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Immunohistochemical profile of the penile urethra and differential expression of GATA3 in urothelial versus squamous cell carcinomas of the penile urethra
Human Pathology (2013) 44, 2760–2767
www.elsevier.com/locate/humpath
Original contribution
Immunohistochemical profile of the penile urethra and differential expression of GATA3 in urothelial versus squamous cell carcinomas of the penile urethra☆ Alcides Chaux MD a,b,⁎, Jeong S. Han MD, PhD a,c , Stephen Lee MD a , Nilda Gonzalez-Roibon MD a , Rajni Sharma PhD a , Arthur L. Burnett MD d , Antonio L. Cubilla MD e , George J. Netto MD a,d,f a
Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD 31231, USA Office of Scientific Research, Norte University, 1614 Asunción, Paraguay c Kaiser Permanente Medical Center, Oakland, CA 94611, USA d Department of Urology, Johns Hopkins Medical Institutions, Baltimore, MD 31231, USA e Instituto de Patología e Investigación, 1817 Asunción, Paraguay f Department of Oncology, Johns Hopkins Medical Institutions, Baltimore, MD 31231, USA b
Received 29 May 2013; revised 9 July 2013; accepted 17 July 2013
Keywords: Penile urethra; Squamous cell carcinoma; Urothelial carcinoma; CK7; CK20; p63; GATA3; Immunohistochemistry
Summary The penile urethra has a distinctive morphology not yet fully characterized by immunohistochemistry. In addition, both urothelial and squamous cell carcinomas have been reported in the penile urethra, and the distinction between these 2 tumors might be difficult. The purposes of this study are to assess the histology and immunohistochemical profile (CK20, CK7, p63, and GATA3) of the penile urethra and to assess the usefulness of Trans-acting T-cell-specific transcription factor (GATA3) and human papillomavirus detection in distinguishing urothelial versus squamous cell carcinomas. Normal penile urethra was evaluated in 11 total penectomies. The penile urethra was lined by 2 cell layers: a superficial single layer of CK7+, CK20−, and p63− columnar cells and a deep stratified layer of CK7−, CK20−, and p63+ cubical cells. Both layers were GATA3+, supporting urothelial differentiation. In addition, 2 tissue microarrays and 6 surgical specimens of primary tumors of the penile urethra (3 urothelial and 3 squamous cell carcinomas) were evaluated for GATA3 expression. In the tissue microarrays, 22 of 25 upper tract urothelial carcinomas and 0 of 38 penile squamous cell carcinomas were GATA3+. In the surgical specimens, GATA3 was positive in all urothelial carcinomas and negative in all squamous cell carcinomas. Human papillomavirus was detected in 2 of 3 squamous cell carcinomas and in 0 of 3 of the urothelial carcinomas. In conclusion, the penile urethra is covered by epithelial cells that are unique in morphology and immunohistochemical profile. In addition, our study suggests that GATA3 and human papillomavirus detection are useful markers for distinguishing urothelial carcinomas from squamous cell carcinomas of the penile urethra. © 2013 Elsevier Inc. All rights reserved.
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Disclosure: 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. Office of Scientific Research, Norte University, Gral. Santos e/25 de Mayo, Asunción, Paraguay. E-mail address: [email protected] (A. Chaux). 0046-8177/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2013.07.023
GATA3 in penile urethra
1. Introduction The penile urethra anatomically represents the transition area between the urothelium covering the urinary tract and the squamous mucosa covering the glans and the inner foreskin. The more proximal portion is lined by urothelium, whereas squamous mucosa lines the fossa navicularis distally. The area in between resembles the urothelium seen elsewhere, although the umbrella cells are replaced by a monolayer of columnar cells. Given its pathology, it has been suggested that the epithelium of the penile urethra is more closely related to the squamous epithelium than to the urothelium [1]. Normal immunohistochemical profile of the usual urothelium found in the urinary tract is well described with a specific staining pattern of cytokeratins: CK20 positivity for the umbrella cells and CK7 positivity for the underlying stratified urothelial cells [2]. However, despite its peculiar morphology, the distinct epithelium lining the penile urethra has not been well characterized immunohistochemically. To the best of our knowledge, only 1 previous study has evaluated the immunohistochemical expression of cytokeratins, not in adult males but in human fetal phallic specimens [3]. Analogous to the gastroesophageal junction and other regions of mucosal transition, the type of penile urethra carcinoma depends on whether the tumor arises from the urothelium or the squamous epithelium. Although primary urethral neoplasms only account for a small proportion of genitourinary neoplasm in males, both urothelial and squamous cell carcinomas have been reported in the penile urethra [4,5]. As there may be considerable morphologic overlapping, the distinction between these 2 tumors on pathologic grounds might be difficult. In this scenario, the identification and validation of biomarkers capable of distinguishing between urothelial and squamous cell carcinomas would be helpful. This distinction is not merely academic but has potential clinical implications as well because urothelial and squamous cell carcinomas may differ in their prognosis and treatment [6-8]. Recently, Trans-acting T-cell-specific transcription factor (GATA3), a transcription factor located on chromosome 10p14 [9], has emerged as a novel, sensitive, and selective marker for urothelial differentiation, as demonstrated by complementary DNA microarrays and immunohistochemical studies [10,11]. In addition, recent studies indicate that human papillomavirus (HPV) infection is not involved in urothelial carcinogenesis [12,13], whereas HPV presence is found in approximately one-half of squamous carcinomas of the penis [14], especially those that bear morphologic resemblance to urothelial carcinomas [15,16]. Thus, defining HPV status could also be useful in the differential diagnosis of urothelial versus squamous cell carcinomas of the penile urethra. The purpose of this study is 2-fold: first, to assess the histology and immunohistochemical profile of the penile urethra via a panel of stains typically used for squamous and urothelial differentiation, including GATA3 as a marker for the latter; second, to assess the usefulness of
2761 GATA3 expression and HPV detection in defining the diagnosis of urothelial versus squamous cell carcinomas of the penile urethra.
2. Materials and methods The current study was approved by the Institutional Review Board at the Johns Hopkins School of Medicine (Baltimore, MD).
2.1. Case selection For the histologic and immunohistochemical evaluation of the normal penile urethra, 11 cases were selected from the pathology files of the Instituto de Patología e Investigación (Asunción, Paraguay). All cases corresponded to patients with squamous cell carcinoma of the penile glans treated by total penectomy. Extension into the urethral mucosa was histologically ruled out in all cases. A representative block was selected from each case for evaluating CK7, CK20, p63, and GATA3 expression by immunohistochemistry. For determining the usefulness of GATA3 in distinguishing urothelial carcinomas from squamous cell carcinomas, 2 sets of tissue microarrays (TMAs) were built at the Johns Hopkins TMA Lab Core (Baltimore, MD) using a previously described procedure [17]. The first TMA consisted of 25 cases of urothelial carcinomas of the upper genitourinary tract gathered from the Johns Hopkins Pathology Department (Baltimore, MD). The second TMA consisted of 38 cases of squamous cell carcinomas of the penis gathered from the Instituto de Patología de Investigación (Asunción, Paraguay), different from the aforementioned 11 cases. For both TMAs, 1 to 4 blocks were selected per case, and 3 tissue cores of 1 mm each were obtained per block. In addition to the 11 penectomies and the 2 TMAs, we also retrieved 6 cases of primary carcinomas of the penile urethra from the pathology files of the Johns Hopkins Hospital (Baltimore, MD). Extension from adjacent urologic sites was excluded on clinical grounds. Patients with a previous history of urothelial or prostatic carcinomas were excluded as well as patients with carcinomas of the penile glans with secondary extension to the penile urethra. All slides were reviewed by 2 urologic pathologists (A. C. and G. N.), and a consensus diagnosis was reached in all cases. A representative block was selected from each case for evaluating GATA3 expression by immunohistochemistry and defining HPV status by in situ hybridization.
2.2. Immunohistochemistry and in situ hybridization Immunohistochemistry was performed on 5-μm-thick formalin-fixed, paraffin-embedded sections. As mentioned before, the following immunohistochemical markers were
2762 used: CK20 (1:500 dilution; DAKO, Carpinteria, CA), CK7 (1:500 dilution; DAKO), p63 (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), and GATA3 (1:100 dilution; Biocare, Concord, CA). Immunohistochemical stains were carried out using automated stainers (BondLeica; Leica Microsystems, Bannockburn, IL, or Ventana Benchmark XT; Ventana Medical Systems, Inc, Tucson, AZ). Briefly, slides were deparaffinized and hydrated, followed by heat-induced antigen retrieval. Incubation with the primary antibody using optimal conditions was followed by development of the immunostaining and counterstaining. The secondary antibody and detection were applied as per the instructions of the manufacturer. The negative controls used were tissue sections with no primary antibody incubation. For each marker, cases were classified as follows, according to the extent of immunohistochemical expression in the tumor cells: 0%, negative; less than 25%, focal positivity; 25% to 75%, extensive positivity; greater than 75%, diffuse positivity. HPV in situ hybridization was performed on automated benchmark XT systems (Ventana Medical Systems, Inc). The INFORM HPV III family 16 probe (B) cocktail, with affinity for high-risk genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66), was applied, and the reaction was developed using a hybrid ready detection kit (ref no. 780-4295 and ref no. 254-2210-2116, respectively; Ventana Medical Systems, Inc).
3. Results 3.1. Histology and immunohistochemical profile of the penile urethra In all cases, the penile urethra was lined by a stratified epithelium, with an underlying lamina propria of loose connective tissue, in which some periurethral glands (Littré glands) were seen (Fig. 1A). Two distinctive cell types were observed in the epithelial lining of the penile urethra (Fig. 1B). The superficial layer was composed of a single layer of tall columnar cells with clear cytoplasm and welldefined cell borders. The underlying layer was composed of stratified cubical cells with scant cytoplasm and inconspicuous cell borders. Near the meatus urethralis, in the fossa navicularis, the columnar stratified epithelium changed abruptly to a nonkeratinizing squamous epithelium. In all cases, the epithelium of the penile urethra was negative for CK20 (Fig. 1C). Cytoplasmic CK7 staining was extensive to diffuse in the superficial columnar cells and negative in the underlying layers (Fig. 1D). In contrast, p63 displayed an inverse pattern of expression, with absence of staining in the superficial columnar cells and diffuse nuclear positivity in the underlying layers (Fig. 1E). With the exception of 1 case, extensive to diffuse GATA3 nuclear positivity was observed in the superficial and the underlying layers of all sections (Fig. 1F). In all positive cases, staining
A. Chaux et al. intensity ranged from moderate to strong; we did not observe weak staining with any immunohistochemical marker. Table 1 shows the distribution of stain intensity by markers.
3.2. Differential GATA3 expression in urothelial and squamous cell carcinomas In the TMAs, GATA3 positivity was observed in 22 of 25 urothelial carcinomas of the upper urinary tract and in none of the 38 squamous cell carcinomas of the penis. Considering the hematoxylin and eosin (H&E) morphology as the criterion standard, GATA3 had a sensitivity of 88% (95% confidence interval [CI], 69%-97%) and a specificity of 100% (95% CI, 91%-100%) in telling apart urothelial carcinomas from squamous cell carcinomas. The positive and negative predictive values of GATA3 immunohistochemistry were 100% (95% CI, 84%-100%) and 93% (95% CI, 80%-98%), respectively.
3.3. GATA3 expression and HPV in situ hybridization in urothelial and squamous cell carcinomas of the penile urethra Of the 6 cases of penile urethra carcinomas, 3 were squamous cell carcinomas, and 3 were urothelial carcinomas. The squamous cell carcinomas corresponded to 1 basaloid carcinoma, 1 warty carcinoma, and 1 warty-basaloid carcinoma. In the warty carcinomas, an adjacent high-grade noninvasive papillary urothelial carcinoma was observed (mixed squamous cell carcinoma–urothelial carcinoma). All 3 squamous cell carcinomas were GATA3 negative (Fig. 2A and B), whereas all 3 urothelial carcinomas showed diffuse and strong nuclear GATA3 positivity (Fig. 2C and D). In the mixed squamous cell carcinoma–urothelial carcinoma, the squamous component was GATA3 negative (Fig. 2E and F), and the urothelial component was GATA3 positive (Fig. 2G and 2H). HPV was detected in 2 of the 3 squamous cell carcinomas (Fig. 3) and in none of the urothelial carcinomas. Table 2 summarizes all these findings.
4. Discussion This study confirms prior findings of a specialized epithelium lining the penile urethra, morphologically distinct from both squamous epithelium and urothelium [1]. The specialized epithelium had 2 distinct layers. The deep layer was composed of a stratified epithelium morphologically and immunohistochemically resembling usual urothelium. However, in contrast to normal urothelium, the surface was composed of a single layer of CK7-positive, CK20-negative columnar cells rather than the typical CK20-positive, CK7negative umbrella cells [2]. Along with all these findings, the positivity for GATA3 suggests that the epithelium lining the
GATA3 in penile urethra
2763
Fig. 1 Morphologic and immunohistochemical features of the penile urethra. A, The penile urethra was lined by a stratified epithelium with an underlying lamina propria of loose connective tissue and periurethral (Littré) glands (×50). B, The epithelium was composed of a superficial layer of tall columnar cells and an underlying stratified cubical epithelium (×200). C, Immunohistochemistry for CK20 was negative throughout the epithelium (×200). D, Immunohistochemistry for CK7 was strongly and diffusely positive in the superficial columnar cells (×200). E, Immunohistochemistry for p63 was positive in the deep cubical cells and negative in the superficial columnar cells (×200). F, Immunohistochemistry for GATA3 was positive in both the superficial and the deep cell layers; positivity was moderate to strong in the columnar cell layer and weak to moderate in the cubical cell layer (×200).
Table 1
Immunohistochemical expression of CK7, CK20, p63, and GATA3 in 11 normal penile urethras CK20 (%)
Negative (0%) Focal (b25%) Extensive (25%-75%) Diffuse (N75%)
CK7 (%)
p63 (%)
GATA3 (%)
Superficial
Deep
Superficial
Deep
Superficial
Deep
Superficial
Deep
11 (100) 0 (0) 0 (0) 0 (0)
11 (100) 0 (0) 0 (0) 0 (0)
0 0 3 8
11 (100) 0 (0) 0 (0) 0 (0)
11 (100) 0 (0) 0 (0) 0 (0)
0 (0) 0 (0) 0 (0) 11 (100)
1 (9) 0 (0) 5 (45.5) 5 (45.5)
1 0 2 8
(0) (0) (27) (73)
(9) (0) (18) (73)
NOTE. “Superficial” and “Deep” refer to the superficial single columnar layer and the deep stratified cubic layer of the urethral epithelium, respectively.
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A. Chaux et al.
Fig. 2 GATA3 immunohistochemistry in squamous cell carcinoma and urothelial carcinoma of the penile urethra. A, Squamous cell carcinoma with basaloid features (H&E, ×50). B, The squamous cell carcinoma was GATA3 negative; note the GATA3 positivity of the adjacent penile urethra (×50). C, High-grade invasive urothelial carcinoma (H&E). D, Tumor cells were strongly and diffusely GATA3 positive (×50). E, Mixed squamous cell carcinoma–urothelial carcinoma showing the squamous component corresponding to a warty carcinoma (H&E, ×50). F, The squamous component was GATA3 negative (×50). G, The urothelial component of the mixed carcinoma corresponded to a high-grade papillary noninvasive urothelial carcinoma (H&E, ×50). H, The urothelial component was strongly and diffusely GATA3 positive (×50).
GATA3 in penile urethra
2765
Fig. 3 HPV status in penile urethra carcinomas: positive HPV detection by in situ hybridization in a squamous cell carcinoma at medium (A, ×200) and high (B, ×400) field powers.
penile urethra corresponds to a modified urothelium rather than to a specialized squamous epithelium. Our results also lend support to the theory of endodermal differentiation of the human penile urethra [3], as opposed to the widely accepted theory of ectodermal ingrowth growth. Nevertheless, considering that there are no umbrella cells and that the columnar cells on the luminal surface did not stain with CK20, we cannot entirely rule out (from the theoretical standpoint) that the epithelium of the penile urethra is a heretofore unrecognized variant of epithelium that is neither squamous epithelium nor urothelium. If this is the case, maybe this epithelium has not been previously described due to the lack for extensive formal study in the past and the application of new methods such as immunohistochemistry. Table 2 Clinicopathologic features, GATA3 immunohistochemistry, and HPV status in 6 primary carcinomas of the penile urethra Case no. Age (y) Diagnosis
GATA3
HPV
1 2 3 4 5 6
Negative Positive Positive Positive Negative Negative/ positive
Positive Negative Negative Negative Negative Positive/ negative
48 69 70 73 60 73
Basaloid SCC Invasive UC Invasive UC Invasive UC Warty-basaloid SCC Warty SCC/HGUC
Abbreviations: SCC, squamous cell carcinoma; UC, urothelial carcinoma; HGUC, high-grade noninvasive papillary urothelial carcinoma.
The second part of this study establishes the potential utility of GATA3 immunohistochemistry and HPV in situ hybridization for the differential diagnosis of urothelial carcinomas versus squamous cell carcinomas of the penile urethra. Positive staining of GATA3 was seen in most urothelial carcinomas, whereas no bona fide squamous carcinoma of the penis was positive for GATA3. Conversely, for primary tumors of the penile urethra, HPV positivity was a strong indicator of a squamous origin because none of the urothelial carcinomas had HPV infection, whereas HPV was present in most squamous cell carcinomas. Urothelial carcinomas arising from the penile urethra may spread to the squamous epithelium of the glans, and squamous cell carcinomas of the glans may involve the urethra continuously or discontinuously. Given that urothelial carcinomas might morphologically overlap with squamous cell carcinomas, the differential diagnosis between both may be challenging. Urothelial markers such as thrombomodulin and uroplakin have relatively low sensitivity and may not be helpful in a considerable number of cases [2]. As mentioned before, the differential diagnosis of urothelial carcinoma versus squamous cell carcinoma may have significant clinical implications because they differ in their prognosis and treatment [6-8]. Although scant data are available specifically for carcinomas of the penile urethra, clinical response to chemotherapy may be different between urothelial carcinomas and squamous cell carcinomas [18-21]. Previous studies give evidence supporting the usefulness of GATA3 as a sensitive and selective marker for urothelial carcinomas. In several recently published series, GATA3 positivity ranged from 78% to 93% in primary urothelial carcinomas of the urinary tract [22-25]. GATA3 positivity was also retained in secondary urothelial carcinomas metastatic to the lungs. Chang et al [23] found that 80% of urothelial carcinomas metastatic to the lungs were GATA3 positive, whereas none of the pulmonary squamous cell carcinomas or non–small cell carcinomas with squamous features showed GATA3 positivity. In another study, Gruver et al [25] found GATA3 positivity in only 23% of pulmonary squamous cell carcinomas. Taken together, these studies suggest the potential role of GATA3 in the differential diagnosis of metastatic urothelial carcinomas versus primary lung carcinomas. GATA3 has been also evaluated in tumors that should be considered in the differential diagnosis of metastatic urothelial carcinomas. In the study by Chang et al, GATA3 was positive in 0% of prostatic adenocarcinomas, 10% of anal squamous cell carcinomas, and 25% of uterine cervical squamous cell carcinomas, with most of positive cases depicting only focal and weak positivity [23]. On the contrary, in our series, GATA3 expression, when positive, was moderate to strong and extensive to diffuse. GATA3 expression was also observed in morphologic variants of urothelial carcinoma such as plasmacytoid urothelial carcinoma [23] and sarcomatoid urothelial carcinoma (unpublished observations). In addition, a recent study by Gulmann et al [24] suggests that GATA3 is a reliable marker of urothelial differentiation in urothelial
2766 carcinomas with squamous differentiation. Finally, in the study by Miyamoto et al [22], GATA3 expression was an independent predictor of poor prognosis in urothelial carcinomas of the urinary bladder. Worth noting, other studies in patients with breast carcinoma have also shown an association between GATA3 and clinical outcome [26,27]. Another useful marker for the differential diagnosis of penile urethra carcinomas is HPV detection. Among the subtypes of squamous cell carcinomas of the penis, basaloid carcinoma is the most challenging differential diagnosis for urothelial carcinoma, given their similarity in architecture and cytologic features. Solid nests of small and uniform basaloid cells may be easily taken as neoplastic urothelial cells, especially when squamous differentiation is not apparent. Basaloid squamous cell carcinoma of the penis is an aggressive and HPV-related tumor that is reported in up to 10% of all penile carcinomas with a metastatic rate between 50% and 100% [28]. Although this tumor predominantly affects the glans of the penis, this study shows that the penile urethra may be primarily involved. Another tumor that may raise the differential diagnosis of urothelial carcinomas in the penile urethra is warty-basaloid carcinoma, especially due to its papillomatous architecture and the presence of invasive nests of basaloid cells [29]. The identification of koilocytic changes and parakeratosis, not usually seen in urothelial carcinomas, might help suggesting a squamous origin in routine sections, but ought not to be taken as the sole basis for diagnosis. Although not seen in the current series and not yet reportedinthepenileurethra,anothersquamouscellcarcinomathat can be confused with urothelial carcinomas is a recently described variant of basaloid carcinoma showing papillary features [16]. In thisvariant,thetumoriscomposedofconspicuouspapillaelinedby basaloid cells, with or without an associated typical basaloid carcinoma or warty-basaloid carcinoma. Morphology can be deceiving in all these variants of squamous cell carcinomas, rendering the differential diagnosis between urothelial and squamous cell carcinoma almost impossible on H&E grounds alone. Defining HPV status can be useful in this context, considering that tumors with warty and basaloid features are usually HPV positive [15], whereas urothelial carcinomas are usually HPV negative [28]. Nevertheless, HPV in situ hybridization alone may not be sufficient to rule out urothelial carcinomas because a significant number of squamous cell carcinomas could also be negative for HPV. However, combined with GATA3 immunohistochemistry, HPV in situ hybridization or any other method for HPV detection such as the polymerase chain reaction may offer an additional tool to further confirm a specific differentiation of penile urethra carcinomas. This suggestion is further supported by the distinctive GATA3/ HPV status in the mixed squamous cell carcinoma/urothelial carcinoma that was found. Finally, the usefulness of surrogates for HPV infection, such as p16INK4a immunohistochemistry [30], combined with markers of urothelial differentiation such as GATA3 also merits further investigation. Three limitations must be acknowledged for the current study: first, the relatively small number of penectomies and
A. Chaux et al. primary urethral carcinomas of the penis; second, the use of TMAs for GATA3 evaluation rather than whole tissue sections; and third, the use of a limited set of squamous cell markers. For the first limitation, proper evaluation of the penile urethra required total penectomies, which were performed in locally advanced cases, cases that most likely also had urethral invasion. Identifying and retrieving total penectomies without tumor invasion of the penile urethra were difficult, moreover, considering the rarity of penile tumors. In addition, identifying and retrieving carcinomas exclusive of the penile urethra were also difficult, given their rarity. Most cases found corresponded to tumors with secondary extension from either the penile glans or the urinary tract. Thus, although the number of cases is relatively small, they are sufficient for establishing the immunohistochemical profile of the normal penile urethra and the potential usefulness of GATA3 and HPV detection in the differential diagnosis of urothelial versus squamous cell carcinomas of the penile urethra. Regarding the second limitation, although it would be preferable to use whole sections in determining the usefulness of a marker, TMAs provide a convenient way to evaluate a considerable number of cases under the same conditions and using the same protocol for immunohistochemistry. This diminishes the impact that external conditions might have in the development of the stain and facilitates the interpretation of the immunohistochemical expression. The consistency that was found between surgical specimens of primary tumors of the penile urethra and TMAs of urothelial and squamous cell carcinomas strengthens the notion that GATA3 is a useful marker in distinguishing these tumors. Finally, the evaluation of additional markers of squamous differentiation, such as CK14 and desmoglein, would be interesting, as shown by some previously discussed studies [24,25]. In conclusion, the penile urethra is covered by epithelial cells that are unique in morphology and immunohistochemical profile. The superficial layer was composed of a single layer of tall columnar cells that were CK7 positive, CK20 negative, and p63 negative. The deep layer was composed of stratified cubical cells that were CK7 negative, CK20 negative, and p63 positive. Both superficial and deep layers of the penile urethra were GATA3 positive, suggesting urothelial origin. In addition, immunohistochemistry for GATA3 was usually positive in urothelial carcinomas and consistently negative in squamous cell carcinomas, whereas the opposite trend was observed for HPV presence by in situ hybridization. Our study suggests that GATA3 and HPV detection are useful markers for distinguishing urothelial carcinomas from squamous cells carcinomas of the penile urethra.
Acknowledgments 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.
GATA3 in penile urethra
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Original contribution
Immunohistochemical profile of the penile urethra and differential expression of GATA3 in urothelial versus squamous cell carcinomas of the penile urethra☆ Alcides Chaux MD a,b,⁎, Jeong S. Han MD, PhD a,c , Stephen Lee MD a , Nilda Gonzalez-Roibon MD a , Rajni Sharma PhD a , Arthur L. Burnett MD d , Antonio L. Cubilla MD e , George J. Netto MD a,d,f a
Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD 31231, USA Office of Scientific Research, Norte University, 1614 Asunción, Paraguay c Kaiser Permanente Medical Center, Oakland, CA 94611, USA d Department of Urology, Johns Hopkins Medical Institutions, Baltimore, MD 31231, USA e Instituto de Patología e Investigación, 1817 Asunción, Paraguay f Department of Oncology, Johns Hopkins Medical Institutions, Baltimore, MD 31231, USA b
Received 29 May 2013; revised 9 July 2013; accepted 17 July 2013
Keywords: Penile urethra; Squamous cell carcinoma; Urothelial carcinoma; CK7; CK20; p63; GATA3; Immunohistochemistry
Summary The penile urethra has a distinctive morphology not yet fully characterized by immunohistochemistry. In addition, both urothelial and squamous cell carcinomas have been reported in the penile urethra, and the distinction between these 2 tumors might be difficult. The purposes of this study are to assess the histology and immunohistochemical profile (CK20, CK7, p63, and GATA3) of the penile urethra and to assess the usefulness of Trans-acting T-cell-specific transcription factor (GATA3) and human papillomavirus detection in distinguishing urothelial versus squamous cell carcinomas. Normal penile urethra was evaluated in 11 total penectomies. The penile urethra was lined by 2 cell layers: a superficial single layer of CK7+, CK20−, and p63− columnar cells and a deep stratified layer of CK7−, CK20−, and p63+ cubical cells. Both layers were GATA3+, supporting urothelial differentiation. In addition, 2 tissue microarrays and 6 surgical specimens of primary tumors of the penile urethra (3 urothelial and 3 squamous cell carcinomas) were evaluated for GATA3 expression. In the tissue microarrays, 22 of 25 upper tract urothelial carcinomas and 0 of 38 penile squamous cell carcinomas were GATA3+. In the surgical specimens, GATA3 was positive in all urothelial carcinomas and negative in all squamous cell carcinomas. Human papillomavirus was detected in 2 of 3 squamous cell carcinomas and in 0 of 3 of the urothelial carcinomas. In conclusion, the penile urethra is covered by epithelial cells that are unique in morphology and immunohistochemical profile. In addition, our study suggests that GATA3 and human papillomavirus detection are useful markers for distinguishing urothelial carcinomas from squamous cell carcinomas of the penile urethra. © 2013 Elsevier Inc. All rights reserved.
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Disclosure: 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. Office of Scientific Research, Norte University, Gral. Santos e/25 de Mayo, Asunción, Paraguay. E-mail address: [email protected] (A. Chaux). 0046-8177/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2013.07.023
GATA3 in penile urethra
1. Introduction The penile urethra anatomically represents the transition area between the urothelium covering the urinary tract and the squamous mucosa covering the glans and the inner foreskin. The more proximal portion is lined by urothelium, whereas squamous mucosa lines the fossa navicularis distally. The area in between resembles the urothelium seen elsewhere, although the umbrella cells are replaced by a monolayer of columnar cells. Given its pathology, it has been suggested that the epithelium of the penile urethra is more closely related to the squamous epithelium than to the urothelium [1]. Normal immunohistochemical profile of the usual urothelium found in the urinary tract is well described with a specific staining pattern of cytokeratins: CK20 positivity for the umbrella cells and CK7 positivity for the underlying stratified urothelial cells [2]. However, despite its peculiar morphology, the distinct epithelium lining the penile urethra has not been well characterized immunohistochemically. To the best of our knowledge, only 1 previous study has evaluated the immunohistochemical expression of cytokeratins, not in adult males but in human fetal phallic specimens [3]. Analogous to the gastroesophageal junction and other regions of mucosal transition, the type of penile urethra carcinoma depends on whether the tumor arises from the urothelium or the squamous epithelium. Although primary urethral neoplasms only account for a small proportion of genitourinary neoplasm in males, both urothelial and squamous cell carcinomas have been reported in the penile urethra [4,5]. As there may be considerable morphologic overlapping, the distinction between these 2 tumors on pathologic grounds might be difficult. In this scenario, the identification and validation of biomarkers capable of distinguishing between urothelial and squamous cell carcinomas would be helpful. This distinction is not merely academic but has potential clinical implications as well because urothelial and squamous cell carcinomas may differ in their prognosis and treatment [6-8]. Recently, Trans-acting T-cell-specific transcription factor (GATA3), a transcription factor located on chromosome 10p14 [9], has emerged as a novel, sensitive, and selective marker for urothelial differentiation, as demonstrated by complementary DNA microarrays and immunohistochemical studies [10,11]. In addition, recent studies indicate that human papillomavirus (HPV) infection is not involved in urothelial carcinogenesis [12,13], whereas HPV presence is found in approximately one-half of squamous carcinomas of the penis [14], especially those that bear morphologic resemblance to urothelial carcinomas [15,16]. Thus, defining HPV status could also be useful in the differential diagnosis of urothelial versus squamous cell carcinomas of the penile urethra. The purpose of this study is 2-fold: first, to assess the histology and immunohistochemical profile of the penile urethra via a panel of stains typically used for squamous and urothelial differentiation, including GATA3 as a marker for the latter; second, to assess the usefulness of
2761 GATA3 expression and HPV detection in defining the diagnosis of urothelial versus squamous cell carcinomas of the penile urethra.
2. Materials and methods The current study was approved by the Institutional Review Board at the Johns Hopkins School of Medicine (Baltimore, MD).
2.1. Case selection For the histologic and immunohistochemical evaluation of the normal penile urethra, 11 cases were selected from the pathology files of the Instituto de Patología e Investigación (Asunción, Paraguay). All cases corresponded to patients with squamous cell carcinoma of the penile glans treated by total penectomy. Extension into the urethral mucosa was histologically ruled out in all cases. A representative block was selected from each case for evaluating CK7, CK20, p63, and GATA3 expression by immunohistochemistry. For determining the usefulness of GATA3 in distinguishing urothelial carcinomas from squamous cell carcinomas, 2 sets of tissue microarrays (TMAs) were built at the Johns Hopkins TMA Lab Core (Baltimore, MD) using a previously described procedure [17]. The first TMA consisted of 25 cases of urothelial carcinomas of the upper genitourinary tract gathered from the Johns Hopkins Pathology Department (Baltimore, MD). The second TMA consisted of 38 cases of squamous cell carcinomas of the penis gathered from the Instituto de Patología de Investigación (Asunción, Paraguay), different from the aforementioned 11 cases. For both TMAs, 1 to 4 blocks were selected per case, and 3 tissue cores of 1 mm each were obtained per block. In addition to the 11 penectomies and the 2 TMAs, we also retrieved 6 cases of primary carcinomas of the penile urethra from the pathology files of the Johns Hopkins Hospital (Baltimore, MD). Extension from adjacent urologic sites was excluded on clinical grounds. Patients with a previous history of urothelial or prostatic carcinomas were excluded as well as patients with carcinomas of the penile glans with secondary extension to the penile urethra. All slides were reviewed by 2 urologic pathologists (A. C. and G. N.), and a consensus diagnosis was reached in all cases. A representative block was selected from each case for evaluating GATA3 expression by immunohistochemistry and defining HPV status by in situ hybridization.
2.2. Immunohistochemistry and in situ hybridization Immunohistochemistry was performed on 5-μm-thick formalin-fixed, paraffin-embedded sections. As mentioned before, the following immunohistochemical markers were
2762 used: CK20 (1:500 dilution; DAKO, Carpinteria, CA), CK7 (1:500 dilution; DAKO), p63 (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), and GATA3 (1:100 dilution; Biocare, Concord, CA). Immunohistochemical stains were carried out using automated stainers (BondLeica; Leica Microsystems, Bannockburn, IL, or Ventana Benchmark XT; Ventana Medical Systems, Inc, Tucson, AZ). Briefly, slides were deparaffinized and hydrated, followed by heat-induced antigen retrieval. Incubation with the primary antibody using optimal conditions was followed by development of the immunostaining and counterstaining. The secondary antibody and detection were applied as per the instructions of the manufacturer. The negative controls used were tissue sections with no primary antibody incubation. For each marker, cases were classified as follows, according to the extent of immunohistochemical expression in the tumor cells: 0%, negative; less than 25%, focal positivity; 25% to 75%, extensive positivity; greater than 75%, diffuse positivity. HPV in situ hybridization was performed on automated benchmark XT systems (Ventana Medical Systems, Inc). The INFORM HPV III family 16 probe (B) cocktail, with affinity for high-risk genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66), was applied, and the reaction was developed using a hybrid ready detection kit (ref no. 780-4295 and ref no. 254-2210-2116, respectively; Ventana Medical Systems, Inc).
3. Results 3.1. Histology and immunohistochemical profile of the penile urethra In all cases, the penile urethra was lined by a stratified epithelium, with an underlying lamina propria of loose connective tissue, in which some periurethral glands (Littré glands) were seen (Fig. 1A). Two distinctive cell types were observed in the epithelial lining of the penile urethra (Fig. 1B). The superficial layer was composed of a single layer of tall columnar cells with clear cytoplasm and welldefined cell borders. The underlying layer was composed of stratified cubical cells with scant cytoplasm and inconspicuous cell borders. Near the meatus urethralis, in the fossa navicularis, the columnar stratified epithelium changed abruptly to a nonkeratinizing squamous epithelium. In all cases, the epithelium of the penile urethra was negative for CK20 (Fig. 1C). Cytoplasmic CK7 staining was extensive to diffuse in the superficial columnar cells and negative in the underlying layers (Fig. 1D). In contrast, p63 displayed an inverse pattern of expression, with absence of staining in the superficial columnar cells and diffuse nuclear positivity in the underlying layers (Fig. 1E). With the exception of 1 case, extensive to diffuse GATA3 nuclear positivity was observed in the superficial and the underlying layers of all sections (Fig. 1F). In all positive cases, staining
A. Chaux et al. intensity ranged from moderate to strong; we did not observe weak staining with any immunohistochemical marker. Table 1 shows the distribution of stain intensity by markers.
3.2. Differential GATA3 expression in urothelial and squamous cell carcinomas In the TMAs, GATA3 positivity was observed in 22 of 25 urothelial carcinomas of the upper urinary tract and in none of the 38 squamous cell carcinomas of the penis. Considering the hematoxylin and eosin (H&E) morphology as the criterion standard, GATA3 had a sensitivity of 88% (95% confidence interval [CI], 69%-97%) and a specificity of 100% (95% CI, 91%-100%) in telling apart urothelial carcinomas from squamous cell carcinomas. The positive and negative predictive values of GATA3 immunohistochemistry were 100% (95% CI, 84%-100%) and 93% (95% CI, 80%-98%), respectively.
3.3. GATA3 expression and HPV in situ hybridization in urothelial and squamous cell carcinomas of the penile urethra Of the 6 cases of penile urethra carcinomas, 3 were squamous cell carcinomas, and 3 were urothelial carcinomas. The squamous cell carcinomas corresponded to 1 basaloid carcinoma, 1 warty carcinoma, and 1 warty-basaloid carcinoma. In the warty carcinomas, an adjacent high-grade noninvasive papillary urothelial carcinoma was observed (mixed squamous cell carcinoma–urothelial carcinoma). All 3 squamous cell carcinomas were GATA3 negative (Fig. 2A and B), whereas all 3 urothelial carcinomas showed diffuse and strong nuclear GATA3 positivity (Fig. 2C and D). In the mixed squamous cell carcinoma–urothelial carcinoma, the squamous component was GATA3 negative (Fig. 2E and F), and the urothelial component was GATA3 positive (Fig. 2G and 2H). HPV was detected in 2 of the 3 squamous cell carcinomas (Fig. 3) and in none of the urothelial carcinomas. Table 2 summarizes all these findings.
4. Discussion This study confirms prior findings of a specialized epithelium lining the penile urethra, morphologically distinct from both squamous epithelium and urothelium [1]. The specialized epithelium had 2 distinct layers. The deep layer was composed of a stratified epithelium morphologically and immunohistochemically resembling usual urothelium. However, in contrast to normal urothelium, the surface was composed of a single layer of CK7-positive, CK20-negative columnar cells rather than the typical CK20-positive, CK7negative umbrella cells [2]. Along with all these findings, the positivity for GATA3 suggests that the epithelium lining the
GATA3 in penile urethra
2763
Fig. 1 Morphologic and immunohistochemical features of the penile urethra. A, The penile urethra was lined by a stratified epithelium with an underlying lamina propria of loose connective tissue and periurethral (Littré) glands (×50). B, The epithelium was composed of a superficial layer of tall columnar cells and an underlying stratified cubical epithelium (×200). C, Immunohistochemistry for CK20 was negative throughout the epithelium (×200). D, Immunohistochemistry for CK7 was strongly and diffusely positive in the superficial columnar cells (×200). E, Immunohistochemistry for p63 was positive in the deep cubical cells and negative in the superficial columnar cells (×200). F, Immunohistochemistry for GATA3 was positive in both the superficial and the deep cell layers; positivity was moderate to strong in the columnar cell layer and weak to moderate in the cubical cell layer (×200).
Table 1
Immunohistochemical expression of CK7, CK20, p63, and GATA3 in 11 normal penile urethras CK20 (%)
Negative (0%) Focal (b25%) Extensive (25%-75%) Diffuse (N75%)
CK7 (%)
p63 (%)
GATA3 (%)
Superficial
Deep
Superficial
Deep
Superficial
Deep
Superficial
Deep
11 (100) 0 (0) 0 (0) 0 (0)
11 (100) 0 (0) 0 (0) 0 (0)
0 0 3 8
11 (100) 0 (0) 0 (0) 0 (0)
11 (100) 0 (0) 0 (0) 0 (0)
0 (0) 0 (0) 0 (0) 11 (100)
1 (9) 0 (0) 5 (45.5) 5 (45.5)
1 0 2 8
(0) (0) (27) (73)
(9) (0) (18) (73)
NOTE. “Superficial” and “Deep” refer to the superficial single columnar layer and the deep stratified cubic layer of the urethral epithelium, respectively.
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A. Chaux et al.
Fig. 2 GATA3 immunohistochemistry in squamous cell carcinoma and urothelial carcinoma of the penile urethra. A, Squamous cell carcinoma with basaloid features (H&E, ×50). B, The squamous cell carcinoma was GATA3 negative; note the GATA3 positivity of the adjacent penile urethra (×50). C, High-grade invasive urothelial carcinoma (H&E). D, Tumor cells were strongly and diffusely GATA3 positive (×50). E, Mixed squamous cell carcinoma–urothelial carcinoma showing the squamous component corresponding to a warty carcinoma (H&E, ×50). F, The squamous component was GATA3 negative (×50). G, The urothelial component of the mixed carcinoma corresponded to a high-grade papillary noninvasive urothelial carcinoma (H&E, ×50). H, The urothelial component was strongly and diffusely GATA3 positive (×50).
GATA3 in penile urethra
2765
Fig. 3 HPV status in penile urethra carcinomas: positive HPV detection by in situ hybridization in a squamous cell carcinoma at medium (A, ×200) and high (B, ×400) field powers.
penile urethra corresponds to a modified urothelium rather than to a specialized squamous epithelium. Our results also lend support to the theory of endodermal differentiation of the human penile urethra [3], as opposed to the widely accepted theory of ectodermal ingrowth growth. Nevertheless, considering that there are no umbrella cells and that the columnar cells on the luminal surface did not stain with CK20, we cannot entirely rule out (from the theoretical standpoint) that the epithelium of the penile urethra is a heretofore unrecognized variant of epithelium that is neither squamous epithelium nor urothelium. If this is the case, maybe this epithelium has not been previously described due to the lack for extensive formal study in the past and the application of new methods such as immunohistochemistry. Table 2 Clinicopathologic features, GATA3 immunohistochemistry, and HPV status in 6 primary carcinomas of the penile urethra Case no. Age (y) Diagnosis
GATA3
HPV
1 2 3 4 5 6
Negative Positive Positive Positive Negative Negative/ positive
Positive Negative Negative Negative Negative Positive/ negative
48 69 70 73 60 73
Basaloid SCC Invasive UC Invasive UC Invasive UC Warty-basaloid SCC Warty SCC/HGUC
Abbreviations: SCC, squamous cell carcinoma; UC, urothelial carcinoma; HGUC, high-grade noninvasive papillary urothelial carcinoma.
The second part of this study establishes the potential utility of GATA3 immunohistochemistry and HPV in situ hybridization for the differential diagnosis of urothelial carcinomas versus squamous cell carcinomas of the penile urethra. Positive staining of GATA3 was seen in most urothelial carcinomas, whereas no bona fide squamous carcinoma of the penis was positive for GATA3. Conversely, for primary tumors of the penile urethra, HPV positivity was a strong indicator of a squamous origin because none of the urothelial carcinomas had HPV infection, whereas HPV was present in most squamous cell carcinomas. Urothelial carcinomas arising from the penile urethra may spread to the squamous epithelium of the glans, and squamous cell carcinomas of the glans may involve the urethra continuously or discontinuously. Given that urothelial carcinomas might morphologically overlap with squamous cell carcinomas, the differential diagnosis between both may be challenging. Urothelial markers such as thrombomodulin and uroplakin have relatively low sensitivity and may not be helpful in a considerable number of cases [2]. As mentioned before, the differential diagnosis of urothelial carcinoma versus squamous cell carcinoma may have significant clinical implications because they differ in their prognosis and treatment [6-8]. Although scant data are available specifically for carcinomas of the penile urethra, clinical response to chemotherapy may be different between urothelial carcinomas and squamous cell carcinomas [18-21]. Previous studies give evidence supporting the usefulness of GATA3 as a sensitive and selective marker for urothelial carcinomas. In several recently published series, GATA3 positivity ranged from 78% to 93% in primary urothelial carcinomas of the urinary tract [22-25]. GATA3 positivity was also retained in secondary urothelial carcinomas metastatic to the lungs. Chang et al [23] found that 80% of urothelial carcinomas metastatic to the lungs were GATA3 positive, whereas none of the pulmonary squamous cell carcinomas or non–small cell carcinomas with squamous features showed GATA3 positivity. In another study, Gruver et al [25] found GATA3 positivity in only 23% of pulmonary squamous cell carcinomas. Taken together, these studies suggest the potential role of GATA3 in the differential diagnosis of metastatic urothelial carcinomas versus primary lung carcinomas. GATA3 has been also evaluated in tumors that should be considered in the differential diagnosis of metastatic urothelial carcinomas. In the study by Chang et al, GATA3 was positive in 0% of prostatic adenocarcinomas, 10% of anal squamous cell carcinomas, and 25% of uterine cervical squamous cell carcinomas, with most of positive cases depicting only focal and weak positivity [23]. On the contrary, in our series, GATA3 expression, when positive, was moderate to strong and extensive to diffuse. GATA3 expression was also observed in morphologic variants of urothelial carcinoma such as plasmacytoid urothelial carcinoma [23] and sarcomatoid urothelial carcinoma (unpublished observations). In addition, a recent study by Gulmann et al [24] suggests that GATA3 is a reliable marker of urothelial differentiation in urothelial
2766 carcinomas with squamous differentiation. Finally, in the study by Miyamoto et al [22], GATA3 expression was an independent predictor of poor prognosis in urothelial carcinomas of the urinary bladder. Worth noting, other studies in patients with breast carcinoma have also shown an association between GATA3 and clinical outcome [26,27]. Another useful marker for the differential diagnosis of penile urethra carcinomas is HPV detection. Among the subtypes of squamous cell carcinomas of the penis, basaloid carcinoma is the most challenging differential diagnosis for urothelial carcinoma, given their similarity in architecture and cytologic features. Solid nests of small and uniform basaloid cells may be easily taken as neoplastic urothelial cells, especially when squamous differentiation is not apparent. Basaloid squamous cell carcinoma of the penis is an aggressive and HPV-related tumor that is reported in up to 10% of all penile carcinomas with a metastatic rate between 50% and 100% [28]. Although this tumor predominantly affects the glans of the penis, this study shows that the penile urethra may be primarily involved. Another tumor that may raise the differential diagnosis of urothelial carcinomas in the penile urethra is warty-basaloid carcinoma, especially due to its papillomatous architecture and the presence of invasive nests of basaloid cells [29]. The identification of koilocytic changes and parakeratosis, not usually seen in urothelial carcinomas, might help suggesting a squamous origin in routine sections, but ought not to be taken as the sole basis for diagnosis. Although not seen in the current series and not yet reportedinthepenileurethra,anothersquamouscellcarcinomathat can be confused with urothelial carcinomas is a recently described variant of basaloid carcinoma showing papillary features [16]. In thisvariant,thetumoriscomposedofconspicuouspapillaelinedby basaloid cells, with or without an associated typical basaloid carcinoma or warty-basaloid carcinoma. Morphology can be deceiving in all these variants of squamous cell carcinomas, rendering the differential diagnosis between urothelial and squamous cell carcinoma almost impossible on H&E grounds alone. Defining HPV status can be useful in this context, considering that tumors with warty and basaloid features are usually HPV positive [15], whereas urothelial carcinomas are usually HPV negative [28]. Nevertheless, HPV in situ hybridization alone may not be sufficient to rule out urothelial carcinomas because a significant number of squamous cell carcinomas could also be negative for HPV. However, combined with GATA3 immunohistochemistry, HPV in situ hybridization or any other method for HPV detection such as the polymerase chain reaction may offer an additional tool to further confirm a specific differentiation of penile urethra carcinomas. This suggestion is further supported by the distinctive GATA3/ HPV status in the mixed squamous cell carcinoma/urothelial carcinoma that was found. Finally, the usefulness of surrogates for HPV infection, such as p16INK4a immunohistochemistry [30], combined with markers of urothelial differentiation such as GATA3 also merits further investigation. Three limitations must be acknowledged for the current study: first, the relatively small number of penectomies and
A. Chaux et al. primary urethral carcinomas of the penis; second, the use of TMAs for GATA3 evaluation rather than whole tissue sections; and third, the use of a limited set of squamous cell markers. For the first limitation, proper evaluation of the penile urethra required total penectomies, which were performed in locally advanced cases, cases that most likely also had urethral invasion. Identifying and retrieving total penectomies without tumor invasion of the penile urethra were difficult, moreover, considering the rarity of penile tumors. In addition, identifying and retrieving carcinomas exclusive of the penile urethra were also difficult, given their rarity. Most cases found corresponded to tumors with secondary extension from either the penile glans or the urinary tract. Thus, although the number of cases is relatively small, they are sufficient for establishing the immunohistochemical profile of the normal penile urethra and the potential usefulness of GATA3 and HPV detection in the differential diagnosis of urothelial versus squamous cell carcinomas of the penile urethra. Regarding the second limitation, although it would be preferable to use whole sections in determining the usefulness of a marker, TMAs provide a convenient way to evaluate a considerable number of cases under the same conditions and using the same protocol for immunohistochemistry. This diminishes the impact that external conditions might have in the development of the stain and facilitates the interpretation of the immunohistochemical expression. The consistency that was found between surgical specimens of primary tumors of the penile urethra and TMAs of urothelial and squamous cell carcinomas strengthens the notion that GATA3 is a useful marker in distinguishing these tumors. Finally, the evaluation of additional markers of squamous differentiation, such as CK14 and desmoglein, would be interesting, as shown by some previously discussed studies [24,25]. In conclusion, the penile urethra is covered by epithelial cells that are unique in morphology and immunohistochemical profile. The superficial layer was composed of a single layer of tall columnar cells that were CK7 positive, CK20 negative, and p63 negative. The deep layer was composed of stratified cubical cells that were CK7 negative, CK20 negative, and p63 positive. Both superficial and deep layers of the penile urethra were GATA3 positive, suggesting urothelial origin. In addition, immunohistochemistry for GATA3 was usually positive in urothelial carcinomas and consistently negative in squamous cell carcinomas, whereas the opposite trend was observed for HPV presence by in situ hybridization. Our study suggests that GATA3 and HPV detection are useful markers for distinguishing urothelial carcinomas from squamous cells carcinomas of the penile urethra.
Acknowledgments 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.
GATA3 in penile urethra
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