Primary Urethral Clear-Cell Adenocarcinoma

Primary clear-cell adenocarcinoma of the urethra, a rare tumor that histomorphologically resembles clear-cell carcinoma of the female genital tract, occurs predominantly in women and is associated with a relatively poor prognosis. The histogenesis of this rare urethral neoplasm has not been completely resolved, but it is thought to arise from either müllerian rests or metaplastic urothelium. Herein, we present comprehensive surgical pathological and cytopathological findings from a patient with primary urethral clear-cell adenocarcinoma and describe next-generation sequencing results for this patient's unique tumor—the first such reported characterization of molecular aberrations in urethral clear-cell adenocarcinoma at the transcriptomic and genomic levels. Transcriptome analysis revealed novel gene fusion candidates, including ANKRD28-FNDC3B. Whole-exome analysis demonstrated focal copy number loss at the SMAD4 and ARID2 loci and 38 somatic mutations, including a truncating mutation in ATM and a novel nonsynonymous mutation in ALK. Primary clear-cell adenocarcinoma of the urethra, a rare tumor that histomorphologically resembles clear-cell carcinoma of the female genital tract, occurs predominantly in women and is associated with a relatively poor prognosis. The histogenesis of this rare urethral neoplasm has not been completely resolved, but it is thought to arise from either müllerian rests or metaplastic urothelium. Herein, we present comprehensive surgical pathological and cytopathological findings from a patient with primary urethral clear-cell adenocarcinoma and describe next-generation sequencing results for this patient's unique tumor—the first such reported characterization of molecular aberrations in urethral clear-cell adenocarcinoma at the transcriptomic and genomic levels. Transcriptome analysis revealed novel gene fusion candidates, including ANKRD28-FNDC3B. Whole-exome analysis demonstrated focal copy number loss at the SMAD4 and ARID2 loci and 38 somatic mutations, including a truncating mutation in ATM and a novel nonsynonymous mutation in ALK. Primary carcinomas of the urethra are rare and, unlike most other genitourinary malignancies, occur more commonly in women than men.1Amin M.B. Young R.H. Primary carcinomas of the urethra.Semin Diagn Pathol. 1997; 14: 147-160PubMed Google Scholar In women, squamous cell carcinoma constitutes the vast majority of urethral carcinomas (approximately 70%), whereas conventional urothelial (transitional cell) carcinoma and conventional adenocarcinoma account for another 20% and 10%, respectively. There is a striking anatomical distribution of these tumors: distal urethral carcinomas are predominantly squamous cell type, whereas proximal urethral carcinomas are more often urothelial or adenocarcinoma type. Compared with other urinary tract malignancies, the prognosis for urethral carcinoma is poor, and patients often present with metastatic disease. The two principal prognostic factors for urethral carcinoma are pathological stage and tumor location, with a tendency for distal tumors (ie, squamous cell carcinoma) to have a better prognosis than proximal tumors (ie, urothelial carcinoma or adenocarcinoma). Although rare variants have been reported, primary urethral adenocarcinoma generally shows either columnar/mucinous or clear-cell histomorphological features.2Meis J.M. Ayala A.G. Johnson D.E. Adenocarcinoma of the urethra in women: a clinicopathologic study.Cancer. 1987; 60: 1038-1052Crossref PubMed Scopus (65) Google Scholar Columnar/mucinous adenocarcinoma resembles well-differentiated colonic or endocervical adenocarcinoma, whereas clear-cell adenocarcinoma is similar to clear-cell carcinoma of the female genital tract. In their seminal report, Oliva and Young3Oliva E. Young R.H. Clear cell adenocarcinoma of the urethra: a clinicopathologic analysis of 19 cases.Mod Pathol. 1996; 9: 513-520PubMed Google Scholar described the clinicopathological characteristics of a large cohort of primary urethral clear-cell adenocarcinomas. These tumors showed a marked sex predilection, with 18 cases in women compared with only 1 case in a man; in addition, most cases apparently arose within abnormally dilated periurethral structures—so-called urethral diverticula. Histomorphologically, these tumors displayed tubulocystic, papillary, and/or solid architecture and were composed of cells with clear cytoplasm, conspicuous nuclear atypia, and numerous mitotic figures. The histogenesis of primary urethral clear-cell adenocarcinoma has not been conclusively determined. Although it was initially thought to arise from mesonephric remnants, the current hypothesis is that clear-cell adenocarcinoma derives from metaplastic urothelium or müllerian rests.1Amin M.B. Young R.H. Primary carcinomas of the urethra.Semin Diagn Pathol. 1997; 14: 147-160PubMed Google Scholar, 3Oliva E. Young R.H. Clear cell adenocarcinoma of the urethra: a clinicopathologic analysis of 19 cases.Mod Pathol. 1996; 9: 513-520PubMed Google Scholar, 4Drew P.A. Murphy W.M. Civantos F. Speights V.O. The histogenesis of clear cell adenocarcinoma of the lower urinary tract: case series and review of the literature.Hum Pathol. 1996; 27: 248-252Abstract Full Text PDF PubMed Scopus (79) Google Scholar, 5Sung M.T. Zhang S. MacLennan G.T. Lopez-Beltran A. Montironi R. Wang M. Tan P.H. Cheng L. Histogenesis of clear cell adenocarcinoma in the urinary tract: evidence of urothelial origin.Clin Cancer Res. 2008; 14: 1947-1955Crossref PubMed Scopus (59) Google Scholar Alternative, but less widely accepted, theories include that the tumor originates from the periurethral (Skene's) glands or results from malignant transformation of nephrogenic adenoma.6Spencer J.R. Brodin A.G. Ignatoff J.M. Clear cell adenocarcinoma of the urethra: evidence for origin within paraurethral ducts.J Urol. 1990; 143: 122-125Abstract Full Text PDF PubMed Scopus (61) Google Scholar, 7Tong G.X. Weeden E.M. Hamele-Bena D. Huan Y. Unger P. Memeo L. O'Toole K. Expression of PAX8 in nephrogenic adenoma and clear cell adenocarcinoma of the lower urinary tract: evidence of related histogenesis?.Am J Surg Pathol. 2008; 32: 1380-1387Crossref PubMed Scopus (94) Google Scholar, 8Reis L.O. Billis A. Ferreira F.T. Ikari L.Y. Stellini R.F. Ferreira U. Female urethral carcinoma: evidences to origin from Skene's glands.Urol Oncol. 2011; 29: 218-223Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar The immunophenotype of this tumor reflects its histogenetic uncertainty.2Meis J.M. Ayala A.G. Johnson D.E. Adenocarcinoma of the urethra in women: a clinicopathologic study.Cancer. 1987; 60: 1038-1052Crossref PubMed Scopus (65) Google Scholar, 3Oliva E. Young R.H. Clear cell adenocarcinoma of the urethra: a clinicopathologic analysis of 19 cases.Mod Pathol. 1996; 9: 513-520PubMed Google Scholar, 4Drew P.A. Murphy W.M. Civantos F. Speights V.O. The histogenesis of clear cell adenocarcinoma of the lower urinary tract: case series and review of the literature.Hum Pathol. 1996; 27: 248-252Abstract Full Text PDF PubMed Scopus (79) Google Scholar, 7Tong G.X. Weeden E.M. Hamele-Bena D. Huan Y. Unger P. Memeo L. O'Toole K. Expression of PAX8 in nephrogenic adenoma and clear cell adenocarcinoma of the lower urinary tract: evidence of related histogenesis?.Am J Surg Pathol. 2008; 32: 1380-1387Crossref PubMed Scopus (94) Google Scholar, 9Dodson M.K. Cliby W.A. Pettavel P.P. Keeney G.L. Podratz K.C. Female urethral adenocarcinoma: evidence for more than one tissue of origin?.Gynecol Oncol. 1995; 59: 352-357Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 10Gilcrease M.Z. Delgado R. Vuitch F. Albores-Saavedra J. Clear cell adenocarcinoma and nephrogenic adenoma of the urethra and urinary bladder: a histopathologic and immunohistochemical comparison.Hum Pathol. 1998; 29: 1451-1456Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 11Sun K. Huan Y. Unger P.D. Clear cell adenocarcinoma of urinary bladder and urethra: another urinary tract lesion immunoreactive for P504S.Arch Pathol Lab Med. 2008; 132: 1417-1422Crossref PubMed Google Scholar, 12Brimo F. Herawi M. Sharma R. Netto G.J. Epstein J.I. Illei P.B. Hepatocyte nuclear factor-1beta expression in clear cell adenocarcinomas of the bladder and urethra: diagnostic utility and implications for histogenesis.Hum Pathol. 2011; 42: 1613-1619Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 13Alexiev B.A. Tavora F. Histology and immunohistochemistry of clear cell adenocarcinoma of the urethra: histogenesis and diagnostic problems.Virchows Arch. 2013; 462: 193-201Crossref PubMed Scopus (14) Google Scholar Tumor cells usually express PAX8, cytokeratin (CK) 7, α-methylacyl-CoA racemase, HNF-1β, and high molecular weight cytokeratin (CK34βE12) proteins; there is often nuclear accumulation of p53; and the Ki-67 index is generally high (>25%). Tumor cells also show variable expression of CA-125 and CK20 but are routinely negative for prostate-specific antigen, estrogen receptor (ER), progesterone receptor (PR), and p63 protein expression. Although Sung et al5Sung M.T. Zhang S. MacLennan G.T. Lopez-Beltran A. Montironi R. Wang M. Tan P.H. Cheng L. Histogenesis of clear cell adenocarcinoma in the urinary tract: evidence of urothelial origin.Clin Cancer Res. 2008; 14: 1947-1955Crossref PubMed Scopus (59) Google Scholar recently demonstrated frequent copy number gain of chromosomes 3, 7, and 17, similar to conventional urothelial carcinoma, by fluorescent in situ hybridization (FISH) in primary urethral clear-cell adenocarcinoma, little else is known about its molecular oncogenesis. Herein, we present a patient with primary urethral clear-cell adenocarcinoma, detailing the relevant surgical pathological and cytopathological findings. We also describe next-generation sequencing results for this patient's tumor (the first such analysis of this type for primary urethral clear-cell adenocarcinoma), including several novel gene fusion candidates, copy number loss at the SMAD4 and ARID2 loci, and somatic mutations in ATM and ALK. This study was approved by the University of Michigan (Ann Arbor) Health Sciences Institutional Review Board. Immunohistochemistry (IHC) was performed by the Department of Pathology at the University of Michigan Health System using a BenchMark ULTRA automated stainer and the ultraView Universal DAB Detection Kit (Ventana Medical Systems, Oro Valley, AZ). The following primary antibodies were used: PAX8 (predilute; Cell Marque, Rocklin, CA), CK7 and CK20 (1:200; Cell Marque), and ER, PR, p63, and p53 (predilute; Ventana Medical Systems). Next-generation sequencing, including transcriptome, copy number, mutation, and gene fusion analysis, was performed as described previously.14Roychowdhury S. Iyer M.K. Robinson D.R. Lonigro R.J. Wu Y.M. Cao X. Kalyana-Sundaram S. Sam L. Balbin O.A. Quist M.J. Barrette T. Everett J. Siddiqui J. Kunju L.P. Navone N. Araujo J.C. Troncoso P. Logothetis C.J. Innis J.W. Smith D.C. Lao C.D. Kim S.Y. Roberts J.S. Gruber S.B. Pienta K.J. Talpaz M. Chinnaiyan A.M. Personalized oncology through integrative high-throughput sequencing: a pilot study.Sci Transl Med. 2011; 3: 111ra121Crossref PubMed Scopus (503) Google Scholar, 15Robinson D.R. Wu Y.M. Kalyana-Sundaram S. Cao X. Lonigro R.J. Sung Y.S. Chen C.L. Zhang L. Wang R. Su F. Iyer M.K. Roychowdhury S. Siddiqui J. Pienta K.J. Kunju L.P. Talpaz M. Mosquera J.M. Singer S. Schuetze S.M. Antonescu C.R. Chinnaiyan A.M. Identification of recurrent NAB2-STAT6 gene fusions in solitary fibrous tumor by integrative sequencing.Nat Genet. 2013; 45: 180-185Crossref PubMed Scopus (550) Google Scholar, 16Roychowdhury S. Chinnaiyan A.M. Advancing precision medicine for prostate cancer through genomics.J Clin Oncol. 2013; 31: 1866-1873Crossref PubMed Scopus (77) Google Scholar, 17Wu Y.M. Su F. Kalyana-Sundaram S. Khazanov N. Ateeq B. Cao X. Lonigro R.J. Vats P. Wang R. Lin S.F. Cheng A.J. Kunju L.P. Siddiqui J. Tomlins S.A. Wyngaard P. Sadis S. Roychowdhury S. Hussain M.H. Feng F.Y. Zalupski M.M. Talpaz M. Pienta K.J. Rhodes D.R. Robinson D.R. Chinnaiyan A.M. Identification of targetable FGFR gene fusions in diverse cancers.Cancer Discov. 2013; 3: 636-647Crossref PubMed Scopus (554) Google Scholar Whole-exome and transcriptome sequencing was performed on a HiSeq 2000 (Illumina, San Diego, CA), and the resulting FASTQ sequence files were processed through in-house pipelines. For whole-exome sequencing, Novoalign Multithreaded software version 2.08.02 (Novocraft Technologies, Selangor, Malaysia) was used to align the sequencing reads to the human reference genome build (hg19/GRCh37). Mutation analysis was performed using VarScan 2 software version 2.3.2 (Washington University, St. Louis, MO),18Koboldt D.C. Zhang Q. Larson D.E. Shen D. McLellan M.D. Lin L. Miller C.A. Mardis E.R. Ding L. Wilson R.K. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing.Genome Res. 2012; 22: 568-576Crossref PubMed Scopus (3044) Google Scholar and pileup files were produced by the SAMtools mpileup program.19Li H. Handsaker B. Wysoker A. Fennell T. Ruan J. Homer N. Marth G. Abecasis G. Durbin R. The Sequence Alignment/Map format and SAMtools.Bioinformatics. 2009; 25: 2078-2079Crossref PubMed Scopus (32884) Google Scholar Indel analysis was performed using Pindel, version 0.2.4 (Washington University),20Ye K. Schulz M.H. Long Q. Apweiler R. Ning Z. Pindel: a pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short 2009; 25: PubMed Scopus Google Scholar and were variants were using K. M. H. of variants from high-throughput sequencing Res. PubMed Scopus Google Scholar For paired-end transcriptome sequencing, reads were using software version of D. G. H. R. of in the of deletions and gene 2013; 14: PubMed Scopus Google Scholar the short version B. M. and of short to the human 2009; PubMed Scopus Google Scholar reads the fusion were with the to the fusion and a sequence of a fusion gene was and for using for gene fusions with were using the to the gain or loss of known in the fusion For ATM somatic mutation ATM was from genomic with and and The The was with and to sequencing at the University of Michigan using the were with software version For gene fusion was from tissue using the Kit and using Technologies, were performed on a System with and gene fusion and and and The patient is a with and for of and In the patient's in was and with to was to women to the of and In was to a clear-cell tumor in women to this in The and the of in the patient's with demonstrated a and a but the most the patient was initially with for a urinary tract of demonstrated urinary bladder and results were for was as with carcinoma, and with showed a cells with variable The patient was for of a bladder a lesion of the bladder was and completely large of the urethra and apparently from within a in the urethral was and of the revealed of tumor with predominantly architecture and the of the of urothelium were evidence of urothelial or conventional urothelial Tumor cells were medium with cell and clear were medium and with nuclear but demonstrated that the tumor cells and PAX8 and CK7 and but were negative for ER, PR, and p63 protein expression not The histomorphological and immunophenotype were with clear-cell demonstrated tumor in the bladder and urethra, and showed clear-cell adenocarcinoma with the bladder with was for cytopathological a was and revealed a composed predominantly of of cells with to nuclear In and in were also present The a to and were with of the cell and with were cell was the in the from the cell revealed of cells clear on the of a of clear-cell adenocarcinoma was of the and showed bladder from the of the to the bladder and the proximal urethra, with and of tumor from the and were was by of the but other evidence of was the and findings were with a primary urethral clear-cell adenocarcinoma with bladder and urinary and to the patient to and pathological analysis of the revealed a primary urethral clear-cell adenocarcinoma in the of the proximal urethra, the periurethral the periurethral and The also through the bladder the bladder with of the bladder and the The tissue was negative for of were by metastatic clear-cell the pathological of patient's tumor was to stage S.B. on Cancer Scholar from tissue were for next-generation number analysis revealed several of copy number gain in tumor including a of and a of and a of and to a of and and a of and to and In addition, there was copy number loss of a of ARID2 and of focal copy number loss on SMAD4 3, and Mutation analysis revealed 38 somatic mutations, including a mutation in ATM and a novel nonsynonymous mutation of in and to in of ATM a at the ATM protein the and This somatic mutation in ATM was by sequencing In the a to in 19 results in of for at this occurs to the in a a somatic of in of results in a at and, similar to the ATM mutation, a protein the or this not by Transcriptome analysis gene fusion of were by and The gene fusion results from of 3, 2 of to of The fusion protein most of the of with type and a This fusion was by analysis The gene fusion is in a of copy number it results from of the of to the of The gene fusion results from of 1 of to within the The gene fusion results from a and the first of to the 18 of to The gene fusion results from a and the first 19 of to the of to of the and 2 showed evidence of or metastatic disease. the high pathological stage and and were and the patient two of and of to the the patient was evidence of tumor Primary urethral clear-cell adenocarcinoma is a tumor that a diagnostic for and a for The for urethral clear-cell metastatic clear-cell carcinoma of the female genital tract, urothelial carcinoma with clear-cell or metastatic clear-cell cell carcinoma and nephrogenic M.B. Young R.H. Primary carcinomas of the urethra.Semin Diagn Pathol. 1997; 14: 147-160PubMed Google Scholar primary urethral clear-cell adenocarcinoma from urothelial carcinoma or metastatic clear-cell is usually on histomorphological characteristics the of conventional urothelial carcinoma) and/or a of including p63 in urothelial carcinoma) and CK7 in clear-cell Although nephrogenic adenoma a and of primary urethral clear-cell M.Z. Delgado R. Vuitch F. Albores-Saavedra J. Clear cell adenocarcinoma and nephrogenic adenoma of the urethra and urinary bladder: a histopathologic and immunohistochemical comparison.Hum Pathol. 1998; 29: 1451-1456Abstract Full Text PDF PubMed Scopus (102) Google Scholar, M. P.A. Epstein J.I. Clear cell adenocarcinoma of the bladder and urethra: cases nephrogenic Pathol. Full Text Full Text PDF PubMed Scopus Google Scholar these two usually on large on or evidence of or or by pathological patient's case the histogenetic of primary urethral clear-cell The theories that these tumors arise from either müllerian rests or metaplastic M.B. Young R.H. Primary carcinomas of the urethra.Semin Diagn Pathol. 1997; 14: 147-160PubMed Google Scholar, 3Oliva E. Young R.H. Clear cell adenocarcinoma of the urethra: a clinicopathologic analysis of 19 cases.Mod Pathol. 1996; 9: 513-520PubMed Google Scholar, 4Drew P.A. Murphy W.M. Civantos F. Speights V.O. The histogenesis of clear cell adenocarcinoma of the lower urinary tract: case series and review of the literature.Hum Pathol. 1996; 27: 248-252Abstract Full Text PDF PubMed Scopus (79) Google Scholar, 5Sung M.T. Zhang S. MacLennan G.T. Lopez-Beltran A. Montironi R. Wang M. Tan P.H. Cheng L. Histogenesis of clear cell adenocarcinoma in the urinary tract: evidence of urothelial origin.Clin Cancer Res. 2008; 14: 1947-1955Crossref PubMed Scopus (59) Google Scholar Although patient's tumor with either of these of its with urethral we that it most arose from metaplastic urothelium. to urinary in recurrent urinary tract or C.R. Female urethral Urol. PubMed Scopus Google Scholar to other of the urinary tract, the resulting and to malignant Although müllerian rests were in the or surgical the immunophenotype and of the tumor in the proximal urethra that a müllerian origin completely In to the surgical pathological findings of primary we have described cytopathological results from a of patient's The demonstrated of cells with and and the cell the tumor histomorphological characteristics in the Although relatively have the cytopathological of primary urethra clear-cell adenocarcinoma, the reported are similar to patient's and cell for PAX8, p63, and Ki-67 to urethral clear-cell B.A. Tavora F. Histology and immunohistochemistry of clear cell adenocarcinoma of the urethra: histogenesis and diagnostic problems.Virchows Arch. 2013; 462: 193-201Crossref PubMed Scopus (14) Google Scholar, S. T. M. K. R. K. T. H. K. G. case of clear cell adenocarcinoma from the urethral utility of urinary and 2012; 9: PubMed Scopus Google Scholar, G. Wang A. A.M. P. of clear cell carcinoma of the urethra and urinary 1996; 14: PubMed Scopus Google Scholar These are not by cytopathological analysis for the of clear-cell The molecular of primary urethral clear-cell adenocarcinoma is not molecular of patient's unique we performed transcriptome, copy number, and mutation by next-generation first such analysis for primary urethral clear-cell described analysis of primary urethral clear-cell adenocarcinoma and showed frequent copy number gain of chromosomes 3, 7, and 17, a pattern similar to conventional urothelial M.T. Zhang S. MacLennan G.T. Lopez-Beltran A. Montironi R. Wang M. Tan P.H. Cheng L. Histogenesis of clear cell adenocarcinoma in the urinary tract: evidence of urothelial origin.Clin Cancer Res. 2008; 14: 1947-1955Crossref PubMed Scopus (59) Google Scholar In analysis of patient's tumor by next-generation sequencing demonstrated copy number gain of chromosomes 7, and The for this is but the of tumor and/or in of the a gene a is associated with of B. S. Su T. M. B. M. S. H. R. of the in cancer through of by 2009; PubMed Scopus Google Scholar copy number loss at the SMAD4 on was also SMAD4 a protein in the protein and as a tumor in a of human most and G. X. in J PubMed Scopus Google Scholar SMAD4 loss of is in these cancer and SMAD4 a rare by and tumors of the tract. we copy number loss at the ARID2 on ARID2 a protein in the of by and recurrent somatic mutations in ARID2 have been in a of human including carcinoma, adenocarcinoma, and cell J.R. The of mutations, in human 2013; PubMed Scopus Google Scholar, G. E. A. A. H. E. K. S. F. J. P. H. mutations of ARID2 in cell J 2013; 132: PubMed Scopus (59) Google Scholar, N. et cancer aberrations in 2012; PubMed Scopus Google Scholar Mutation analysis revealed 38 somatic in the including a truncating ATM mutation and a novel nonsynonymous The ATM is a protein with a in the to of cell and as a tumor in a of human D. more than at the J. 2011; PubMed Scopus Google Scholar The ATM mutation results in a ATM protein (ie, the and to the of the This is in of the of nuclear p53 accumulation in patient's is a of primary urethral clear-cell adenocarcinomas. p53 expression in tumor cells usually results from the accumulation of p53 somatic alteration of the of nuclear p53 accumulation in patient's tumor is with next-generation sequencing ATM and in the and a negative ATM and mutations has been reported in a large cohort of L. G. Mardis E.R. McLellan M.D. K. et mutations in 2008; PubMed Scopus Google Scholar This that mutation of ATM in patient's tumor for of the a nonsynonymous mutation of was in the of the nuclear receptor have this gene to for L. P. et study cancer loci on chromosomes and Genet. 42: PubMed Scopus Google Scholar Transcriptome analysis the novel gene fusion ANKRD28-FNDC3B. This gene fusion 2 of to of and protein of the is present on is in a diverse number of human cancer and of has been in and of of including and protein M. X. Q. J. J. of cancer and tumor of the 2012; PubMed Scopus (61) Google Scholar In carcinoma, for and of the Lin P.H. Lin Chen Wu Chen M.T. Y.S. copy number in cancer cancer in PubMed Scopus Google Scholar In we have described the surgical and molecular pathological findings from a patient with primary urethral clear-cell patient's tumor demonstrated of the and histomorphological of this and the cytopathological results were with in the Next-generation sequencing revealed a of novel about the molecular of this unique cancer type, including of and and in and These molecular the of to rare tumors in the current of medicine and to the molecular of primary urethral clear-cell with a cohort of tumors molecular clear-cell tumors of the urinary and female genital or the of the histogenesis of primary urethral clear-cell We and the of University of Michigan (Ann Arbor) Health for and for with with files with files with files with files