Pediatric and Developmental Pathology 18, 117-121, 2015 DOI: 10.2350/14-08-1539-OA.1 © 2015 Society for Pediatric Pathology
Gonadoblastoma in Patients with UllrichTurner Syndrome G abriela Z e laya , 1* J essica M . L opez M M
a r ta
a r ti , 2
Ro x a n a M
a r in o , 3
M
a r ia
T . G arc ia
de
D a v il a , 2
and
S. G a l l e g o 1
’ Department o f Genetics, Laboratory o f Cytogenetics, Hospital Nacional de Pediatria J.P Garrahan, Combate de los Pozos 1881 (CP 1245) Buenos Aires, Argentina 2Departement of Pathology, Hospital Nacional de Pediatria J. P Garrahan, Combate de los Pozos 1881 (CP 1245) Buenos Aires, Argentina 3Department o f Endocrinology, Laboratory o f Molecular Biology, Hospital Nacional de Pediatria J. P Garrahan, Combate de los Pozos 1881 (CP 1245) Buenos Aires, Argentina
Received August 19, 2014; accepted December 22, 2014; published online December 23, 2014.
ABSTRACT Ullrich-Turner syndrome (UTS) is a common chromo somal abnormality caused by partial or complete X chromosome monosomy. One half of the patients have a 45,X karyotype, whereas the remaining patients display other X chromosome anomalies. In 6% to 11% of UTS, a normal or partly deleted Y chromosome has been found. A 10% to 30% risk of developing gonadoblastoma was found in the latter patients. The aim of this study was to evaluate the prevalence of Y chromosome-derived material, the occurrence of gonadoblastoma, and the incidence of possible neoplasms in patients with UTS. Of 217 patients studied with UTS and chromosome analysis of peripheral-blood lymphocytes, Y chromosome material was found in 20 patients. Fluorescence in situ hybridiza tion (FISH) testing was performed to characterize the structurally abnormal Y chromosome in 13 cases. Molecular analysis of the SRY gene could only be performed in 20 patients with 45,X karyotype. Two patients had the SRY genomes. Of the 20 patients with Y chromosome-derived material, 17 underwent gonadectomy. The incidence of gonadoblastoma development in our series was 35.5%. Furthermore, 1 patient also showed a pure dysgerminoma, and another showed a mixed dysgerminoma and embryonal carcinoma. We emphasize the importance of complete processing of the gonadectomy specimen, including step sections, molecular studies, and FISH, in addition to the classic cytogenetic searching for Y chromosome sequences, in patients who present with a nonmosaic 45,X karyotype. Finally, we propose to routinely collect a sample for storage in the tumor bank for future studies.
* Corresponding author, e-mail: [email protected]
Key words: gonadal dysgenesis, gonadectomy, gonado blastoma, mosaicism, Ullrich-Turner syndrome, Y chromosome
INTRODUCTION The Ullrich-Turner syndrome (UTS) is one of the most common chromosomal abnormalities presenting in humans, with a frequency of 1 in 2500 live-bom females. It is a disorder caused by partial or complete X chromosome monosomy and characterized by short stature, ovarian failure, and specific somatic abnormalities [1,2]. Approx imately one half of patients with UTS have a 45,X karyotype, whereas the remaining patients display a variety of other chromosomal anomalies of the X chromosome, including several mosaicisms [3]. A normal or partly deleted Y chromosome or Y-derived marker chromosome has been found in 6% to 11% of patient with UTS using standard cytogenetic techniques. Occult Y chromosome mosaicism detected by techniques other than standard cytogenetics varies according to study and methodology used [4-6], The early detection of Y-derived sequences in the genome of individuals with UTS is of great importance because of the relatively high risk (10%-30%) of developing gonadal tumors (ie, gonadoblastoma or dysger minoma) [7,8]. A gonadoblastoma is a neoplasm composed of germ cells and sex-cord elements with an excellent prognosis if detected early. However, gonadoblastoma may progress to dysgerminoma with metastatic potential, justifying early gonadectomy [9]. A gonadoblastoma susceptibility locus has been proposed for the pericentromeric region of the Y chromosome [10,11]. The neoplasm does not appear to correlate with the presence of SRY. The aim of this study was to evaluate the prevalence of Y chromosome-derived material, the occurrence of gonadoblastoma, and the incidence of possible neoplasia in patients with UTS.
Table 1.
Cytogenetic and in situ hybridization studies
Patient
Karyotype
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
mos 45,X[15]/46,X, del(Y)(ql 1.2) [5] mos 45,X[30]/46,X,idic(Y)(pl 1.3).ish idic(Y)(p 11.3 )(DYZ l++,SRY++)[ 10] mos 45,X[88]/46,X,+mar.ish idic(Y)(pll.3)(DYZ3++)[6] mos 46, X,i(Y)(p 10) [22]/45,X[8], ish idic(Y)(qll.2(DYZ3++) mos 45,X[17]/46,X,idic(Y)(ql 1.2).ish idic(Y)(ql 1)(SRY++)[13] mos 45,X[47]/46,X,i(Y)(ql0).ish i(Y)(q 10)(wcpY+,SRY—)[3] mos 46,X,+mar [24]/45,X[6].ish i(Y)(pl l.lql 1.1 )(DYZ3+)(SRY++) mos 46,X,idic(Y)(ql 1.2).ish idic(Y)(q 11,2)(SRY++)[19]/45,X[ 11] mos 45,X[39]/46,X,idic(Y)(ql l).ish idic(Y)(ql 1.2)(SRY++,DYZ3++)[5] mos 45,X[26]/ 45,X,dic(Y; 15)(Ypter->Yq 11.2:: 15q 13-> 15q)[7] mos 45,X[30]/46,X,+mar.ish der(Y)(wcpY+)[15] mos 45,X[30]/46,X,i(Y)(pl0).ish idic(Y)(ql l)(SRY++,DYZ3++)[3] mos 46,X,idic(Y)(ql 1.2).ish idic(Y)(q 11,2)(SRY++,DYZ3++)[12]/45,X[8] mos 45,X[14]/46,XY[6] mos 45,X[19]/46,XY[11] mos 45,X[40]/46,XY[34] mos 45,X[7]/46,XY[5] mos 45,X[23]/46,XY[7] mos 45,X[25]/46,XY[4] mos 45,X[9]/46,XY[21]
METHODS
Two hundred seventeen patients with phenotypic UTS were diagnosed between January 1989 and December 2012 .
Chromosome analysis of peripheral blood lympho cytes was performed with GTG banding (G-banding with trypsin and Giemsa) according to standard methods. A minimum of 30 metaphase cells was analyzed in cases with a nonmosaic 45,X karyotype or a minimum of 20 metaphase cells when mosaicism was identified within the first 20 cells. Fluorescence in situ hybridization (FISH) analysis using commercially available centromeric probes (DXZ1/ DYZ3), SRY, and whole Y chromosome painting (WCP Y) (Vysis, Inc, Abbott Molecular, Downers Grove, IL, USA) was performed in peripheral blood samples obtained from the patients following the manufacturer’s protocol. Genomic DNA was isolated from peripheral-blood lymphocytes according to standard protocols. To identify the presence of Y chromosome in 20 patients with 45,X karyotype, a 472-base pair (bp) fragment of SRY, including the DNA-binding motif, was amplified by duplex polymerase chain reaction (PCR) using primers 118
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al.
specific for the SRY gene and SMA gene (exon 7), previously reported [12,13], as an internal control for amplification (226-bp fragment). Moreover, 2 fragments of the amelogenin gene (AMELX and AMELY), a 800-bp fragment located on chromosome Y and a 997-bp fragment located on chromosome X, were amplified by PCR using the same primers [14], After gonadectomy, right and left gonadal tissues were fixed in formalin buffer and processed in paraffin with 2 to 4 step sections of approximately 3 pm from each block and stained with hematoxylin and eosin (H&E). Interphase FISH assay was performed using the centromeric probes (DXZ1/DYZ3). Signals were scored in at least 200 nonoverlapping intact nuclei. The ratio of Y:X signals was calculated and was used to express the frequency of Y-positive cells. RESULTS
The karyotype distribution among 217 patients was 45,X (44.2%, n = 96), X structural abnormalities (39.6%, n = 86), Y structural abnormalities (6%, n = 13), 45,X/46,XX (5.1%, n = 11), 45,X/46,XY (3.2%, n = 7), 45,X/46,XX/ 47,XXX (1.4%, n = 3), 45,X/47,XXX (0.5%, n = 1). The FISH testing was performed to characterize the structurally abnormal Y chromosome in 13 cases (Table l).We also investigated the presence of 2 Ypspecific sequences (SRY and AMELY) and 1 Xp-specific sequence (AMELX) by PCR in 20 patients with UTS and a 45,X karyotype. As expected, all patients had a positive amplification for the Xp-specific sequence AMELX. Interestingly, 2 of the patients had the Y SRY and AMELY sequences in their genomes. Gonadectomy was performed in 17 patients with ages ranging from 2 to 18 years (mean, 11.5 years). We studied the histologic characteristics in all specimens of dysgenetic gonads, especially in those that had undergone neoplastic transformation, and compared those findings with the features of the gonadal tissue adjacent to and within the gonadoblastoma samples. We found 2 main patterns of dysgenetic gonads. The first pattern, considered the classic streak gonad pattern, consisted of whorls and bundles of fibrous connective tissue, ovarian-like stroma devoid of germ cells, with isolated inclusion cysts lined by cuboidal epithelium. This pattern was found in 9 cases (patients 1-3, 8, and 10-14). In 1 case (patient 1) only, it was associated with gonadoblastoma and dysgerminoma. (Fig. 1 and Table 2) The second pattern, with epithelial cord-like struc tures, consisted of ovarian, cortex-like stroma with epithelial cord-like structures occasionally forming an anastomosing network. These structures contained 2 types of cells: small cells with hyperchromatic nuclei and germ cells. This pattern was found in 7 cases (patients 4-7, 9, 15, and 17). In 3 cases, it was associated with gonadoblastoma, and in 1 case, it was associated with gonadoblastoma and a malignant mixed germ cell tumor (patient 7).
Figure 1. P atient 15. Streak g onad w ith small foci o f g o n ad ob lasto m a. A. H em atoxylin and eosin (H&E), X20. B. H&E, X40.
In an attempt to assess dysgenetic gonads, we performed additional step sections and found microscopic foci of gonadoblastoma in 2 additional cases (patients 5 and 6). In patient 8, the gonadal tissue was a streak testis. Six patients (35.3%) showed bilateral gonadoblas toma. In 4 of these cases, the tumor consisted of microscopic foci that could be detected by step section ing. In addition to gonadoblastoma, dysgerminoma was detected in the other 2 cases. Interestingly, in 1 of these cases, the tumor coexisted, in the right gonad, with an embryonic carcinoma. Macroscopically, the tumor had a maximum diameter of 5 cm (Fig. 2). Interphase FISH analysis performed in gonadal tissue showed Y chromosomal material in all cases, coinciding with the cytogenetic findings in peripheral lymphocytes. DISCUSSION
The detection of Y chromosome mosaicism is clinically important because of the risk of tumorigenesis, especially gonadoblastoma [15], or other nontumoral androgenproducing lesions [16] in the dysgenetic gonads of patients with UTS and a normal, structurally abnormal, or hidden Y chromosome. The gonadoblastoma is a benign neoplasm composed of undifferentiated cells similar to those seen in gonadal
development admixed with sex-cord derivatives. It was first described by Scully in 1953 [17]. Although gonadoblastoma is a benign tumor, in 60% of cases, it can turn into an invasive dysgerminoma and may also progress to other forms of malignant germ-cell tumors [18]. This could be the result of a disturbance in the germ cell maturation. Therefore, early prophylactic gonadectomy is indicated in all cases when the association with Y chromosome sequences is confirmed. Page [19] postulated the GBY locus as an oncogene in the dysgenetic gonads. Five genes have been described in this region. The TSPY (testis-specific protein Yencoded) gene might be involved in the initial selection of tumorigenic cells, possibly by having a role in cellcycle regulation or cell division, even though its function is still unclear [20], Octamer-binding transcription factor 4 (OCT4) (also know OCT3 or POU5FI; OMIM 164177) is a transcription factor essential to the maintenance of pluripotentiality of embryonic stem cells and primordial germ cells [21,22]. It is considered a useful marker of germ cell tumor, such as gonadoblastoma, dysgerminoma, seminoma, and others. Expression of OCT4 is detected by immunohistochemistry in 100% of cases of gonadoblas toma [23], In our series, the prevalence of mosaicism with a normal or abnormal Y chromosome was 9.2% detected by conventional cytogenetics. We investigated the presence of Y chromosome sequences by PCR in only 20 patients with UTS and a 45,X karyotype and found 2 patients with Yp-specific sequences (10%). Seventeen of these patients (85%) were gonadectomized, and gonadoblastoma was found in 6 (35.3%). According to the classification of streak gonads [24-26], in our series, we found 9 cases of classic streak gonads, 7 of streak gonads with epithelial cord-like structures, and 1 streak testis. In the first group, we found an association between gonadoblastoma and dysgermino ma in 1 case, and in the second group, we found 5 gonadoblastomas and the coexistence in 1 of these cases of a dysgerminoma and an embryonal carcinoma. Based on these findings, we hypothesize that gonadoblastoma could originate from surviving germ cells in areas of streak gonadal tissue, mainly present in the cord-like structures within the dysgenetic gonad. In the presence of streak gonads, it is important to perform immunostain studies to detect germ cells to rule out neoplastic foci at an early stage. Complete processing included step sectioning of the gonadectomy specimen, is recommended by the Turner Syndrome Society of the United States [27], It allowed for the detection of small neoplastic foci in 2 patients. Gonadoblastoma was associated with dysgerminoma in 2 cases, progressing to embryonal carcinoma in 1 case (patient 7). In that patient, metastasis was found in the lungs and the omentum at the time of diagnosis, and the prognosis for that patient was poor. On the other hand, it has been reported that gonadoblastoma are thought to arise prenatally [28-30] G onadoblastoma in P atients
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Table 2. Patient
Histologic findings of resected gonadal tissue A ge (years)
R ight gonad
Left gonad
1
15
2 3 4 5
18 9 14
C lassic streak gonad G B L dysgerm inom a C lassic streak gonad C lassic streak gonad Streak w ith epithelial cord-like structures Streak w ith epithelial cord-like structures GBL
6
2
7
14
8 9 10 11 12
16 13 10 2 18
13 14 15
17 6 17
16 17
7 4
C lassic streak gonad G BL C lassic streak gonad C lassic streak gonad Streak w ith epithelial Streak w ith epithelial G BL Streak w ith epithelial G BL Streak w ith epithelial GBL Streak testis Streak w ith epithelial C lassic streak gonad C lassic streak gonad C lassic streak gonad C lassic streak gonad C lassic streak gonad Streak w ith epithelial G BL C lassic streak gonad Streak w ith epithelial G BL
14
Streak w ith epithelial cord-like structures GBL Streak w ith epithelial cord-like structures G BL dysgerm inom a em bryonal carcinom a Streak testis Streak w ith epithelial cord-like structures C lassic streak gonad C lassic streak gonad C lassic streak gonad C lassic streak gonad C lassic streak gonad Streak w ith epithelial cord-like structures GBL C lassic streak gonad Streak w ith epithelial cord-like structures GBL
cord-like structures cord-like structures cord-like structures cord-like structures
cord-like structures
cord-like structures
cord-like structures
GBL indicates gonadoblastoma.
and that the risk of tumor increases after puberty. Although the natural history of gonadoblastoma at the prepubertal age has not been determined, our data suggest that the age at appearance and the possibility of malignant degeneration may be in early life. In our study, the incidence of Y-positive nuclei in gonadal tissue correlated with that found in blood samples. The finding of a gonadoblastoma in dysgenetic
gonadal tissue of patients with UTS and Y-chromosome material confirms the usefulness of genetic techniques in the identification of risk of tumor development. Some authors suggest that the development of gonadoblastoma with and without malignant germ-cell tumors would be a multistep and multifactorial process. The aberrant expression of TSPYis likely to predispose to dysgenetic gonads and to other oncogenic events in the
Figure 2. P atient 7. A. R igh t g onad w ith a solid and heterogeneous tu m o r m easuring 8 cm. B. G onadoblastom a (h e m a to xylin and eosin [H&E], X40). C. D ysgerm inom a and em b ryo na l carcinom a (H&E, X10). D. D ysgerm inom a (H&E, X20). E. Em bryonal carcinom a (H&E, x 4 0 ). F. Im m u n o sta in in g w ith CD30 in th e e m bryonal carcinom a c o m p o n e n t (X20).
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malignant transition of a gonadoblastoma to a dysgerminoma, strongly suggesting the need for genetic screening for TSPY in patients with UTS. Moreover, Barros and colleagues [31] discuss the importance of an immunohistochemical study with OCT4 in all gonads of patients with UTS, in addition to the histopathologic analysis, to establish the risk of tumor development. Considering the need of material for future studies, it is important to establish a protocol for the routine storage of a mirror section of the gonadal tissue. In conclusion, we found a 35.3% incidence of gonadoblastoma development in patients with UTS and Y chromosome-derived material in their karyotype. Based on the finding of microscopic tumor foci, we emphasize complete processing of the gonadectomy specimen, including step sections. We point out the importance of performing molec ular, classic cytogenetics, and FISH studies searching for Y chromosome sequences in patients who present with a nonmosaic 45,X karyotype. Furthermore, the incorpora tion of the analysis of the genes implicated in the development of gonadoblastoma will be useful in the evaluation of these patients. Finally, we propose to routinely collect a sample for storage in the tumor bank for future studies. REFERENCES 1. Zinn AR, Page DC, Fisher EM. Turner syndrome: the ease of the missing sex chromosome. Trends Genet 1993;9:90-93. 2. Lopez M, Canto P, Aguinaga M. Frequency of Y chromosomal material in Mexican patients with Ullrich-Tumer syndrome. Am J Med Genet 1998;76:120-124. 3. Mathur A, Stekol L, Schatz D, MacLaren NK, Scott ML, Lippe B. The parental origen of the single X chromosome in Turner syndrome: lack of correlation with parental age or clinical phenotype. Am J Hum Genet 1991;48:682-686. 4. Jacobs P, Dalton P, James R, et al. Turner syndrome: a cytogenetic and molecular study. Ann Hum Genet 1997:61:471^183. 5. Kokova M, Siegel SF, Wenger SL, Lee PA, Trucco M. Detection of Y chromosome sequences in Turner’s syndrome by Southern blot analysis of amplified DNA. Lancet 1993;342:140-143. 6. Wiktor AE, Van Dyke DL. Detection of low level sex chromosome mosaicism in Ullrich-Tumer syndrome patients. Am J Med Genet A 2005;138A:259-261. 7. Verp MS, Simpson JL. Abnormal sexual differentiation and neoplasia. Cancer Genet Cytogenet 1987;25:191-218. 8. Gravholt CH, Fedder J, Naeraa RW, Muller J. Occurrence of gonadoblastoma in females with Turner syndrome and Y chromo some material: a population study. J Clin Endocrinol Metab 2000; 85:3199-3202. 9. Cools M, Stoop H, Kersemaekers AM, et al. Gonadoblastoma arising in undifferentiated gonadal tissue within dysgenetic gonads. J Clin Endocrinol Metab 2006;91:2404-2413. 10. Tsuchiya K, Reijo R, Page DC, Disteche CM. Gonadoblastoma: molecular definition of the susceptibility region on the Y chromosome. Am J Hum Genet 1995;57:1400-1407. 11. Lau YF. Gonadoblastoma, testicular and prostate cancers, and the TPSY gene. Am J Hum Genet 1999;64:921-927.
12. Goto E, Toral JF, Menendez M.I, et al. PCR-based study of the presence of Y-chromosome sequences in patients with UllrichTumer syndrome. Am J Med Genet 1995;57:393-396. 13. van der Steege G, Grootscholten PM, van der Vlies P, et al. PCRbased DNA test to confirm clinical diagnosis of autosomal recessive spinal muscular atrophy. Lancet 1995;345:985-986. 14. Dardis A, Saraco N, Mendilaharzu H, Rivarola MA, Belgorosky A. Report of an XX male with hypospadias and pubertal gynecomastia, SRY gene negative in blood leukocytes but SRY gene positive in testicular cells. Horm Res 1997;47:85-88. 15. Scully RE. Gonadoblastoma: a review of 74 cases. Cancer 1970;25: 1340-1356. 16. Bianco B, Nunes Lipay MV, Guedes AD, Verrerschi IT. Clinical implications of the detection Y-chromosome mosaicism in Turner’s syndrome: report of 3 cases. Fertil Steril 2008;90:1197.el7-20. 17. Scully RE. Gonadoblastoma: a gonadal tumor related to the dysgemiinoma (seminoma) and capable of sex-hormone production. Cancer 1953;6:455-463. 18. Saenger P. Clinical review 48: the current status of diagnosis and therapeutic intervention in Turner’s syndrome. J Clin Endocrinol Metab 1993;77:297-301. 19. Page DC. Hypothesis: a Y-chromosomal gene causes gonadoblas toma in dysgenetic gonads. Development 1987;101:151-155. 20. Vogt PH, Affara N, Davery P, et al. Report on the Third International Workshop on Y Chromosome Mapping 1997: Heidel berg, Germany, April 13-16, 1997. Cytogenet Cell Genet 1997;79: 1- 20 .
21. Rosenfeld RG, Tesch LG, Rodriguez-Rigau LJ, et al. Recommen dations for diagnosis, treatment and management of individuals with Turner syndrome. Endocrinologist 1994;4:351-358. 22. Nichols J, Zevnik B, Anastassiadis K, et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcriptional factor OCT4. Cell 1998;95:379-391. 23. Donovan PJ. High Oct-ane fuel powers the stem cell. Nat Genet 2001;29:246-247. 24. Cheng L, Sung M-T, Cossu-Rocca P, et al. OCT4: biological functions and clinical applications as a marker of germ cell neoplasia. J Pathol 2007;211:1-9. 25. Nistal M, Paniagua R, Gonzalez-Peramato P, Reyes-Mugica M. Gonadal dysgenesis. Pediatr Dev Pathol in press. 26. Nistal M, Paniagua R, Gonzalez-Peramato P, Disorders of sex development: nonneoplastic diseases of the testis. In: Bostwick DG, Cheng L eds. Urologic Surgical Pathology, 3rd ed. Philadelphia: Elsevier, 2014;629-632. 27. Nistal M, Garcia-Fernandez E, Marino-Enriquez A, Serrano A, Regadera J, Gonzalez- Permato P. Diagnostic value of the gonadal biopsy in the disorders of sex development [in Spanish], Actas Urol Esp 2007;31:1056-1075. 28. Skakkebaaik NE, Berthelsen JG, Giwercman A, Muller J. Carcinoma-in-situ of the testis: possible origin from gonocytes and precursor of all types of germ cell tumours except spermatocytoma. Int J Androl 1987;10:19-28. 29. Verp MS, Simpson JL. Abnormal sexual differentiation and neoplasia. Cancer Genet Cytogenet 1987;25:191-218. 30. Jorgensen N, Muller J, Jaubert F, Clausen OP, Skakkebatek NE. Heterogeneity of gonadoblastoma germ cells: similarities with immature germ cells, spermatogonia and testicular carcinoma in situ cells. Histopathology 1997;30:177-186. 31. Manuel M, Katayama KP, Jones HW Jr. The age of occurrence of gonadal tumors in intersex patients with a Y chromosome. Am J Obstet Gynecol 1976;124:293-300. 32. Barros BA, Maraes SG, Coeli FB, et al. OCT4 iinmunohistochemistry may be necessary to identify the real risk of gonadal tumors in patients with Turner syndrome and Y chromosome sequences. Hum Reprod 2011;26:3450-3455.
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Gonadoblastoma in Patients with UllrichTurner Syndrome G abriela Z e laya , 1* J essica M . L opez M M
a r ta
a r ti , 2
Ro x a n a M
a r in o , 3
M
a r ia
T . G arc ia
de
D a v il a , 2
and
S. G a l l e g o 1
’ Department o f Genetics, Laboratory o f Cytogenetics, Hospital Nacional de Pediatria J.P Garrahan, Combate de los Pozos 1881 (CP 1245) Buenos Aires, Argentina 2Departement of Pathology, Hospital Nacional de Pediatria J. P Garrahan, Combate de los Pozos 1881 (CP 1245) Buenos Aires, Argentina 3Department o f Endocrinology, Laboratory o f Molecular Biology, Hospital Nacional de Pediatria J. P Garrahan, Combate de los Pozos 1881 (CP 1245) Buenos Aires, Argentina
Received August 19, 2014; accepted December 22, 2014; published online December 23, 2014.
ABSTRACT Ullrich-Turner syndrome (UTS) is a common chromo somal abnormality caused by partial or complete X chromosome monosomy. One half of the patients have a 45,X karyotype, whereas the remaining patients display other X chromosome anomalies. In 6% to 11% of UTS, a normal or partly deleted Y chromosome has been found. A 10% to 30% risk of developing gonadoblastoma was found in the latter patients. The aim of this study was to evaluate the prevalence of Y chromosome-derived material, the occurrence of gonadoblastoma, and the incidence of possible neoplasms in patients with UTS. Of 217 patients studied with UTS and chromosome analysis of peripheral-blood lymphocytes, Y chromosome material was found in 20 patients. Fluorescence in situ hybridiza tion (FISH) testing was performed to characterize the structurally abnormal Y chromosome in 13 cases. Molecular analysis of the SRY gene could only be performed in 20 patients with 45,X karyotype. Two patients had the SRY genomes. Of the 20 patients with Y chromosome-derived material, 17 underwent gonadectomy. The incidence of gonadoblastoma development in our series was 35.5%. Furthermore, 1 patient also showed a pure dysgerminoma, and another showed a mixed dysgerminoma and embryonal carcinoma. We emphasize the importance of complete processing of the gonadectomy specimen, including step sections, molecular studies, and FISH, in addition to the classic cytogenetic searching for Y chromosome sequences, in patients who present with a nonmosaic 45,X karyotype. Finally, we propose to routinely collect a sample for storage in the tumor bank for future studies.
* Corresponding author, e-mail: [email protected]
Key words: gonadal dysgenesis, gonadectomy, gonado blastoma, mosaicism, Ullrich-Turner syndrome, Y chromosome
INTRODUCTION The Ullrich-Turner syndrome (UTS) is one of the most common chromosomal abnormalities presenting in humans, with a frequency of 1 in 2500 live-bom females. It is a disorder caused by partial or complete X chromosome monosomy and characterized by short stature, ovarian failure, and specific somatic abnormalities [1,2]. Approx imately one half of patients with UTS have a 45,X karyotype, whereas the remaining patients display a variety of other chromosomal anomalies of the X chromosome, including several mosaicisms [3]. A normal or partly deleted Y chromosome or Y-derived marker chromosome has been found in 6% to 11% of patient with UTS using standard cytogenetic techniques. Occult Y chromosome mosaicism detected by techniques other than standard cytogenetics varies according to study and methodology used [4-6], The early detection of Y-derived sequences in the genome of individuals with UTS is of great importance because of the relatively high risk (10%-30%) of developing gonadal tumors (ie, gonadoblastoma or dysger minoma) [7,8]. A gonadoblastoma is a neoplasm composed of germ cells and sex-cord elements with an excellent prognosis if detected early. However, gonadoblastoma may progress to dysgerminoma with metastatic potential, justifying early gonadectomy [9]. A gonadoblastoma susceptibility locus has been proposed for the pericentromeric region of the Y chromosome [10,11]. The neoplasm does not appear to correlate with the presence of SRY. The aim of this study was to evaluate the prevalence of Y chromosome-derived material, the occurrence of gonadoblastoma, and the incidence of possible neoplasia in patients with UTS.
Table 1.
Cytogenetic and in situ hybridization studies
Patient
Karyotype
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
mos 45,X[15]/46,X, del(Y)(ql 1.2) [5] mos 45,X[30]/46,X,idic(Y)(pl 1.3).ish idic(Y)(p 11.3 )(DYZ l++,SRY++)[ 10] mos 45,X[88]/46,X,+mar.ish idic(Y)(pll.3)(DYZ3++)[6] mos 46, X,i(Y)(p 10) [22]/45,X[8], ish idic(Y)(qll.2(DYZ3++) mos 45,X[17]/46,X,idic(Y)(ql 1.2).ish idic(Y)(ql 1)(SRY++)[13] mos 45,X[47]/46,X,i(Y)(ql0).ish i(Y)(q 10)(wcpY+,SRY—)[3] mos 46,X,+mar [24]/45,X[6].ish i(Y)(pl l.lql 1.1 )(DYZ3+)(SRY++) mos 46,X,idic(Y)(ql 1.2).ish idic(Y)(q 11,2)(SRY++)[19]/45,X[ 11] mos 45,X[39]/46,X,idic(Y)(ql l).ish idic(Y)(ql 1.2)(SRY++,DYZ3++)[5] mos 45,X[26]/ 45,X,dic(Y; 15)(Ypter->Yq 11.2:: 15q 13-> 15q)[7] mos 45,X[30]/46,X,+mar.ish der(Y)(wcpY+)[15] mos 45,X[30]/46,X,i(Y)(pl0).ish idic(Y)(ql l)(SRY++,DYZ3++)[3] mos 46,X,idic(Y)(ql 1.2).ish idic(Y)(q 11,2)(SRY++,DYZ3++)[12]/45,X[8] mos 45,X[14]/46,XY[6] mos 45,X[19]/46,XY[11] mos 45,X[40]/46,XY[34] mos 45,X[7]/46,XY[5] mos 45,X[23]/46,XY[7] mos 45,X[25]/46,XY[4] mos 45,X[9]/46,XY[21]
METHODS
Two hundred seventeen patients with phenotypic UTS were diagnosed between January 1989 and December 2012 .
Chromosome analysis of peripheral blood lympho cytes was performed with GTG banding (G-banding with trypsin and Giemsa) according to standard methods. A minimum of 30 metaphase cells was analyzed in cases with a nonmosaic 45,X karyotype or a minimum of 20 metaphase cells when mosaicism was identified within the first 20 cells. Fluorescence in situ hybridization (FISH) analysis using commercially available centromeric probes (DXZ1/ DYZ3), SRY, and whole Y chromosome painting (WCP Y) (Vysis, Inc, Abbott Molecular, Downers Grove, IL, USA) was performed in peripheral blood samples obtained from the patients following the manufacturer’s protocol. Genomic DNA was isolated from peripheral-blood lymphocytes according to standard protocols. To identify the presence of Y chromosome in 20 patients with 45,X karyotype, a 472-base pair (bp) fragment of SRY, including the DNA-binding motif, was amplified by duplex polymerase chain reaction (PCR) using primers 118
G. Z elaya et
al.
specific for the SRY gene and SMA gene (exon 7), previously reported [12,13], as an internal control for amplification (226-bp fragment). Moreover, 2 fragments of the amelogenin gene (AMELX and AMELY), a 800-bp fragment located on chromosome Y and a 997-bp fragment located on chromosome X, were amplified by PCR using the same primers [14], After gonadectomy, right and left gonadal tissues were fixed in formalin buffer and processed in paraffin with 2 to 4 step sections of approximately 3 pm from each block and stained with hematoxylin and eosin (H&E). Interphase FISH assay was performed using the centromeric probes (DXZ1/DYZ3). Signals were scored in at least 200 nonoverlapping intact nuclei. The ratio of Y:X signals was calculated and was used to express the frequency of Y-positive cells. RESULTS
The karyotype distribution among 217 patients was 45,X (44.2%, n = 96), X structural abnormalities (39.6%, n = 86), Y structural abnormalities (6%, n = 13), 45,X/46,XX (5.1%, n = 11), 45,X/46,XY (3.2%, n = 7), 45,X/46,XX/ 47,XXX (1.4%, n = 3), 45,X/47,XXX (0.5%, n = 1). The FISH testing was performed to characterize the structurally abnormal Y chromosome in 13 cases (Table l).We also investigated the presence of 2 Ypspecific sequences (SRY and AMELY) and 1 Xp-specific sequence (AMELX) by PCR in 20 patients with UTS and a 45,X karyotype. As expected, all patients had a positive amplification for the Xp-specific sequence AMELX. Interestingly, 2 of the patients had the Y SRY and AMELY sequences in their genomes. Gonadectomy was performed in 17 patients with ages ranging from 2 to 18 years (mean, 11.5 years). We studied the histologic characteristics in all specimens of dysgenetic gonads, especially in those that had undergone neoplastic transformation, and compared those findings with the features of the gonadal tissue adjacent to and within the gonadoblastoma samples. We found 2 main patterns of dysgenetic gonads. The first pattern, considered the classic streak gonad pattern, consisted of whorls and bundles of fibrous connective tissue, ovarian-like stroma devoid of germ cells, with isolated inclusion cysts lined by cuboidal epithelium. This pattern was found in 9 cases (patients 1-3, 8, and 10-14). In 1 case (patient 1) only, it was associated with gonadoblastoma and dysgerminoma. (Fig. 1 and Table 2) The second pattern, with epithelial cord-like struc tures, consisted of ovarian, cortex-like stroma with epithelial cord-like structures occasionally forming an anastomosing network. These structures contained 2 types of cells: small cells with hyperchromatic nuclei and germ cells. This pattern was found in 7 cases (patients 4-7, 9, 15, and 17). In 3 cases, it was associated with gonadoblastoma, and in 1 case, it was associated with gonadoblastoma and a malignant mixed germ cell tumor (patient 7).
Figure 1. P atient 15. Streak g onad w ith small foci o f g o n ad ob lasto m a. A. H em atoxylin and eosin (H&E), X20. B. H&E, X40.
In an attempt to assess dysgenetic gonads, we performed additional step sections and found microscopic foci of gonadoblastoma in 2 additional cases (patients 5 and 6). In patient 8, the gonadal tissue was a streak testis. Six patients (35.3%) showed bilateral gonadoblas toma. In 4 of these cases, the tumor consisted of microscopic foci that could be detected by step section ing. In addition to gonadoblastoma, dysgerminoma was detected in the other 2 cases. Interestingly, in 1 of these cases, the tumor coexisted, in the right gonad, with an embryonic carcinoma. Macroscopically, the tumor had a maximum diameter of 5 cm (Fig. 2). Interphase FISH analysis performed in gonadal tissue showed Y chromosomal material in all cases, coinciding with the cytogenetic findings in peripheral lymphocytes. DISCUSSION
The detection of Y chromosome mosaicism is clinically important because of the risk of tumorigenesis, especially gonadoblastoma [15], or other nontumoral androgenproducing lesions [16] in the dysgenetic gonads of patients with UTS and a normal, structurally abnormal, or hidden Y chromosome. The gonadoblastoma is a benign neoplasm composed of undifferentiated cells similar to those seen in gonadal
development admixed with sex-cord derivatives. It was first described by Scully in 1953 [17]. Although gonadoblastoma is a benign tumor, in 60% of cases, it can turn into an invasive dysgerminoma and may also progress to other forms of malignant germ-cell tumors [18]. This could be the result of a disturbance in the germ cell maturation. Therefore, early prophylactic gonadectomy is indicated in all cases when the association with Y chromosome sequences is confirmed. Page [19] postulated the GBY locus as an oncogene in the dysgenetic gonads. Five genes have been described in this region. The TSPY (testis-specific protein Yencoded) gene might be involved in the initial selection of tumorigenic cells, possibly by having a role in cellcycle regulation or cell division, even though its function is still unclear [20], Octamer-binding transcription factor 4 (OCT4) (also know OCT3 or POU5FI; OMIM 164177) is a transcription factor essential to the maintenance of pluripotentiality of embryonic stem cells and primordial germ cells [21,22]. It is considered a useful marker of germ cell tumor, such as gonadoblastoma, dysgerminoma, seminoma, and others. Expression of OCT4 is detected by immunohistochemistry in 100% of cases of gonadoblas toma [23], In our series, the prevalence of mosaicism with a normal or abnormal Y chromosome was 9.2% detected by conventional cytogenetics. We investigated the presence of Y chromosome sequences by PCR in only 20 patients with UTS and a 45,X karyotype and found 2 patients with Yp-specific sequences (10%). Seventeen of these patients (85%) were gonadectomized, and gonadoblastoma was found in 6 (35.3%). According to the classification of streak gonads [24-26], in our series, we found 9 cases of classic streak gonads, 7 of streak gonads with epithelial cord-like structures, and 1 streak testis. In the first group, we found an association between gonadoblastoma and dysgermino ma in 1 case, and in the second group, we found 5 gonadoblastomas and the coexistence in 1 of these cases of a dysgerminoma and an embryonal carcinoma. Based on these findings, we hypothesize that gonadoblastoma could originate from surviving germ cells in areas of streak gonadal tissue, mainly present in the cord-like structures within the dysgenetic gonad. In the presence of streak gonads, it is important to perform immunostain studies to detect germ cells to rule out neoplastic foci at an early stage. Complete processing included step sectioning of the gonadectomy specimen, is recommended by the Turner Syndrome Society of the United States [27], It allowed for the detection of small neoplastic foci in 2 patients. Gonadoblastoma was associated with dysgerminoma in 2 cases, progressing to embryonal carcinoma in 1 case (patient 7). In that patient, metastasis was found in the lungs and the omentum at the time of diagnosis, and the prognosis for that patient was poor. On the other hand, it has been reported that gonadoblastoma are thought to arise prenatally [28-30] G onadoblastoma in P atients
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Table 2. Patient
Histologic findings of resected gonadal tissue A ge (years)
R ight gonad
Left gonad
1
15
2 3 4 5
18 9 14
C lassic streak gonad G B L dysgerm inom a C lassic streak gonad C lassic streak gonad Streak w ith epithelial cord-like structures Streak w ith epithelial cord-like structures GBL
6
2
7
14
8 9 10 11 12
16 13 10 2 18
13 14 15
17 6 17
16 17
7 4
C lassic streak gonad G BL C lassic streak gonad C lassic streak gonad Streak w ith epithelial Streak w ith epithelial G BL Streak w ith epithelial G BL Streak w ith epithelial GBL Streak testis Streak w ith epithelial C lassic streak gonad C lassic streak gonad C lassic streak gonad C lassic streak gonad C lassic streak gonad Streak w ith epithelial G BL C lassic streak gonad Streak w ith epithelial G BL
14
Streak w ith epithelial cord-like structures GBL Streak w ith epithelial cord-like structures G BL dysgerm inom a em bryonal carcinom a Streak testis Streak w ith epithelial cord-like structures C lassic streak gonad C lassic streak gonad C lassic streak gonad C lassic streak gonad C lassic streak gonad Streak w ith epithelial cord-like structures GBL C lassic streak gonad Streak w ith epithelial cord-like structures GBL
cord-like structures cord-like structures cord-like structures cord-like structures
cord-like structures
cord-like structures
cord-like structures
GBL indicates gonadoblastoma.
and that the risk of tumor increases after puberty. Although the natural history of gonadoblastoma at the prepubertal age has not been determined, our data suggest that the age at appearance and the possibility of malignant degeneration may be in early life. In our study, the incidence of Y-positive nuclei in gonadal tissue correlated with that found in blood samples. The finding of a gonadoblastoma in dysgenetic
gonadal tissue of patients with UTS and Y-chromosome material confirms the usefulness of genetic techniques in the identification of risk of tumor development. Some authors suggest that the development of gonadoblastoma with and without malignant germ-cell tumors would be a multistep and multifactorial process. The aberrant expression of TSPYis likely to predispose to dysgenetic gonads and to other oncogenic events in the
Figure 2. P atient 7. A. R igh t g onad w ith a solid and heterogeneous tu m o r m easuring 8 cm. B. G onadoblastom a (h e m a to xylin and eosin [H&E], X40). C. D ysgerm inom a and em b ryo na l carcinom a (H&E, X10). D. D ysgerm inom a (H&E, X20). E. Em bryonal carcinom a (H&E, x 4 0 ). F. Im m u n o sta in in g w ith CD30 in th e e m bryonal carcinom a c o m p o n e n t (X20).
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malignant transition of a gonadoblastoma to a dysgerminoma, strongly suggesting the need for genetic screening for TSPY in patients with UTS. Moreover, Barros and colleagues [31] discuss the importance of an immunohistochemical study with OCT4 in all gonads of patients with UTS, in addition to the histopathologic analysis, to establish the risk of tumor development. Considering the need of material for future studies, it is important to establish a protocol for the routine storage of a mirror section of the gonadal tissue. In conclusion, we found a 35.3% incidence of gonadoblastoma development in patients with UTS and Y chromosome-derived material in their karyotype. Based on the finding of microscopic tumor foci, we emphasize complete processing of the gonadectomy specimen, including step sections. We point out the importance of performing molec ular, classic cytogenetics, and FISH studies searching for Y chromosome sequences in patients who present with a nonmosaic 45,X karyotype. Furthermore, the incorpora tion of the analysis of the genes implicated in the development of gonadoblastoma will be useful in the evaluation of these patients. Finally, we propose to routinely collect a sample for storage in the tumor bank for future studies. REFERENCES 1. Zinn AR, Page DC, Fisher EM. Turner syndrome: the ease of the missing sex chromosome. Trends Genet 1993;9:90-93. 2. Lopez M, Canto P, Aguinaga M. Frequency of Y chromosomal material in Mexican patients with Ullrich-Tumer syndrome. Am J Med Genet 1998;76:120-124. 3. Mathur A, Stekol L, Schatz D, MacLaren NK, Scott ML, Lippe B. The parental origen of the single X chromosome in Turner syndrome: lack of correlation with parental age or clinical phenotype. Am J Hum Genet 1991;48:682-686. 4. Jacobs P, Dalton P, James R, et al. Turner syndrome: a cytogenetic and molecular study. Ann Hum Genet 1997:61:471^183. 5. Kokova M, Siegel SF, Wenger SL, Lee PA, Trucco M. Detection of Y chromosome sequences in Turner’s syndrome by Southern blot analysis of amplified DNA. Lancet 1993;342:140-143. 6. Wiktor AE, Van Dyke DL. Detection of low level sex chromosome mosaicism in Ullrich-Tumer syndrome patients. Am J Med Genet A 2005;138A:259-261. 7. Verp MS, Simpson JL. Abnormal sexual differentiation and neoplasia. Cancer Genet Cytogenet 1987;25:191-218. 8. Gravholt CH, Fedder J, Naeraa RW, Muller J. Occurrence of gonadoblastoma in females with Turner syndrome and Y chromo some material: a population study. J Clin Endocrinol Metab 2000; 85:3199-3202. 9. Cools M, Stoop H, Kersemaekers AM, et al. Gonadoblastoma arising in undifferentiated gonadal tissue within dysgenetic gonads. J Clin Endocrinol Metab 2006;91:2404-2413. 10. Tsuchiya K, Reijo R, Page DC, Disteche CM. Gonadoblastoma: molecular definition of the susceptibility region on the Y chromosome. Am J Hum Genet 1995;57:1400-1407. 11. Lau YF. Gonadoblastoma, testicular and prostate cancers, and the TPSY gene. Am J Hum Genet 1999;64:921-927.
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