozaki K. Tsuruta T. Furuta M. Morita K. Imoto I. Omura K. Inazawa J. The tumor suppressive microRNA miR-218 targets the mTOR component Rictor and inhibits AKT phosphorylation in oral cancer.Cancer Res. 2011; 71: 5765-5778Crossref PubMed Scopus (220) Google Scholar, 27Dickinson R.E. Dallol A. Bieche I. Krex D. Morton D. Maher E.R. Latif F. Epigenetic inactivation of SLIT3 and SLIT1 genes in human cancers.Br J Cancer. 2004; 91: 2071-2078Crossref PubMed Scopus (115) Google Scholar Indeed, all of the cases analyzed in the present study were unmethylated. miR-218 has antiproliferative and proapoptotic activity in vitro.23Uesugi A. Kozaki K. Tsuruta T. Furuta M. Morita K. Imoto I. Omura K. Inazawa J. The tumor suppressive microRNA miR-218 targets the mTOR component Rictor and inhibits AKT phosphorylation in oral cancer.Cancer Res. 2011; 71: 5765-5778Crossref PubMed Scopus (220) Google Scholar, 26Wu D.W. Cheng Y.W. Wang J. Chen C.Y. Lee H. Paxillin predicts survival and relapse in non-small cell lung cancer by microRNA-218 targeting.Cancer Res. 2010; 70: 10392-10401Crossref PubMed Scopus (118) Google Scholar Several cell targets have been identified for this miRNA, including ROBO1, PXN, and LASP1,26Wu D.W. Cheng Y.W. Wang J. Chen C.Y. Lee H. Paxillin predicts survival and relapse in non-small cell lung cancer by microRNA-218 targeting.Cancer Res. 2010; 70: 10392-10401Crossref PubMed Scopus (118) Google Scholar, 28Tie J. Pan Y. Zhao L. Wu K. Liu J. Sun S. Guo X. Wang B. Gang Y. Zhang Y. Li Q. Qiao T. Zhao Q. Nie Y. Fan D. miR-218 inhibits invasion and metastasis of gastric cancer by targeting the Robo1 receptor.PLoS Genet. 2010; 6: e1000879Crossref PubMed Scopus (392) Google Scholar which are implicated in tumor invasion and metastasis, and the mTOR component RICTOR,23Uesugi A. Kozaki K. Tsuruta T. Furuta M. Morita K. Imoto I. Omura K. Inazawa J. The tumor suppressive microRNA miR-218 targets the mTOR component Rictor and inhibits AKT phosphorylation in oral cancer.Cancer Res. 2011; 71: 5765-5778Crossref PubMed Scopus (220) Google Scholar which has antiproliferative activity. Moreover, the SLIT2 gene has tumor suppressor activity and, together with pRb and p53, is frequently mutated, inactivated, or down-regulated in human cancer, indicating its driving role in oncogenesis.29Peifer M. Fernández-Cuesta L. Sos M.L. George J. Seidel D. Kasper L.H. et al.Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer.Nat Genet. 2012; 44: 1104-1110Crossref PubMed Scopus (1007) Google Scholar In the present study, expression of miR-218 was lower in HR-HPV+ PSCCs and in HR-HPV− PSCCs without p53 immunostaining than in those positive for p53. This suggests that miR-218 is down-regulated independently of p53 and might represent an important molecular event in penile oncogenesis, in addition to HR-HPV transformation and p53 inactivation. We also addressed HPV detection and typing in penile tumors and correlation with biomarkers. In accord with the literature,2Miralles-Guri C. Bruni L. Cubilla A.L. Castellsagué X. Bosch F.X. de Sanjosé S. Human papillomavirus prevalence and type distribution in penile carcinoma.J Clin Pathol. 2009; 62: 870-878Crossref PubMed Scopus (254) Google Scholar, 3Backes D.M. Kurman R.J. Pimenta J.M. Smith J.S. Systematic review of human papillomavirus prevalence in invasive penile cancer.Cancer Causes Control. 2009; 20: 449-457Crossref PubMed Scopus (309) Google Scholar, 4Cubilla A.L. Lloveras B. Alejo M. Clavero O. Chaux A. Kasamatsu E. Velazquez E.F. Lezcano C. Monfulleda N. Tous S. Alemany L. Klaustermeier J. Muñoz N. Quint W. de Sanjose S. Bosch F.X. 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: 104-114Crossref PubMed Scopus (89) Google Scholar HPV16 was the most commonly found HR-HPV type, confirming the relevant role of this HPV type in penile carcinogenesis. In our series of 54 invasive PSCC cases, one was HPV45+ and another was HPV68+ (Table 2). In addition to HPV infection, other underlying molecular mechanisms of carcinogenesis are suggested by the negative p16INK4a immunostaining in the HPV45+ case and p53 overexpression in the HPV68+ case. Although p16INK4a protein expression was a very good marker of HR-HPV infection in PSCCs, p53 immunostaining did not correlate with HR-HPV infection, which is in accord with previous report.30Stankiewicz E. Prowse D.M. Ktori E. Cuzick J. Ambroisine L. Zhang X. Kudahetti S. Watkin N. Corbishley C. Berney D.M. The retinoblastoma protein/p16 INK4A pathway but not p53 is disrupted by human papillomavirus in penile squamous cell carcinoma.Histopathology. 2011; 58: 433-439Crossref PubMed Scopus (23) Google Scholar Stratification of PSCCs according to HR-HPV infection, p16INK4a and p53 immunostaining, tumor grade, and tumor subtype demonstrated that tumors with HR-HPV infection overexpressed p16INK4a, were lower grade, and in some cases were characterized by basaloid features, whereas p53+ immunostaining was more common in cases with high histological grade. In this regard, p16INK4a overexpression and HR-HPV infection are considered markers of good prognosis in PSCC,31Lont A.P. Kroon B.K. Horenblas S. Gallee M.P. Berkhof J. Meijer C.J. Snijders P.J. Presence of high-risk human papillomavirus DNA in penile carcinoma predicts favorable outcome in survival.Int J Cancer. 2006; 119: 1078-1081Crossref PubMed Scopus (174) Google Scholar, 32Gunia S. Erbersdobler A. Hakenberg O.W. Koch S. May M. p16(INK4a) is a marker of good prognosis for primary invasive penile squamous cell carcinoma: a multi-institutional study.J Urol. 2012; 187: 899-907Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar whereas p53 expression predicts poor prognosis.33Gunia S. Kakies C. Erbersdobler A. Hakenberg O.W. Koch S. May M. Expression of p53, p21 and cyclin D1 in penile cancer: p53 predicts poor prognosis.J Clin Pathol. 2012; 65: 232-236Crossref PubMed Scopus (51) Google Scholar Basaloid PSCCs are usually associated with a worse prognosis.7Eble J.N. Sauter G. Epstein J.I. Sesterhenn I.A. Pathology and Genetics of Tumours of the Urinary System and Male Genital Organs. World Health Organization Classification of Tumours. IARC press, Lyon2004: 279-298Google Scholar Interestingly, in our series 3/4 basaloid cases were HR-HPV+, which should have indicated a better prognosis; however, two of these cases were also p53+, which could explain the generally worse outcome of this subtype of PSCCs. This issue should be further addressed in a numerically appropriate series of basaloid PSCCs. Here, we have reported for the first time the analysis of miRNA expression in PSCC. miR-218 was significantly down-regulated in HR-HPV+ PSCCs and in those without immunohistochemical detection of p53 as a consequence of epigenetic silencing of the SLIT2 promoter. Silencing of miR-218 and SLIT2 appears to be an important molecular event involved in PSCC and SCC oncogenesis." @default.
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• W2044256105 title "Profiling of Expression of Human Papillomavirus–Related Cancer miRNAs in Penile Squamous Cell Carcinomas" @default.
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