Report From the International Society of Urological Pathology (ISUP) Consultation Conference on Molecular Pathology of Urogenital Cancers: IV: Current and Future Utilization of Molecular-Genetic Tests for Testicular Germ Cell Tumors

Abstract

The International Society of Urological Pathology (ISUP) organized a Consultation Conference in March 2019 dealing with applications of molecular pathology in Urogenital Pathology, including testicular tumors (with a focus on germ cell tumors [GCTs]), preceded by a survey among its members to get insight into current practices in testicular germ cell tumor (TGCT) diagnostics and adoption of the ISUP immunohistochemical guidelines published in 2014. On the basis of the premeeting survey, the most commonly used immunomarker panel includes OCT3/4, placental alkaline phosphate, D2-40, SALL4, CD117, and CD30 for GCTs and the documentation of germ cell neoplasia in situ (GCNIS). Molecular testing, specifically 12p copy gain, is informative to distinguish non-GCNIS versus GCNIS related GCTs, and establishing germ cell origin of tumors both in the context of primary and metastatic lesions. Other molecular methodologies currently available but not widely utilized for TGCTs include genome-wide and targeted approaches for specific genetic anomalies, P53 mutations, genomic MDM2 amplification, and detection of the p53 inactivating miR-371a-3p. The latter also holds promise as a serum marker for malignant TGCTs. This manuscript provides an update on the classification of TGCTs, and describes the current and future role of molecular-genetic testing. The following recommendations are made: (1) Presence of GCNIS should be documented in all cases along with extent of spermatogenesis; (2) Immunohistochemical staining is optional in the following scenarios: identification of GCNIS, distinguishing embryonal carcinoma from seminoma, confirming presence of yolk sac tumor and/or choriocarcinoma, and differentiating spermatocytic tumor from potential mimics; (3) Detection of gain of the short arm of chromosome 12 is diagnostic to differentiate between non-GCNIS versus GCNIS related GCTs and supportive to the germ cell origin of both primary and metastatic tumors.

FIGURE 1

Schematic representation of the various entities of TGCTs. The timeline is indicated on the left side. The TGCTs include the non-GCNIS related TGCTs (left panel) and GCNIS-related TGCTs (right panel). The non-GCNIS related TGCTs are subcategorized into the prepubertal TE (being diploid) and YST as well as the spermatocytic tumors. These are also referred to as type I and III, respectively. The GCNIS-related TGCTs are histologically (and clinically) subdivided into the seminomas (SE) and the various elements of nonseminomatous TGCTs, being embryonal carcinoma (EC), YST, choriocarcinoma, and TE. Note the overlapping histology between the prepubertal TE/YST and the TE and YST elements in the GCNIS-related nonseminomas (being underlined). However, they have a separate (and independent) pathogenesis (see text for further details). The precursors are indicated when known (preinvasive), while specifically, the benign and malignant behavior of the pediatric TE and YST is highlighted. Besides, the most prominent and recurrent molecular genetic changes are indicated, of putative interest to be used for molecular pathologic approaches. These include total genomic anomalies, like polyploid/aneuploid, specific chromosomal imbalances like losses (−), and gains (+), as well; as recurrent mutations (italics). Also, the methylation status is indicated, as well as the possible use of miR-371a-3p as a liquid biopsy molecular biomarker (underlined). All malignant histologic elements, independent of age, are identified by this biomarker (except TE). The WNT pathway is specifically involved in the YST components, independent of age and as such also of pathogenesis.

FIGURE 2

A, Microscopic example of a prepubertal pure testicular teratoma (3 y of age) (a, b, hematoxylin & eosin [H&E]) after enucleation to leave the testis function intact (top panel). No GCNIS was demonstrated in the adjacent parenchyma. Also, no chromosomal imbalances were identified using EPIC methylation profiling, transferred to CNV (lower panel) (c); B, Example of a pure prepubertal YST (YST) (7 mo of age) macroscopically (a) as well as microscopically (b, c, H&E) and IHC for AFP (d). A mixed GCT displaying prepubertal YST admixed with teratoma component in a 4 months old boy (e, f, H&E). Note immature seminiferous tubules without GCNIS or active spermatogenesis (f). GPC3 highlighted the YST component (g). The chromosomal imbalances are determined based on EPIC methylation profiling data, showing loss of 1p and 6q (right lower panel) (h).

FIGURE 3

Examples of a spermatocytic tumor, macroscopically (A, B), and microscopically (hematoxylin & eosin [H&E]) (C, D), and IHC for DMRT1 (E), and direct alkaline phosphatase (dAP) (G). Macroscopically, the tumor appears homogenous on cut surface, whitish to yellow-tan nodular neoplasm replacing the testicular parenchyma, possibly multifocal. Note the unique presence of 3 variants of cells, being small, intermediate, and large. The precursor, known as intratubular spermatocytic tumor, is shown (D). Also, spectral karyotyping (SKY) (F) and array comparative genomic hybridization are shown (H), demonstrated gain of the whole chromosome 9.

FIGURE 4

Representative examples of GCNIS and gonadoblastoma. GCNIS (top 2 rows) (postpubertal patient with an invasive GCT) stained using hematoxylin & eosin (H&E) (A) and IHC for TSPY (B), OCT3/4 (C), KITLG (D), SOX9 (sertoli cell marker, lower magnification) (E), as well as direct alkaline phosphatase (dAP) (F); Gonadoblastoma (lower row) stained using H&E (G) and IHC for OCT3/4 (H), and (I) FOXL2 (granulosa cell marker). Inset represents the complete overview of the sample slide, the higher magnification area is indicated in the red box.

FIGURE 5

Representative example of a mixed germ cell tumor composed of seminoma and embryonal carcinoma (and a small teratoma component and GCNIS [not shown]) (patient 25 y of age) macroscopically (A), stained using hematoxylin & eosin (H&E) (B) and IHC for OCT3/4 (C), TSPY (D), SOX17 (highlighting seminoma) (E) and SOX2 (highlighting embryonal carcinoma) (F). No double-positive cells for SOX2 and SOX17 are identified, while all are positive for OCT3/4.

FIGURE 6

Illustrations of chromosomal anomalies (including 12p) as identified by karyotyping and FISH with a centromere—and 12p- specific probes (A). Results of chromosomal comparative genomic hybridization of GCNIS and various invasive components (B), showing the specificity of gain of 12p for the latter. Also, spectral karyotyping (SKY) of a tumor metaphase spread is shown (C), and an example of CNV (based) based on EPIC methylation profiling is demonstrated (D).,