0023-6837/02/8202-159$03.00/0 LABORATORY INVESTIGATION Vol. 82, No. 2, p. 159, 2002 Copyright © 2002 by The United States and Canadian Academy of Pathology, Inc. Printed in U.S.A. VASA Is a Specific Marker for Both Normal and Malignant Human Germ Cells Anne-Marie Zeeman, Hans Stoop, Marjan Boter, Ad J. M. Gillis, Diego H. Castrillon, J. Wolter Oosterhuis, and Leendert H. J. Looijenga Department of Pathology/Laboratory for Experimental Patho-Oncology (A-MZ, HS, MB, AJMG, JWO, LHJL), University Hospital of Rotterdam/Daniel, Josephine Nefkens Institute, Erasmus University Rotterdam, The Nether- lands; and Women’s and Perinatal Pathology Division, Department of Pathology (DHC), Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts SUMMARY: VASA is so far the only known gene in mammals whose expression is specific for the germ cell lineage. We investigated the presence of VASA mRNA and protein in a series of germ cell tumors of different histologic subtypes and anatomic location, as well as in nongerm cell tumors such as testicular lymphomas and Leydig cell tumors. We detected VASA mRNA (by quantitative RT-PCR) and protein (by immunohistochemical staining) in normal spermatogenesis, seminoma (both classic and spermatocytic), carcinoma in situ (the precursor of classic seminoma and nonseminoma), dysgerminoma, and gonadoblastoma. VASA immunostaining was relatively weak in seminomas and dysgerminomas compared with spermatocytic seminomas, despite similar mRNA levels, suggesting that VASA is regulated in part by post-transcriptional mechanisms. A higher staining intensity compared with the invasive counterparts was observed in the precursor lesions (ie, carcinoma in situ and gonadoblastoma). No VASA mRNA or protein was detectable in nonseminomatous germ cell tumors (such as embryonal carcinoma, teratoma, and yolk sac tumor) and derived cell lines, or nongerm cell tumors such as lymphoma or Leydig cell tumor. These results provide direct evidence that some germ cell tumors retain germ cell characteristics, whereas other tumors of germ cell origin result from differentiation and loss of germ cell identity. Furthermore, these findings suggest that VASA is likely to serve as a useful and highly specific biomarker for germ cell tumors, particularly classic and spermatocytic seminoma/dysgerminoma, including their precursor stages. (Lab Invest 2002, 82:159–166). he evolutionary highly conserved VASA genes of germ cells. The mouse homolog of VASA, known as T are specifically expressed in the germ cell lin- mvh (mouse Vasa homolog) was recently identified. Its eage, both in vertebrates and invertebrates (for review, expression is also restricted to the germ cell lineage see Raz, 2000). Members of this family have been (Tanaka et al, 2000). Although male knock-out mice for identified in C. elegans, Drosophila, Xenopus, ze- mvh form primordial germ cells, they subsequently brafish, chicken, rainbow trout, rat, and mouse. The develop testicular atrophy because of abnormal pro- Drosophila vasa gene encodes a DEAD-box protein liferation and colonization of the primordial germ cells, with ATP-dependent RNA-helicase activity (Hay et al, as well as death of zygotene spermatocytes, and are 1988; Lasko and Ashburner 1988). The helicase activ- infertile. ity is required for the translation of at least two mRNAs The human VASA gene was recently cloned. VASA involved in germ cell migration and development, ie, mRNA and protein are abundantly and specifically nanos (Forbes and Lehmann 1998; Gavis et al, 1996; expressed in germ cells in both sexes throughout Kobayashi et al, 1996) and gurken (Gonzalez-Reyes et development. In contrast to mice, where the protein is al, 1995; Neuman-Silberberg and Schupbach 1993; induced after the primordial germ cells reach the Roth et al, 1995; Styhler et al, 1998; Tomancak et al, genital ridge, immunohistochemistry demonstrated 1998). VASA is present in the developing oocyte and is that in humans, the VASA protein is already present in part of the germ plasm, a poorly understood ribonu- migrating primordial germ cells. During normal sper- cleoprotein complex crucial for germ cell determina- matogenesis, VASA staining is relatively weak to in- tion. In Drosophila, vasa is essential for the formation termediate in spermatogonia, strong in spermato- cytes/spermatids, and absent in spermatozoa (Castrillon et al, 2000). In the human adult testis, two Received September 24, 2001. biologically and clinically distinct types of germ cell- This research was supported by the Dutch Cancer Society (KWF-DDHK derived tumors are recognized. These are distin- 99-1968). guished by epidemiologic characteristics, histology, Address reprint requests to: Dr. L. H. J. Looijenga, Department of Pathol- clinical behavior, and chromosomal constitution (Looi- ogy/Laboratory for Experimental Patho-Oncology, University Hospital Rot- terdam/Daniel, Josephine Nefkens Institute, Erasmus University Rotter- jenga and Oosterhuis, 1999 for review). The first dam, Building Be, Room 430b, PO Box 1738, 3000 DR Rotterdam, The comprises the seminomas and nonseminomas, here Netherlands. E-mail: [email protected] referred to as testicular germ cell tumors of adoles- Laboratory Investigation • February 2002 • Volume 82 • Number 2 159 Zeeman et al cents and adults (TGCT), and represent the most Results common neoplasms in young Caucasian males (Adami et al, 1994; Pottern et al, 1998; Swerdlow Immunohistochemistry for VASA and GCAP/PLAP 1993). Although it is most likely that the initiating event To study the presence of VASA protein in different in the development of TGCT occurs during embryonic human tumors, we performed immunohistochemistry development (Jørgensen et al, 1995; Møller, 1989, for on formalin-fixed paraffin-embedded tissue sections review), affecting a primordial germ cell, leading to using previously described polyclonal antibodies carcinoma in situ (CIS) (Skakkebæk 1972), also known (Castrillon et al, 2000). The tested samples include as intratubular germ cell neoplasm (ITGCN), the tu- gonadoblastoma (n ϭ 5), dysgerminoma (n ϭ 5), mors only become clinically manifest after puberty. normal testicular parenchyma (n ϭ 5) (containing CIS cells, like primordial germ cells, are characterized spermatogenesis), primary testicular lymphomas (n ϭ by a high glycogen content (Mostofi and Sesterhenn 3), Leydig cell tumors (n ϭ 3), testicular parenchyma 1998), membranous staining for the stem cell factor containing different amounts of CIS-cells and Sertoli receptor c-KIT (Rajpert-De Meyts and Skakkebæk only tubules (n ϭ 14), seminomas (n ϭ 13), nonsemi- 1994), and germ cell/placental alkaline phosphatase nomas (n ϭ 12), and spermatocytic seminomas (n ϭ (GCAP/PLAP) (Roelofs et al, 1999, for review). CIS 4). The results are summarized in Table 1. Represen- cells can progress to seminoma, an invasive tumor in tative examples are shown in Figure 1. which the malignant cells still show, like CIS cells, Relatively weak staining for VASA was found in morphologic and immunohistochemical characteris- spermatogonia, whereas stronger staining was de- tics of primordial germ cells. CIS cells can also tected in later developmental stages. Spermatozoa progress to nonseminomas, tumors of germ cell origin were negative (Fig. 1A). These results are in accor- that have lost their primordial germ cell characteris- dance with earlier data (Castrillon et al, 2000). CIS/ tics, and recapitulate embryonic development (Looi- seminoma and gonadoblastoma/dysgerminoma cells jenga et al, 1997, for review). Nonseminomatous germ were identified by their positive staining for GCAP/ cell tumors exhibit morphologic and immunohisto- PLAP (see Fig. 1, B and C). Double immunohisto- chemical evidence of differentiation into various so- chemistry for GCAP/PLAP and VASA demonstrated matic (teratoma) and extraembryonic lineages (yolk that all of these malignant cells are positive for VASA. sac tumor and choriocarcinoma). In addition, embry- Although the overall intensity of the staining was in the onal carcinoma can be found. Each of these histologic same order as found for spermatogonia, the preinva- subtypes can be found in a pure form or in combina- sive stage (ie, gonadoblastoma and CIS), showed a tions with other types (Mostofi and Sesterhenn 1998). more intense staining compared with their invasive Seminomas and nonseminomas can also be found in counterparts (ie, dysgerminoma and seminoma; see the ovary and dysgenetic gonads (Oosterhuis et al, 1993, for review). The ovarian counterpart of semi- noma is known as dysgerminoma and is histologically Table 1. Summary of the Results of indistinguishable from seminoma. The CIS-like lesion Immunohistochemistry for VASA on Germ Cell Tumors of that arises in dysgenetic gonads is referred to as Different Histology and Anatomical Localization, as well gonadoblastoma (Scully 1970; Jørgensen et al, 1997, as Nongerm Cell Tumors and Normal Testicular Parenchyma for review). The second category of testicular tumors derived Histology Cases tested Staininga from the germ cell lineage are the spermatocytic seminomas. These tumors develop usually at a rela- Testicular parenchyma 19 Ϫ tively advanced age, rarely metastasize, and are as- Leydig cells Ϫ sumed to be derived from a later developmental stage Sertoli cells Ϫ of spermatogenesis (spermatocytes) than the precur- Stromal cells ϩϩ sor cells of TGCT (Burke and Mostofi, 1993; Eble, Spermatogonia ϩϩϩ 1994; Muller et al, 1987; Romanenko and Persidskii, Spermatocytes Spermatids ϩϩϩ 1983;