Establishing a germ cell origin for metastatic tumors using OCT4 immunohistochemistry

Authors

  • Liang Cheng M.D.

    Corresponding author
    1. Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana
    • Department of Pathology and Laboratory Medicine, Indiana University Medical Center, University Hospital 3465, 550 North University Boulevard, Indianapolis, IN 46202
    Search for more papers by this author
    • Fax: (317) 274-5346


  • Presented in part at the 93rd annual meeting of the United States and Canadian Academy of Pathology, Vancouver, British Columbia, Canada, March 6–12, 2004.

Abstract

BACKGROUND

There are diverse morphologic manifestations of metastatic tumors. The determination of tumor origin is critical for patient management, and it is especially important when the differential diagnosis includes metastatic germ cell tumor, which is a highly treatable condition. OCT4 is a nuclear transcription factor that is expressed in pluripotent embryonic germ cells. In this study, the author sought to determine the usefulness of OCT4 immunohistochemistry in the diagnosis of metastatic germ cell tumors.

METHODS.

Sixty-two retroperitoneal lymph node dissection specimens from patients with histories of testicular germ cell tumors were stained using the antibodies against OCT4. In addition, 84 metastatic, nongerm cell lesions from men with known primary tumors were studied in parallel for OCT4 immunohistochemistry.

RESULTS.

All embryonal carcinoma components (n = 29 specimens) and seminoma components (n = 18 specimens) from retroperitoneal lymph node dissection specimens showed strong, intense, diffuse nuclear staining for OCT4. Yolk sac tumors (n = 12 tumors), choriocarcinomas (n = 4 tumors), mature teratomas (n = 16 tumors), and primitive neuroectodermal tumors (n = 5 tumors) were negative for OCT4 staining. Metastatic, nonsmall cell carcinomas from the lung (n = 14 tumors), colon (n = 12 tumors), stomach (n = 5 tumors), pancreas (n = 7 tumors), prostate (n = 12 tumors), kidney (n = 3 tumors), and urinary bladder (n = 15 tumors) all were found to be negative immunohistochemically for OCT4, as were metastatic small cell carcinomas (n = 4 tumors) and metastatic melanomas (n = 7 tumors). In addition, malignant lymphomas (n = 5 tumors) also were negative for OCT4.

CONCLUSIONS.

Immunohistochemical detection of OCT4 is highly sensitive and specific for the diagnosis of seminoma and embryonal carcinoma metastatic from the testis. Establishing germ cell origin for metastatic tumors has important implications for assessing patient prognosis and treatment options. Cancer 2004 © 2004 American Cancer Society.

Carcinoma of the testis is the most common malignancy in men ages 15–35 years, with an estimated 8980 new cases and 360 deaths expected in 2004.1 With the advent of cisplatin combination chemotherapy, > 90% of all newly diagnosed patients will be cured.2 With time, some develop metastatic tumors. Accurate determination of tumor origin is critical for the assessment of a patient's prognosis and planning of subsequent treatment, because somatic malignancies (nongerm cell tumors) typically do not respond to germ cell tumor chemotherapy.

There are diverse morphologic manifestations of metastatic germ cell tumors. The diagnosis of metastatic germ cell tumor in an adult, with or without a history of testicular germ cell tumors, can be challenging.3 It may be difficult to distinguish metastatic germ cell tumors from nongerm cell tumors morphologically. Therefore, there is a need for a sensitive and specific germ cell tumor marker.

OCT4 is a nuclear transcription factor that is expressed in early embryonic cells and germ cells.4–7 OCT4 regulates the expression of a 1.5-kilobase (kb) alternative platelet-derived growth factor α receptor, and it has been shown that OCT4 is essential for self-renewal for embryonic stem cells and for early mouse development.8, 9 Targeted disruption of the OCT4 gene is lethal to early mouse embryos, because the inner cell mass cells from OCT4-deficient mouse embryos are not pluripotent; they differentiate exclusively into trophoblasts.8 OCT4 has been detected in neoplastic pluripotent germ cells, specifically those of testicular seminoma and embryonal carcinoma.10 The objective of the current study was to determine the usefulness of OCT4 immunohistochemistry in the diagnosis of metastatic germ cell tumors.

MATERIALS AND METHODS

Sixty-two retroperitoneal lymph node dissection specimens from patients who were diagnosed previously with testicular germ cell tumors were studied. Metastatic germ cell tumors were present in all specimens, and these tumors were classified according to accepted criteria.11 In addition, 84 metastatic carcinomas from male patients with known nongerm cell primary tumors were studied for OCT4 immunoreactivity in parallel. These included metastatic nonsmall cell carcinoma specimens from the lung (n = 14 tumors), colon (n = 12 tumors), stomach (n = 5 tumors), pancreas (n = 7 tumors), prostate (n = 12 tumors), kidney (n = 3 tumors), and urinary bladder (n = 15 tumors) as well as metastatic small cell carcinoma (n = 4 tumors) and metastatic melanoma (n = 7 tumors). In addition, samples of malignant lymphoma (three diffuse large B-cell lymphomas and two Hodgkin lymphomas) also were evaluated. This research was approved by the Indiana University Institutional Review Board.

Five-micrometer-thick sections of appropriately selected, formalin-fixed, paraffin-embedded surgical pathology specimens were used for immunohistochemical staining. OCT4 immunostaining was accomplished with a polyclonal goat anti-OCT4 antibody (C20, sc 8629; Santa Cruz Biotechnology, Santa Cruz, CA; at 1:500 dilution for 30 minutes at room temperature) directed toward the − COOH terminus of the protein. Antigen retrieval was performed by heating sections in 1 mmol/L of ethylenediamine tetraacetic acid, pH 8.0, for 30 minutes. Endogenous peroxidase activity was inactivated by incubation in 3% hydrogen peroxide for 15 minutes. Nonspecific binding sites were blocked using Protein Block (Dako Corporation, Carpinteria, CA) for 20 minutes.

The percentages of cells that stained positively for OCT4 were estimated, and the staining intensity was classified as negative (0), weak (1+), moderate (2+), or strong (3+), as described previously.12–22 OCT4 is a nuclear transcription factor that is involved in gene regulation; thus, only nuclear staining is considered a positive result.

RESULTS

All embryonal carcinoma components (n = 29 samples) and seminoma components (n = 18 samples) from retroperitoneal lymph node dissection specimens showed strong (3+), intense, diffuse nuclear staining for OCT4 (Fig. 1). Yolk sac tumors (n = 12 tumors), choriocarcinomas (n = 4 tumors), mature teratomas (n = 16 tumors), and primitive neuroectodermal tumors (n = 5 tumors) were negative for OCT4 staining (Fig. 1). Metastatic nonsmall cell carcinomas from the lung (n = 14 tumors), colon (n = 12 tumors), stomach (n = 5 tumors), pancreas (n = 7 tumors), prostate (n = 12 tumors), kidney (n = 3 tumors), and urinary bladder (n = 15 tumors) all were found to be immunohistochemically negative for OCT4, as were metastatic small cell carcinomas (n = 4 tumors) and metastatic melanomas (n = 7 tumors). In addition, malignant lymphomas (n = 5 tumors) also were negative for OCT4.

Figure 1.

Diagnosing metastatic germ cell tumor using OCT4 immunohistochemistry. (A) Metastatic seminoma is seen in a necrotic background. A reliable diagnosis was difficult to determine in limited biopsy materials. (B) OCT4 immunostaining highlights seminoma cells. (C) An embryonal carcinoma is intermixed with a myxoid yolk sac tumor. (D) An embryonal carcinoma was positive for OCT4. The adjacent yolk sac tumor is negative for OCT4. (E) A yolk sac tumor with a glandular pattern is shown. The tumor mimics embryonal carcinoma. (F) Negative OCT4 immunostaining confirmed the diagnosis of yolk sac tumor. (G,H) Choriocarcinoma is negative for OCT. (I,J) This primitive neuroectodermal tumor was negative for OCT4.

DISCUSSION

At times, the histogenesis and site of origin of malignancies encountered in clinical practice are enigmatic. The differential diagnosis in such cases may include metastatic germ cell tumor. Resolution of the clinical and pathologic dilemma is critical because patients with metastatic germ cell tumors have a favorable prognosis after treatment with surgical resection and cisplatin-based chemotherapy regimens. Therefore, it is crucial to determine whether a metastatic tumor is of germ cell origin, especially in young adult men with primary tumors of unknown origin. Regression of testicular germ cell tumors has been well documented.23, 24 Some patients may present with metastatic germ cell tumors without a prior history of testicular carcinoma.24

Accurate histologic assessment of metastatic lesions is critical to identify tumor origin. Nonetheless, it may be difficult to ascertain the primary site of origin of a tumor without additional studies. Moertel et al. estimated that approximately 10% of patients in a general oncology practice presented with metastatic tumors of unknown primary origins.25 It is highly desirable to identify a tumor marker that is sensitive and specific and that can be applied readily in routine surgical pathology practice.26 Accurately diagnosing metastatic tumors of germ cell origin is critical for patient management because effective and even curative therapy is available for these patients. Previous studies have examined the utility of placental-like alkaline phosphatase (PLAP) as a diagnostic tool for establishing germ cell origin. PLAP is a highly sensitive but nonspecific biomarker for germ cell differentiation. Immunoreactivity for PLAP has been observed in nongerm cell tumors.27–35 In a survey of 482 somatic malignancies, Wick et al. found that 62 tumors (13%) showed positive immunostaining for PLAP.34 Recent studies indicated that CD30 is a useful biomarker for the diagnosis of embryonal carcinoma.36 Nonetheless, caution is warranted in interpreting the results. CD30 immunoreactivity can be observed in hematopoietic tumors,37 malignant melanoma,38 and other germ cell tumors.39 Approximately 65% of metastatic embryonal carcinomas will lose CD30 expression after chemotherapy.40 The identification of ≥ 1 copies of i(12p) establishes the diagnosis of germ cell tumor.41–43 However, approximately 20% of germ cell tumors will be i(12p)-negative. The methodology and criteria for identification of i(12p) have not been standardized and vary in different laboratories. Fresh tissue is not always available for conventional cytogenetic studies. Although it may be feasible, interphase fluorescence in situ hybridization for i(12p) detection remains a technical challenge.

OCT4 (also known as POU5F1, OCT3, or OTF3) is a POU-domain, octamer-binding nuclear transcription factor that is expressed in undifferentiated, pluripotent cells, including human embryonic stem and germ cells.4–7 The oct4 (pou5f1) gene is mapped to the human chromosome 6p21.344 and encodes a protein that is involved in normal development through the regulation of many downstream target genes.45 It has been detected in testicular seminomas and embryonal carcinomas10, 22 and in ovarian dysgerminomas.17 To our knowledge, the utility of OCT4 immunohistochemistry in diagnosing metastatic germ cell tumors has not been evaluated previously. We found that OCT4 was highly sensitive and specific for the diagnosis of certain metastatic germ cell tumors. OCT4 immunohistochemical staining identified metastatic embryonal carcinomas and seminomas in all 62 patients with testicular carcinoma who underwent retroperitoneal lymph node dissection. In contrast, none of the 84 metastatic nongerm cell tumors analyzed showed immunoreactivity for OCT4.

Long-term survival in patients with testicular carcinoma has been achieved using modern chemotherapy.2 These patients may develop secondary nongerm cell tumors. Spontaneous regression of testicular germ cell tumors may occur in some patients who present with metastatic germ cell tumor but without clinical evidence of a primary testicular tumor. The correct diagnosis of metastatic tumor of germ cell origin has important implications for the patient's prognosis and therapeutic strategies. The use of OCT4 immunohistochemistry may prove to be a very useful adjunct in confirming the germ cell origin of a metastatic tumor.

Ancillary