TRAPDOORS IN TESTICULAR PATHOLOGY

Transcription

1 TRAPDOORS IN TESTICULAR PATHOLOGY Daniel Berney, M.B.B.S. FRCPath Consultant Histopathologist Department of Cellular Pathology The Orchid Tissue Laboratory, Barts and The London NHS Trust The Royal London Hospital Whitechapel, London E1 2ES, UK. Muhammad T. Idrees, M.D. Department of Pathology and Laboratory Medicine Indiana University School of Medicine IU Health Pathology Laboratory 350 West 11th Street, 4th Floor Indianapolis, IN

2 Introduction We present to you this morning ten trapdoors in testicular pathology. The unrivalled complexity of testicular neoplasms makes it an area where it is all too easy to be guided by a pathological pattern that mimics another or by variable immunochemistry. As well as this, the rarity of some entities means that many pathologists will encounter some entities only once or twice in their lifetime. The cases we have chosen are all from our diagnostic practice from the last two years, or from consult cases. We do not attempt to be comprehensive: indeed this would be impossible in a half-day session, but we have attempted to demonstrate cases that are not merely of academic interest, but illustrate variations that have prognostic or diagnostic import, or may lead to misdiagnosis.

3 CASE 1: Speaker: Daniel Berney A 24-year-old man presented to his GP complaining of a lump in the right testis. Examination showed a 4 cm mass. Ultrasound confirmed this was a suspicious lump and tumor markers were not raised. He underwent a right orchiectomy. Macroscopy revealed a cream and mottled 4 cm mass in the testicular parenchyma. Images from this follow. Diagnosis: Classical seminoma, tubular variant. Trapdoors in Seminoma Seminoma may be both the easiest and most difficult of diagnoses in testicular pathology. It is the most common tumor of the testis and, therefore, might be thought to be the most familiar. The classic appearances of seminoma are well known. Broad fibrous bands and an associated lymphoplasmacytic infiltrate are readily identified at low power. At higher power, the cytology is characteristic with pleomorphic cells possessing clear cytoplasm, well-defined cellular boundaries, and very characteristic nuclei with an almost polygonal appearance. The large nucleoli are frequently rather ill defined. Outside this classic appearance, however, there are many variants which may be misdiagnosed as nonseminoma, or even nonneoplastic conditions. The Inflammatory Infiltrate is Variable. The extreme variation that may be seen in seminoma is a cause for diagnostic misinterpretation. Although rare, occasional seminomas may lack a lymphoplasmacytic infiltrate, leading to diagnostic confusion. However a more common problem is when the inflammatory infiltrate overwhelms the tumor cells, so that rare seminoma cells are studded in a dense inflammatory infiltrate. The pale cytoplasm of the seminoma cells may make the neoplastic cells stand out like stars on a winter s night, in a similar way that macrophages stand out in a lymphoblastic lymphoma; so, in an inflammatory testicular lesion, it is important to examine these areas with care. Granulomatous inflammation may also be present and overwhelm the tumor. These cases should be treated with caution and a careful hunt made for typical seminoma cells. Tuberculosis, sarcoidosis, leprosy, and other even rarer granulomatous infections may affect the testis, but ruling out seminoma is vital. Therefore, in all inflammatory testes, the question should be asked Am I missing a seminoma? Intertubular Seminoma. Seminoma cells usually form a solid sheet and displace the seminiferous tubules. However, the cells may show an intertubular pattern of spread where single cells infiltrate in an insidious pattern between the seminiferous tubules. Thus, the overall architecture of the testis is preserved, and at low power, no neoplasm may be identified. This pattern is usually mixed with more solid appearing seminoma but rarely in early stage disease, where there is plentiful IGCNU and it may be missed. Clues towards the presence of intertubular seminoma include an association with inflammation in the stroma and a rather more plentiful parenchyma between the seminiferous tubules.

4 Morphological variants of seminoma cells themselves have been described relatively recently. Occasionally a seminoma may mimic a nonseminoma with a tubular morphology and mimic nonseminomatous components, especially the pseudoglandular components of embryonal carcinoma. Cytology will usually differentiate between the two as the cells retain the classic cytomorphology of seminoma, rather than yolk sac tumor or embryonal carcinoma. In extreme difficulty, immunohistochemistry for CD30 (negative in seminoma, positive in embryonal carcinoma) and CD117 (positive in seminoma, negative in embryonal carcinoma) is helpful. A signet ring morphology in seminoma has also been described. This may mimic a metastasis, but the foci of signet ring morphology have always been mixed with more typical areas, making this a less challenging diagnosis. Seminoma with Trophoblast. Some seminomas will have scattered syncytiotrophoblastic cells. They will usually be associated with a raised serum betahcg. Although mentioned as a variant of seminoma in the WHO classification, unfortunately the diagnostic criteria are not well defined. Many seminomas will have occasional syncytiotrophoblastic cells if searched for, and more will be found on immunohistochemistry. My practice is to check the serum hcg clinically if at all possible (and I believe that knowledge of the serum markers is crucial in the diagnosis of testicular tumors). The diagnosis of seminoma with choriocarcinoma should only be made with caution as it has immense implications in terms of therapy and prognosis. In these rare cases, the syncytiotrophoblastic cells should be creating masses with cytotrophoblast and not admixed with seminoma cells. These cases will also usually show hemorrhage and necrosis. Anaplastic Seminoma Anaplastic seminoma is a term we do not recommend. Occasionally, seminomas have been shown to present unusual features, in terms of nuclear morphology, mitotic rate, or inflammatory infiltrate. The excellent prognosis of seminoma, however, means that a study involving many thousands of patients and standardized care would be necessary to identify, with certainty, such an entity and to prove it was viable and standardized. Mimics of Seminoma Seminoma can be mimicked by other tumors, notably by malignant Sertoli cell tumors which are described elsewhere. Metastases can also be deceptive, particularly renal or prostate. The solid variant of yolk sac tumor may also strongly resemble seminoma. Its more monomorphic appearance as well as the presence of hyaline globules will help morphologically. Again the knowledge of serum markers will be helpful, with a raised alpha fetoprotein being highly suspicious. Immunohistochemistry can be of great help here: cytokeratin positivity as well as glypican 3 positivity and OCT3/4 negativity are indicative of yolk sac tumor. AFP may be less sensitive. Treatment of Seminomas Seminomas are extremely sensitive to radiation and chemotherapy, and these modalities usually represent the primary forms of treatment following orchiectomy, although there is considerable interest in surveillance management of clinical stage I patients. There are many ongoing surveillance protocols for seminoma with varying imaging schedules. As even relapsed seminoma has such a favorable prognosis, the strategy of active surveillance is now established in many countries. Many patients

5 with clinical stage I seminoma receive radiation directed at the ipsilateral inguinal and iliac lymph nodes. Cure rates exceeding 98% can be expected for these patients. Recurrence is almost always outside of the radiated field. In many countries adjuvant carboplatin has replaced radiation for adjuvant treatment. Although it appears equivalent, the long term outcome has not been established. Prognostic Factors As treatment choices are available, it would appear that tumor characteristics might help guide appropriate therapy. Unfortunately, as the overall survival is so favorable, the dissection of histological prognostic factors has proved fraught. The largest series have shown that maximum tumor diameter is important. Some studies have emphasized rete testis invasion, but often do not comment on whether it is of pagetoid or interstitial type. Vascular invasion is more difficult to identify in seminomas from contamination and is not validated in all studies. References 1. Ulbright TM, Young RH. Seminoma with tubular, microcystic, and related patterns: a study of 28 cases of unusual morphologic variants that often cause confusion with yolk sac tumor. Am J Surg Pathol 2005;29: Young RH, Finlayson N, Scully RE. Tubular seminoma. Report of a case. Arch Pathol Lab Med 1989;113: Zavala-Pompa A, Ro JY, El-Naggar AK, et al. Tubular seminoma: an immunohistochemical and DNA flow cytometric study of four cases. Am J Clin Pathol 1994;102: Ulbright TM, Young RH. Seminoma with conspicuous signet ring cells: a rare, previously uncharacterized morphologic variant. Am J Surg Pathol 2008;32: Henley JD, Young RH, Wade CL, et al. Intertubular pattern of growth in seminoma: a report of 12 clinically and grossly inconspicuous tumors. Am J Surg Pathol 2004;28: Oliver RT, Mason MD, Mead GM et al. Radiotherapy versus single-dose carboplatin in adjuvant treatment of stage I seminoma: a randomised trial. Lancet 2005;366; International Germ Cell Consensus Classification: a prognostic factor-based staging system for metastatic germ cell cancers. International Germ Cell Cancer Collaborative Group. J Clin Oncol 1997;15; Wittekind LHSMKGC. TNM Classification of Malignant Tumors 7th Edition. New York: Wiley-Blackwell, Rajab R, Berney DM. Ten testicular trapdoors. Histopathology 2008;53; Ulbright TM. The most common, clinically significant misdiagnoses in testicular tumor pathology, and how to avoid them. Adv Anat Pathol 2008;15;18-27.

6 10. Nazeer T, Ro JY, Kee KH, Ayala AG. Spermatic cord contamination in testicular cancer. Mod Pathol 1996;9; Warde P, Specht L, Horwich A et al. Prognostic factors for relapse in stage I seminoma managed by surveillance: a pooled analysis. J Clin Oncol 2002;20; Thomas GM. Surveillance in stage I seminoma of the testis. Urol Clin North Am 1993;20; Groll RJ, Warde P, Jewett MA. A comprehensive systematic review of testicular germ cell tumor surveillance. Crit Rev Oncol Hematol 2007;64; Mostofi FK. Proceedings: Testicular tumors. Epidemiologic, etiologic, and pathologic features. Cancer 1973;32; Zuckman MH, Williams G, Levin HS. Mitosis counting in seminoma: an exercise of questionable significance. Hum Pathol 1988;19; Tickoo SK, Hutchinson B, Bacik J et al. Testicular seminoma: a clinicopathologic and immunohistochemical study of 105 cases with special reference to seminomas with atypical features. Int J Surg Pathol 2002;10; Ruf CG, Linbecker M, Port M et al. Predicting metastasized seminoma using gene expression. BJU Int;110;E Hori K, Uematsu K, Yasoshima H, Sakurai K, Yamada A. Contribution of cell proliferative activity to malignancy potential in testicular seminoma. Pathol Int 1997;47; Parker C, Milosevic M, Panzarella T et al. The prognostic significance of the tumor infiltrating lymphocyte count in stage I testicular seminoma managed by surveillance. Eur J Cancer 2002;38;

7 CASE 2: Speaker: Daniel Berney A 17-year-old presented to a urologist with a 6 month history of a lump in the testis. Tumor markers were within normal limits. Ultrasound showed a cystic mass, and he underwent a partial orchiectomy. Diagnosis: Benign teratoma of the testis. The biology of teratomas has undergone considerable revision in the past 10 years. This has led to some diagnostic and treatment conundrums which continue to cause debate in the testicular pathology community. Pure teratomas of the testis are unusual. Most are intermixed with other tumor elements. In spite of their composition from mature tissues, they are capable of metastasis. Almost inevitably they are associated with intratubular germ cell neoplasia, unclassified (IGCNU). However, the lesion presented in this meeting has a number of features which suggest it will have an entirely benign outcome. No IGCNU is present in the lesion, there is no evidence of immaturity, and there is no scarring or evidence of regression in the background testis. Taken together these features suggest that this is not a teratoma, but a dermoid cyst A number of entities have been previously described in the testis, related to teratomas, which appear to have a benign natural history. Epidermoid Cyst. So called epidermoid cysts of the testis are composed of laminated keratin, usually in a circumscribed nodule. They are usually identified easily, macroscopically resembling epidermal cysts in the skin. Occasionally, the cyst may have ruptured leading to a less well-circumscribed appearance. Microscopically, they have a typical appearance of concentric laminated keratin surrounded by a simple keratinizing epithelium, though giant cell reactions may be seen if the cyst has ruptured. Systematic blocking is necessary to exclude other germ cell elements. The background testis tissue shows normal spermatogenesis and no IGCNU. They may be treated conservatively with little followup. Dermoid Cyst. Similar to epidermoid cysts, dermoid cysts within the testis are rare but benign entities of unknown pathogenesis where there are skin appendigeal elements present as well as the epidermoid components of an epidermal cyst. However, it is vital to distinguish these benign tumors from teratoma, which is capable of metastasis. Dermoid cysts are not associated with surrounding IGCNU, which is almost inevitably present around a teratoma capable of malignant spread. As mentioned above, other features indicative of a malignant lesion are atrophic features with regressive changes in the stroma. Dermoid cysts are usually associated with normal adjacent spermatogenesis. The hallmarks of these lesions are those of an epidermis-like lining containing pilosebaceous units. These tumors are usually diploid and homozygous whereas mature teratomas show complex chromosomal abnormalities and maybe hypodiploid or hyperdiploid.

8 Benign Teratomas. Benign teratomas are not characterized in the WHO classification from However, a small subset of teratomas has been described in the literature which may show other mixed mature elements including bone cartilage or even neural tissue. They also lack the typical features seen in potentially malignant lesions: IGCNU, immaturity, and regressive features. Further evidence to differentiate these entities comes from recent i12p data, showing that both dermoid cysts and benign teratomas lack the typical i12p chromosomal abnormalities seen in teratomas capable of metastasis. They also occur in a younger age group than other nonseminomatous germ cell tumors and it has been suggested that they are in fact tumors which have persisted from prepubertal teratomas, which are known to have an indolent clinical behavior. Practically, it is difficult to know whether these tumors can be spared the repeated imaging studies of a more typical testicular teratoma. But certainly followup can be conservative and reassurance of the patient seems reasonable of the available data. References 1. Shah KH, Maxted WC, Chun B. Epidermoid cysts of the testis: a report of three cases and an analysis of 141 cases from the world literature. Cancer 1981;47: Price EBJr. Epidermoid cysts of the testis: a clinical and pathologic analysis of 69 cases from the testicular tumor registry. J Urol 1969;102: Burt AD, Cooper G, MacKay C, et al. Dermoid cyst of the testis. Scott Med J 1987;32: Dockerty MB, Priestly JT. Dermoid cysts of the testis. J Urol 1942;48: Ulbright TM, Srigley JR. Dermoid cyst of the testis: a study of five postpubertal cases, including a pilomatrixoma-like variant, with evidence supporting its separate classification from mature testicular teratoma. Am J Surg Pathol 2001;25: Kooijman CD. Immature teratomas in children. Histopathology 1988;12: Leibovitch I, Foster RS, Ulbright TM, et al. Adult primary pure teratoma of the testis. The Indiana experience. Cancer 1995;75: Simmonds PD, Lee AH, Theaker JM, et al. Primary pure teratoma of the testis. J Urol 1996;155: Young RH, Talerman A. Testicular tumors other than germ cell tumors. Semin Diagn Pathol 1987;4: Dieckmann KP, Loy V. Epidermoid cyst of the testis: a review of clinical and histogenetic considerations. Br J Urol 1994;73: Kendall TJ, Featherstone JM, Mead GM, et al. Adult testicular dermoid tumors - mature teratoma or pre-pubertal teratoma? Int Urol Nephrol 2006;38: Zhang C, Berney DM, Hirsch MS, Cheng L, Ulbright TM.Am J Surg Pathol Jun;37(6): Evidence supporting the existence of benign teratomas of the

9 postpubertal testis: a clinical, histopathologic, and molecular genetic analysis of 25 cases. 13. Ramsay AK, Gurun M, Berney DM, Nairn R.Dermoid cyst of the testis with neural tissue in an adult.urology Feb;79(2):e Wang WP, Guo C, Berney DM, Ulbright TM, Hansel DE, Shen R, Ali T, Epstein JI. 15. Primary carcinoid tumors of the testis: a clinicopathologic study of 29 cases. Am J Surg Pathol Apr;34(4):

10 Case 3 A 24-years-old male presented with a bulky testicular tumor (7.4 cm). An orchiectomy was performed histologically displaying yolk sac 60%, teratoma 25%, embryonal carcinoma 12%, and choriocarcinoma 3%. Standard platinum-based chemotherapy was performed. Additionally, postchemotherapy retroperitoneal lymph node dissection (RPLND) was performed. The RPLND sections showed viable tumor as depicted in the virtual slide. Final Diagnosis: Cystic Trophoblastic Tumor and Teratoma Cystic trophoblastic tumor (CTT) is an uncommon lesion mostly seen in postchemotherapy patients for testicular germ cell tumors in retroperitoneal lymph node dissections. Patients with metastatic, nonseminomatous germ cell tumors of the testis often undergo excision of residual masses after cisplatin-based chemotherapy. Further management is based on the findings in such masses and accurate diagnosis is crucial. Presence of teratoma, necrosis, and/or fibroxanthomatous reaction denotes favorable prognosis and no further management is necessary. While patients with residual nonteratomatous germ cell tumor (e.g., seminoma, embryonal carcinoma, yolk sac tumor, choriocarcinoma) usually receive additional chemotherapy due to worse prognosis. It is possible that the entity previously described as atypical choriocarcinoma (choriocarcinoma-like lesions) actually represent CTT. In prior studies, CTT has been described mostly in association with teratoma in postchemotherapy retroperitoneal lymph node dissection specimens; however, in our experience it can be seen in other metastatic sites as well as in primary testicular germ cell neoplasm with or without chemotherapy. In the most comprehensive study, by Ulbright et al, only 7% of patients with followup developed recurrent choriocarcinoma after excision of a CTT, and overall 73% of patients were disease free on extended followup. These statistics are similar to those seen in advanced stage patients whose postchemotherapy resections contain only teratoma. Morphology of cystic trophoblastic tumor The cystic trophoblastic tumors are usually multifocal small cysts, typically measuring less than 3 mm (mean, 2.7 mm; range, mm) without infiltrative pattern and borders. The cysts are lined by trophoblast cells with abundant eosinophilic cytoplasm that vary from a single cell layer to several in thickness. Intracystic papillary tufts or cribriform arrangements can be seen in few cases. The majority of cyst lining cells are mononucleated without a biphasic appearance. The nuclei appear wrinkled and many have a smudged chromatin pattern. Occasional multinucleated cells are sometimes observed with prominent cytoplasmic lacunae. Overall, the cells appear squamoid, although no extracellular keratin production or intercellular bridges are seen. Mitotic activity is limited. The central portion of the cysts usually contain eosinophilic, acellular, and fibrinoid material with eventual resorption of the latter probably resulting in a cystic appearance. Fibrinoid deposits are also occasionally seen admixed with the lining cells. The background stroma is hypocellular and fibrous. Adjacent mature teratoma is present in almost all cases without other nonteratomatous germ cell tumor elements in postchemotherapy

11 RPLND patients. Hemorrhage is characteristically absent. Some of these lesions may be predominantly solid with minimal cystic change. Pathogenesis Two hypotheses have been proposed. The first is that this trophoblastic proliferation is a consequence of chemotherapy-induced cystification of choriocarcinoma. The second may be a relation to teratoma with trophoblastic differentiation or alternatively teratomatous differentiation of choriocarcinoma. However, the distinction is arbitrary and CTT represents a continuous spectrum from those with a teratomatous appearance and sometimes in continuity with teratomatous epithelium to those that are more clearly derived from chemotherapeutically altered choriocarcinoma. We think that choriocarcinoma may, on occasion, differentiate toward teratoma. No consistent elevation of hcg is seen in these cases, which supports the above observations. The occurrence of fibrinoid material in the central regions of some cases may well reflect a chemotherapy-induced change that eventually progresses to the mostly empty cysts. Retroperitoneal Lymph Node Dissection Specimens Retroperitoneal lymph node dissection (RPLND) is both a staging and a therapeutic procedure for patients with clinical stage I testicular cancer. It is also used in patients who have residual retroperitoneal masses after cisplatin-based chemotherapy. After radical orchiectomy, germ cell tumor patients undergo evaluation to determine serum AFP and hcg levels. In addition, periodic CT scans of the abdomen, pelvis, and chest are performed. Patients who have clinical stage I disease will harbor occult metastases in 30% of cases. The cure rate for these patients is 99% with either surveillance or nerve-sparing RPLND. Templates for removal of lymphatic tissue in the paracaval, precaval and interaortocaval (right sided primary), and left paraaortic and preaortic areas (left sided primary tumor) in patients with stage I disease have been published. Patients with advanced-stage disease undergo a full bilateral RPLND instead of a template-type dissection. Postchemotherapy RPLNDs may necessitate removal of other organs. The lymph nodes are removed en bloc because testis cancer can implant in the peritoneum if tumor is spilled during the procedure. The pathologist should attempt to dissect the individual lymph nodes in an area in order to determine the number present and the number involved by metastatic tumor. Sometimes, especially in postchemotherapy lymph node dissections, nodes may be matted together by residual tumor or fibrous tissue; this occurrence should be reflected in the pathology report. It is important to sample each lymph node, with thorough sampling of all areas that appear abnormal on gross examination. Many mixed germ cell tumors have a variegated gross appearance, and numerous sections are necessary in order to avoid overlooking residual small areas of either germ cell tumor or a nongerm cell tumor arising from the germ cell tumor. Postchemotherapy RPLNDs may contain necrosis, fibrosis, teratoma, or other persistent germ cell tumor. The pathology report should state the number of lymph nodes identified in each site and the types of residual tumor identified. The presence of necrosis or fibrosis consistent with chemotherapy effect should also be noted. Clinical Significance of Diagnosing CTT in PCRPLND

12 CTT versus Choriocarcinoma. The distinction between CTT and choriocarcinoma has important implications for patient management and prognosis. Patients with residual choriocarcinoma after chemotherapy have a poor prognosis, and aggressive treatment is warranted, in contrast to CTT. Misclassification of CTT as choriocarcinoma may subject the patient to inappropriate chemotherapy. CTT is distinguished from choriocarcinoma by the lack of a biphasic pattern of admixed cytotrophoblast and syncytiotrophoblast cells, lack of infiltrative growth, lack of necrosis, lower mitotic rate, cystic appearance, and lack of hemorrhagic background. Additionally, choriocarcinoma has more widespread immunoreactivity for hcg, reflecting its more abundant component of differentiated syncytiotrophoblast cells compared with CTT. This differential diagnosis is also facilitated by serum levels of hcg, with many cases of choriocarcinoma having elevations exceeding several thousands of international units per liter. CTT versus Teratoma. Teratoma also represents a significant differential diagnostic consideration. Like CTT, many teratomatous foci in postchemotherapy resections are cystic and demonstrate cytologic atypia, with nuclear enlargement and hyperchromasia but generally low mitotic rates. As mentioned above, this distinction may be arbitrary in individual cases, since it is our opinion that some CTTs have evidence of teratomatous transformation. In general, however, the absence of differentiated somatic-type epithelium in CTT is helpful; CTTs lack goblet cells, ciliated cells, and other specialized epithelial types that are common in teratoma. The teratomatous elements that CTTs are most apt to be confused with are squamous nests or cysts because of the shared prominence of cellular stratification and eosinophilic cytoplasm. CTT, however, lacks keratohyaline granules and central keratin, often has intracytoplasmic lacunae, and may show limited reactivity for hcg. Furthermore, this distinction has no known clinical significance. CTT versus Other Trophoblastic Derived Tumors in RPLND. In a few recent publications, other trophoblastic derived tumors have been reported including epithelioid trophoblastic tumor (ETT), monophasic choriocarcinoma, as well as placental site trophoblastic tumor (PSTT). The key diagnostic differences are cystic nature, fibrinoid material, less pleomorphism, no prominent mitotic activity, noninvasive growth pattern, and nonhemorrhagic background. Only two cases of monophasic choriocarcinoma in relation to testis have been reported. It should be noted that these tumors are preferably termed as unclassified trophoblastic tumors. Nodules of tumor are usually separated by a fibromuscular stroma. A sheet-like arrangement of cells in an eosinophilic edematous matrix is characteristic. Small clusters of lymphocytes and rare syncytiotrophoblastic cells can be noticed. Almost all the tumor cells are mononucleated, with oval to irregular, variably sized nuclei and ill-defined eosinophilic to clear cytoplasm. Mitotic figures can be seen in variable numbers, up to 18 per 10 high-power fields. Small areas of hemorrhage and necrosis may be noticed; however, diffuse hemorrhage and necrosis of typical choriocarcinoma is not observed. Rare foci with the characteristic biphasic pattern of choriocarcinoma with small zones of hemorrhagic necrosis have been documented. Additionally, small tumor nodules may display microcystic foci associated with hyaline globules a pattern characteristic of yolk sac tumor. Only two examples of PSTT have been reported, one in testis and one in a late recurrence. PSTT consists of tumor cells resembling implantation site intermediate

13 trophoblastic cells and are different from typical biphasic choriocarcinoma consisting of cytotrophoblast and syncytiotrophoblast. The large trophoblastic cells in PSTT are mostly mononucleated, and some of these display multiclefted nuclei. Multiclefted nuclei are a distinct feature of intermediate trophoblastic cells of the implantation site. Bi- and trinucleated cells can be seen. Few syncytiotrophoblastic giant cells are usually present. Vascular wall invasion of tumor cells is another typical feature of PSTT. The background stroma is usually edematous. Foci of hemorrhage are usually present but without necrosis. p63 and MIB1 immunohistochemical stains in addition to trophoblastic markers may contribute in differentiating these entities. See Table. CTT ETT PSTT Choriocarcinoma Inhibin /- p hcg HPLC ++ +/ Ki67 <5% >10% >8% >%50% CTT versus Somatic Malignancies Arising in Teratoma. It should be noted that rarely these tumors can mimic somatic malignancies arising in association with teratoma. In at least one of our consult cases, a CTT was misinterpreted as squamous cell carcinoma arising from teratoma in postchemotherapy RPLND. In summary, patients with CTT after chemotherapy do not show the high frequency of progressive disease that is associated with other forms of persistent, nonteratomatous germ cell tumor in this setting. Its significance is similar to that of residual teratoma. The finding of CTT in postchemotherapy resections does not warrant additional chemotherapy, although it is important to continue to carefully monitor these patients on followup. If CTT is present, provide a note regarding its significance, otherwise overtreatment is likely. References 1. Little JS Jr, Foster RS, Ulbright TM, Donohue JP. Unusual neoplasms detected in testis cancer patients undergoing post-chemotherapy retroperitoneal lymphadenectomy. J Urol 1994 Oct;152(4): Ulbright TM, Henley JD, Cummings OW, Foster RS, Cheng L. Cystic trophoblastic tumor: a nonaggressive lesion in postchemotherapy resections of patients with testicular germ cell tumors. Am J Surg Pathol 2004 Sep;28(9):1212-6

14 3. Hartmann JT, Schmoll HJ, Kuczyk MA, et al. Postchemotherapy resections of residual masses from metastatic nonseminomatous testicular germ cell tumors. Ann Oncol 1997;8: Donohue JP, Roth LM, Zachary JM, et al. Cytoreductive surgery for metastatic testis cancer: tissue analysis of retroperitoneal masses after chemotherapy. J Urol 1982;127: Fossä SD, Aass N, Ous S, et al. Histology of tumor residuals following chemotherapy in patients with advanced nonseminomatous testicular cancer. J Urol 1989;142: Fox EP, Weathers TD, Williams SD, et al. Outcome analysis for patients with persistent nonteratomatous germ cell tumor in postchemotherapy retroperitoneal lymph node dissections. J Clin Oncol 1993;11: Stenning SP, Parkinson MC, Fisher C, et al. Postchemotherapy residual masses in germ cell tumor patients: content, clinical features, and prognosis. Medical Research Council Testicular Tumor Working Party. Cancer 1998;83: Ulbright TM, Loehrer PJ. Choriocarcinoma-like lesions in patients with testicular germ cell tumors: two histologic variants. Am J Surg Pathol 1988;12: Mosharafa AA, Foster RS, Koch MO et al. Complications of post-chemotherapy retroperitoneal lymph node dissection for testic cancer. J Urol 2004;171: Donohue JP, Thornhill JA, Foster RS et al. Retroperitoneal lymphadenectomy for clinical stage A testis cancer (1965 to 1989): modifications of technique and impact on ejaculation. J Urol 1993;149: Colls BM, Harvey VJ, Skelton L, et al. Late results of surveillance of clinical stage I nonseminoma germ cell testicular tumors: 17 years experience in a national study in New Zealand. BJU Int 1999;83: Foster R, Bihrle R. Current status of retroperitoneal lymph node dissection and testicular cancer: when to operate. Cancer Control 2002;9: Suurmeijer AJ, Gietema JA, Hoekstra HJ. Placental site trophoblastic tumor in a late recurrence of a nonseminomatous germ cell tumor of the testis. Am J Surg Pathol 2004 Jun;28: Ulbright TM, Young RH, Scully RE. Trophoblastic tumors of the testis other than classic choriocarcinoma: "monophasic" choriocarcinoma and placental site trophoblastic tumor: a report of two cases. Am J Surg Pathol 1997;21: Wan SK, Lam PW, Pau MY, et al. Multiclefted nuclei: a helpful feature for identification of intermediate trophoblastic cells in uterine curetting specimens. Am J Surg Pathol 1992;16: Young RH, Kurman RJ, Scully RE. Proliferations and tumors of intermediate trophoblast of the placental site. Semin Diagn Pathol 1988;5: Shih IM, Kurman RJ. Ki-67 labeling index in the differential diagnosis of exaggerated placental site, placental site trophoblastic tumor, and

15 choriocarcinoma: a double immunohistochemical staining technique using Ki-67 and Mel-CAM antibodies. Hum Pathol 1998;29: Shih IM, Kurman RJ. Immunohistochemical localization of inhibin-alpha in the placenta and gestational trophoblastic lesions. Int J Gynecol Pathol 1999;18: Shih IM, Kurman RJ. The pathology of intermediate trophoblastic tumors and tumor-like lesions. Int J Gynecol Pathol 2001;20:31 47.

16 Case 4: The patient is a 48-year-old Caucasian male with a past medical history significant for nonseminomatous germ cell tumor that was treated with 3 cycles of BEP approximately 12 years ago. The patient recently presented to the emergency room complaining of persistent abdominal pain. A CT was obtained and revealed a 3 cm aortocaval mass. It should be noted that a CT scan obtained approximately 6 months earlier was negative for any retroperitoneal lymphadenopathy. The patient was consequently referred for RPLND. Diagnosis: Solid Yolk Sac Tumor, Late Recurrence of Germ Cell Tumor. Yolk sac tumors may exhibit numerous patterns, including microcystic/reticular, papillary, glandular, and solid, most of them elucidated by Teilum in his classic writings on this neoplasm. He commented to a limited degree on a solid pattern, but this pattern has not been subjected to a detailed study. This particular morphology is prone to misinterpretation, most commonly as seminoma and to lesser extent with other germ cell and nongerm cell tumors, particularly in small biopsy specimens from metastatic sites. Distinguishing solid pattern yolk sac tumor from seminoma is of critical importance since these neoplasms are often treated differently; the former either by retroperitoneal lymph node dissection with or without chemotherapy, or by surveillance, the latter usually with low-dose radiation following orchiectomy. A solid pattern of yolk sac tumor was described by Teilum in his seminal writings as "compact aggregates of undifferentiated embryonal cells." Jacobsen found solid growth in 27% of yolk sac tumors, and Talerman reported that solid foci occasionally contain microcysts and recognized that they may pose a challenge in the differential diagnosis with seminoma. Nonetheless, despite these comments, it is our own impression that many pathologists are not aware that solid foci may be seen in yolk sac tumors. The deceptive nature of solid growth is lessened by more typical patterns of yolk sac neoplasia, such that recognizing the tumor as a pure yolk sac tumor is often not problematic. However, when solid foci are extensive, or the tumor is purely solid, or its sampling is limited, for instance in a needle biopsy from a retroperitoneal metastasis, or the tumor displays features typical of seminomas, such as fibrovascular septa or lymphocytic infiltrates, the misdiagnosis of seminoma is very realistic. The fact that patients with early clinical stage seminoma are often treated with low-dose radiation following orchiectomy, whereas this treatment is contraindicated in those with yolk sac tumors, who often receive retroperitoneal lymph node dissection, emphasizes the importance of this differential diagnosis. Solid Yolk Sac Tumor versus Seminoma. The most important differential diagnosis is seminoma as indicated above. With careful evaluation, it is possible to resolve the differential in majority of cases. Several features characteristic of seminoma are absent or uncommon in solid pattern yolk sac tumors. Remaining cases can be resolved with the help of immunohistochemistry as discussed below. Closely packed, compact solid nests of cells (solid alveolar pattern and extensive stromal hyalinization that characterize many seminomas are usually lacking in solid yolk sac tumor. The infrequency of fibrovascular septa and lymphocytic infiltrates in solid yolk sac tumors contrasts with their high frequency (80 90% and 99%, respectively) in seminomas. A myxoid background occurring in YST is not a common feature of seminomas. Nuclear pleomorphism is a very helpful clue to the possibility of solid

17 yolk sac tumor as opposed to seminoma. Significant nuclear pleomorphism occurs in a substantial number of solid yolk sac tumors (65%) with nuclei varying from small and ovoid with inconspicuous nucleoli to large and polygonal with variable nucleolar prominence. This contrasts with the uniformly large, polygonal nuclei with prominent nucleoli that characterize seminoma. Most of the solid YSTs (75%) display intercellular band-like deposits of basement membrane, evidence of parietal differentiation of the tumor, as well as hyaline globules; whereas, seminomas lack this feature. Most cases (67%) also demonstrated occasional microcysts; this is not a specific feature of yolk sac tumor vis-à-vis seminoma since some seminomas form well-developed microcysts; nonetheless, it should raise concern for yolk sac tumor. Solid Yolk Sac tumor versus Other Germ Cell Tumors. The presence of yolk sac tumor in a nonseminomatous germ cell tumor limited to the testis may be associated with a decreased incidence of relapse; whereas, significant areas of embryonal carcinoma indicate more aggressive disease. On the other hand, yolk sac tumor in patients with advanced stage testicular cancer is associated with a poor prognosis. It appears that yolk sac tumor has less metastatic potential than embryonal carcinoma, but yolk sac tumor is also less sensitive to germ cell tumor chemotherapy. Yolk sac tumor does not display the marked nuclear crowding and extreme nuclear abnormalities seen in embryonal carcinoma. While both may display cytokeratin and AFP staining, CD30 and OCT4 decorate embryonal carcinoma but not yolk sac tumor. Glandular variants of yolk sac tumor are distinguished from teratoma by the absence of other teratomatous elements such as muscle and by positive AFP staining in the former. Some of the solid YSTs may display small, ovoid cells with hyperchromatic nuclei and scant cytoplasm, giving the solid foci a blastematous appearance; however, they are usually associated with microcystic and myxoid patterns. Teilum described this variant as "compact aggregates of undifferentiated embryonal cells." It may be considered as teratomatous in origin; however, its immunostaining properties are typical of yolk sac tumor. Immunohistochemical Features of Solid Yolk Sac Tumor. In those tumors where the light microscopic features remain ambiguous, immunohistochemical staining should be utilized. The single immunostain with the greatest utility in this differential diagnosis is OCT3/4, which is negative in yolk sac tumors and is positive in seminomas. Both AE1/AE3 cytokeratin and glypican 3 stains are useful, as they are generally positive in YST. AE1/AE3 has a generally diffuse, easily appreciated distribution. In contrast, biopsy specimens of seminoma are negative for AE1/AE3 cytokeratin in 80% of cases. Although glypican 3 positivity occurred in our recent observations in 97% of our yolk sac tumors, the intensity of staining was weaker and the distribution more patchy than in adjacent foci with other patterns, making it important to set a relatively low threshold for a positive result in solid yolk sac tumors. In contrast, glypican 3 is uniformly negative in seminomas. Podoplanin also has utility in this differential diagnosis since it is uncommonly found in solid yolk sac tumor (3%) and it is positive in seminomas. CD117 has no value in this differential diagnosis, with majority of the solid yolk sac tumors showing some degree of positivity and most seminomas also being positive. Based on the features discussed, we recommend a concise panel of AE1/AE3 cytokeratin, glypican 3, and OCT3/4 when the differential diagnosis is solid yolk sac tumor versus seminoma.

18 It should be noted that the traditional yolk sac tumor marker, AFP, can be insensitive for the solid pattern and completely fail to stain almost 40% of these cases. In prior studies, AFP had been reported to stain positively in 55 94% of yolk sac tumors, although often patchily so. In more recent studies, employing immunohistochemical method, over 90% of conventional yolk sac tumors stained positive for AFP. There appears, therefore, to be disproportionate insensitivity of AFP for the solid pattern since weakly staining or negative foci of solid yolk sac tumor occurred adjacent to intensely staining foci of other patterns including microcystic, macrocystic, and hepatoid. One therefore cannot rule out a diagnosis of yolk sac tumors based on a negative immunostain for AFP, especially in a tumor with a solid pattern of growth. Table: Immunoreactivity of solid yolk sac tumors IHC stains Number stained % positive Staining Score (E x I) ± SD AE1/AE ± 2.3 Glypican ± 2.5 AFP ± 2.3 CD ± 1.9 Podoplanin ± 0.5 OCT3/ Late Recurrence of Testicular Germ Cell Tumors There was a very significant improvement in the survival of patients with testicular germ cell tumors (GCTs) when cisplatin-based chemotherapy was introduced in the late 1970s. The combination of modern chemotherapy and surgical excision now results in survival of more than 90% of all patients with nonseminomatous testicular GCTs. Two decades ago, patients were considered to be cured after a complete remission lasting two years. Most relapses are detected in the first two years after diagnosis, and recurrence after this length of survival had been considered rare. We now know that a small number of patients, about 3% of GCT patients, will experience recurrences of GCTs or other tumors derived from GCTs at a later time, up to 30 years after a complete remission. Late recurrences are refractory to chemotherapy and associated with a low survival rate. A recent, large study of the histology of late recurrences showed that teratoma is the most common type of tumor in late recurrences (60% of patients), although other types of recurrent tumor are often present in addition to the teratoma. Next to teratoma, yolk sac tumor is the most common type of tumor seen in late recurrences (46% of patients). Yolk sac tumors also may occur along with other tumor types in late recurrences. The frequency of yolk sac tumor in late recurrences of testicular GCT may be due to its relative chemoresistance compared to other types of germ cell neoplasms. Long-term disease-free survival of patients treated with cisplatin-

19 based chemotherapy for advanced-stage testicular cancer containing yolk sac tumor (33%) is significantly poorer than that for patients whose advanced disease does not contain yolk sac tumor (65%). It is also noteworthy that the frequency of yolk sac tumor at autopsy in patients who die with testicular germ cell tumors increased significantly after introduction of cisplatin-based chemotherapy. Since effective chemotherapy eradicates other types of GCTs, the relatively more chemoresistant yolk sac tumor now has a poorer prognosis and may persist after destruction of more chemosensitive GCT elements. Baniel et al studied 81 patients with late recurrences that developed 2 32 years (median, 6.2 years) after complete remission. Sixty percent of late recurrences occurred at least five years after complete remission. Stage at initial presentation, primary tumor histology, and the presence of elevated serum markers (AFP, hcg) were not associated with late recurrences. Most patients had been treated with cisplatin-based chemotherapy at the time of their initial tumor diagnosis. The most common site for late recurrence was the retroperitoneum. Chemotherapy alone left only two patients continuously disease-free after late recurrence; these two patients had not previously received cisplatin-based chemotherapy, indicating that late recurrences are associated with chemoresistance. Treatment for late recurrences is generally surgical. Yolk sac tumor in late recurrences often displays unusual patterns, including glandular, clear cell, and parietal types. In contrast, the microcystic pattern, which is the most common type of yolk sac tumor in primary testicular tumors, is infrequent in late recurrences. Damjanov et al described transformation of microcystic and pseudopapillary yolk sac tumor of the ovary into disseminated, chemoresistant, parietal yolk sac tumor after cisplatin-based chemotherapy. The change in morphology was attributed to selective outgrowth of a chemoresistant clone of yolk sac tumor, a hypothesis that is consistent with the appearance of unusual forms of yolk sac tumor in late recurrences of testicular GCTs. Familiarity with these variants of yolk sac tumor is important in order to avoid misdiagnosis in patients with a remote history of testicular GCTs. Glandular, clear cell, parietal, and hepatoid patterns are especially common in late recurrences. Glandular yolk sac tumor is characterized by glands lined by columnar epithelium with clear cytoplasmic vacuoles, similar to endometrioid-like yolk sac tumor. Many late recurrences display substantial solid areas, which may contain numerous clear cells. Hepatoid yolk sac tumor seen in late recurrences displays polygonal cells with densely eosinophilic cytoplasm and occasional intercellular sinusoids. Parietal yolk sac tumor is frequently seen in late recurrences; it has eosinophilic basement-membrane material between nests of cells. Some late recurrences contain yolk sac tumor with an unusual degree of nuclear pleomorphism. No festoon or polyvesicular vitelline patterns of yolk sac tumor were identified in a large study of these neoplasms. A smaller number of late recurrences display either nonteratomatous GCTs other than yolk sac tumor or somatic-type malignancies arising from germ cell tumors. These patients have a very poor prognosis unless nongerm cell tumors can be completely excised. The rarity of seminoma and embryonal carcinoma in late

20 recurrences probably reflects their marked sensitivity to chemotherapy in the initial neoplasm. Patients whose late recurrences contain only teratoma have a good prognosis, with 79% free of disease at followup. In contrast, only 35% of patients with nongerm cell tumors, 37% of patients with yolk sac tumor, and 36% of patients with nonteratomatous GCTs other than yolk sac tumor were free of disease. All patients reported in the literature with late recurrences containing tumor besides teratoma fared poorly and very few had responded to chemotherapy. Late recurrences should be treated surgically if at all possible. Schematic Immunohistochemical Approach in Germ Cell Tumors Points to Ponder: Immunohistochemistry is not a diagnostic tool but a differential diagnostic tool. We emphasize the need to correlate immunohistochemistry with morphologic and clinical features. Differential algorithms for specific differential diagnoses should be effectively utilized. Apply immunohistochemistry conservatively. No prognostic immunohistochemistry markers for testicular neoplasms. Ask for help. Useful markers: OCT4, CD117, podoplanin, SOX17, CD30, AE1/AE3, SOX2, glypican 3, AFP, hcg, PLAP, SALL4. Scheme. A reasonable and efficient initial panel is: OCT4, CD117, CD30, and glypican 3. This panel may be reduced depending on the light microscopic differential. For instance, omitting OCT4 and glypican 3 if the question by morphology is limited to seminoma versus embryonal carcinoma. SALL4 can be used as a general marker for germ cell neoplasms for distinction from sex cord stromal or metastasis. Caution should be used when using this marker as few other neoplasms show expression and further validation is required.

21 Immunohistochemistry Algorithm for Testicular Germ Cell Tumor Subtyping. In conclusion, solid foci of yolk sac tumor may lead to an erroneous diagnosis of seminoma. Awareness of this pattern and other variant yolk sac morphologies in postchemotherapy RPLND and observation of routine light microscopic differences from seminoma and immunohistochemical studies in particularly problematic cases will result in a correct interpretation and appropriate treatment. References 1. Kao CS, Idrees MT, Young RH, Ulbright TM.. Solid pattern yolk sac tumor: a morphologic and immunohistochemical study of 52 cases. Am J Surg Pathol 2012; 36: Bosl GJ, Motzer RJ. Testicular germ-cell cancer. New Engl J Med 1997;337: de Riese WT, Albers P, Walker EB, et al. Predictive parameters of biologic behavior of early stage nonseminomatous testicular germ cell tumors. Cancer 1994;74: Eglen DE, Ulbright TM. The differential diagnosis of yolk sac tumor and seminoma: usefulness of cytokeratin, alpha-fetoprotein, and alpha-1-antitrypsin immunoperoxidase reactions. Am J Clin Pathol 1987;88: Friedman NB, Moore RA. Tumors of the testis: a report on 922 cases. Milit Surgeon 1946;99:

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