Positive
Negative
Germ cell tumors
Seminoma
Spermatocytic tumor
Embryonal carcinomas
Yolk sac tumors
Choriocarcinoma
Teratomas (epithelial elements)
Non germ cell tumors
GISTs, angiomyolipoma, mastocytosis, thymic carcinomas, lung carcinomas, etc.
Solitary fibrous tumor, alveolar soft part sarcoma, desmoplastic small round cell tumor, glomus tumor, leiomyoma, etc.
Fig. 4.1
CD117 membrane and cytoplasmic staining in classic seminoma
4.1.2 OCT4
4.1.2.1 Nomenclature
OCT4 is also known as POU5F1, OCT3, or OTF3 and has been used previously as OCT3/4 but is more recently known as OCT4 (octamer-binding transcription factor 4).
4.1.2.2 Gene Function and Chromosomal Location
OCT4 represents a mammalian nuclear transcription factor belonging to the POU domain encoded by a gene located in the human chromosome 6p21.3, being essential in blastocyst differentiation [22].
OCT4 protein expression is the sum of different isoforms (and pseudogenes) of which only the OCT4A can be directly linked functionally to pluripotency. For this reason, care should be taken as to the type of antibody used when stem cells of somatic tumors are targeted. In these cases, mRNA levels should be detected in order to confirm their stemness [23]. As a key protein and component in the regulatory network that maintains pluripotency in normal development and in GCT pathogenesis, OCT4 is extensively reviewed in Chap. 3.
4.1.2.3 Recommended Clones and Practical Considerations
Antibody
The most widely used is a mouse monoclonal antibody (clone C-10) raised against amino acids 1–134 of OCT4 of human origin that specifically recognizes the OCT4A isoform. Alkaline HIER is recommended.
Controls
Well-fixed sections of testicular parenchyma with unequivocal changes of GCNIS are recommended as positive controls. Due to the thick fibrous tunica albuginea and unexpected delays in sectioning of orchidectomy specimens, generally the most superficial tumor cells will show more intense staining, while those located in deeper areas of the sample will show weaker nuclear staining and more cytoplasmic background. Fresh sections should be stained as soon as possible to prevent oxidation and loss of antigenicity.
Staining pattern
OCT4 predominantly produces a strong nuclear staining with only minimal cytoplasmic staining.
4.1.2.4 Expression
Focal OCT4 staining can be demonstrated in various normal tissues due to the presence of a population of progenitor cells [24]. Its expression is absent in the majority of somatic tumors [25, 26], but recent studies have shown it in high-grade carcinomas of the thyroid, gastrointestinal tract, or lung, being generally associated with distant metastases and poor response to conventional treatment [27–32]. Granular cytoplasmic staining in both normal tissue and neuroendocrine neoplasms, such as Merkel cell carcinomas of the skin, has been noted. This is probably related to cross-reactivity of some antibodies with the OCT4B isomer. The staining weakens as the differentiation grade increases, and it is inversely related with the Ki67 index [33].
In the first trimester of gestation, OCT4 is expressed in embryonal germ cells, while in the second trimester, secondary to the formation of oocytes and spermatogonia, its expression disappears [34, 35]. However, in newborns, it can be identified in testicular germ cells located within the luminal space as well as in gonads showing germ cell maturation delay [36].
In pathological conditions, it is re-expressed in the preinvasive germ cell lesions such as GCNIS and gonadoblastoma [37–39], as well as in seminoma (Fig. 4.2a) and EC [37, 40, 41–46]. It may also be expressed in isolated epithelial cells of ovarian immature teratomas [47], especially in high-grade tumors [48, 49]. OCT4 is expressed by epithelial areas in cases of GCT patterns originating from somatic neoplasms (see Chap. 6).
Fig. 4.2
(a) OCT4 nuclear staining with cytoplasmic background in classic seminoma. (b) OCT4 expresses in embryonal carcinoma, while areas of microcystic yolk sac tumor are negative
Albeit a more sensitive and specific marker for both primary and metastatic seminomas and EC [44, 50] than their classical markers [43, 45, 51], it must be borne in mind that rare cases of OCT4-negative, chemoresistant EC have been reported [52].
OCT4 does not differentiate seminomas from EC, and it is not expressed in solid areas of choriocarcinoma, yolk sac tumor (YST) (Fig. 4.2b), or ST. Positive staining in cells delineating papillary projections of EC, helps to differentiate them from the Schiller-Duval perivascular structures of YST [53]. In summary, OCT4 is an antibody that differentiates more primitive, pluripotential GCT from differentiated ones.
Positive | Negative | |
|---|---|---|
Germ cell tumors | Seminoma Embryonal carcinoma Primitive GCT areas in somatic tumors | Yolk sac tumor Choriocarcinoma Teratomas Spermatocytic tumor |
Non-germ cell tumors | Virtually none | Generally weak staining and in isolated cells of undifferentiated carcinomas |
4.1.3 SALL4
4.1.3.1 Nomenclature
Sal-like transcription factor 4
4.1.3.2 Gene Function and Chromosomal Location
SALL4 is a nuclear zinc finger transcription factor, part of the Spalt-like family genes, encoded by a gene located on the chromosomal region 20q13.2 and expressed in early developmental stages [54]. Together with OCT4, NANOG, and SOX2, it is part of a transcriptional core network of genes that maintains the pluripotent properties and self-renewal capacities of ESC [55–58]. SALL4 regulates transcription of OCT4 [57]. In comparison with Oct4 and Sox2, it is the only one required for the development of the murine primitive endoderm from the inner cell mass in addition to the epiblast, while the development of trophoblastic lineage is not impaired by the lack of Sall4 [59].
4.1.3.3 Recommended Clones and Practical Considerations
Antibody
Mouse monoclonal antibodies EE-30 or 6E3, developed against recombinant proteins similar to the whole or just a fragment of SALL4 human protein, are both suitable for its detection. Alkaline HIER is recommended.
Controls
Spermatogonia of normal testis (weak–moderate staining) or GCNIS (intense staining) might be used as a positive control.
Staining pattern
As a nuclear transcription factor, SALL4 highlights the nuclei of the tumor cells.
4.1.3.4 Expression
SALL4 is detected in the primordial germ cells and interacts with other transcription factors in the development of the anorectal region, kidney, heart, limbs, and brain. SALL4 nuclear staining has been reported in the proximal renal tubules of the renal cortex, some neural tube elements, intestine, and hepatocytes [60]. Only weak to moderate expression can be seen in the oocytes and spermatogonia [61, 62].
As a master controller of pluripotency maintenance, its nuclear expression can be detected in all primitive and immature GCT with variable results in choriocarcinoma and immature teratoma areas. In trophoblastic tumors, the staining is generally restricted to more immature trophoblastic cells, while the differentiated syncytiotrophoblasts are negative (Fig. 4.3a). Pseudoglandular (Fig. 4.3b) and tubular-neuroectodermal elements of teratomas (Fig. 4.3c) show moderate to weak staining [53, 61–65].
Fig. 4.3
(a) SALL4 shows diffuse and strong staining in embryonal carcinoma and yolk sac tumor, while a weak, focal staining in teratoma is seen. Areas of trophoblastic differentiation are negative in this case. (b) Moderate staining in immature glands of teratoma while epithelial cells of yolk sac tumor are strongly stained. (c) Immature neuroectodermal cells show partial and weak nuclear staining. (d) Moderate positivity in small and intermediate cells of spermatocytic tumor
Compared with the weak–moderate positivity of spermatogonia of the normal seminal epithelium, the atypical cell of GCNIS, as well as the medium and small cells of spermatocytic tumors, presents a stronger nuclear stain (Fig. 4.3d) [62]. Dysregulation of SALL4 might be a common factor in the pathogenesis of all genetic groups of testicular GCT [62, 66]; however, positivity can occur in generally high-grade, poorly differentiated non-GCT, such as ovarian serous carcinomas and undifferentiated urothelial carcinomas [67, 68]. In nonepithelial malignant tumors, SALL4 may be positive in rhabdoid tumors, Wilms’ tumors, melanomas, and rhabdomyosarcomas, among others [68]. The expression in lymphoid neoplasms is controversial, and only isolated cases of precursor B-cell lymphoblastic leukemia/lymphomas showed elevated mRNA levels [67].
Positive | Negative | |
|---|---|---|
Germ cell tumors | Seminoma Embryonal carcinoma Yolk sac tumors Choriocarcinoma (cytotrophoblast) Immature teratomas (immature elements) Spermatocytic tumor | Mature teratomas (epithelial elements) |
Non-germ cell tumors | Ovarian serous carcinomas, urothelial carcinomas, hepatoid AFP-secreting and intestinal-type gastric adenocarcinomas, etc. | All with exceptions (see left) |
4.1.4 SOX2
4.1.4.1 Nomenclature, Gene Function, and Chromosomal Location
SRY (sex-determining region Y-box 2), also known as SOX2, is one of the three members of SOXB1 subgroup of transcription factors encoded by a gene located in chromosome 3q26.33 [69].
In early implantation stages, it has a major role in trophectoderm differentiation; knockout embryos arrest at the morular stage and fail to cavitate [70]. Moreover, a reduction of SOX2 expression in human ESC induces trophectodermal and partial endodermal differentiation [71]. The correct balance between SOX2 and OCT4 might be a key of pluripotency. On murine models, it has been demonstrated that together with Sox3, Sox2 is expressed in the epiblast and extraembryonic ectoderm and is restricted to the forthcoming neural plate and chorion at gastrulation [72]. During early somitogenesis, all three genes are expressed in the neuroectoderm, and Sox2 and Sox3 are also expressed in the primitive streak ectoderm, gut endoderm, and prospective sensory placodes [72]. Consecutively, they may play an important role in maintaining neural crest stem cell multipotency [73, 74] and neuronal formation [75], while its upregulation may contribute to development of supratentorial PNET [76].
4.1.4.2 Recommended Clones and Practical Considerations
Antibody
Rabbit polyclonal antibodies (such as AF2018 or AB5603) or mouse monoclonal E-4 have been the most frequently used for recent assays. From our personal experience, we recommend rabbit clone SP76; it works well with a pH 8 buffer for HIER, 10–20 minutes incubation of the antibody, and with a two-step polymer-based detection system.
Controls
In our assay, embryonal neural tissue was used as positive control.
Staining pattern
The antibody stains the nucleus with little or no cytoplasmic staining.
4.1.4.3 Expression
SOX2 positive staining is mainly reported in squamous cell carcinomas, correlating with amplifications of the 3q26 chromosomal region [30, 77–80]. Other pulmonary non-small cell carcinomas, excluding neuroendocrine ones, also frequently express SOX2 [81–84] as do gastric carcinomas [83, 85], pancreatic and biliary tumors [86], high-grade gliomas [86], and primitive neuroectodermal tumors [87]. Breast carcinomas express SOX2 predominantly in high-grade, HER2-positive tumors and those with a basal-like phenotype. Expression has been reported in metastases of otherwise SOX2-negative primary breast carcinomas [88].
Up to 60.5 % of ovarian carcinomas express SOX2, with a higher percentage of positivity in high-grade tumors and in more advanced stages [89]. Interestingly, a high level of SOX2 is associated with a better prognosis, probably due to a better response to platinum-based therapies [89].
Testicular parenchyma does not express SOX2 during its development [90] but occurs in Sertoli cells associated with GCNIS lesions [91, 92]. Even if it is considered as another important transcription factor in the induction and maintenance of pluripotency [70, 93, 94], in contrast with OCT4 and especially SALL4, SOX2 positivity in GCT is restricted to ECs, immature neuroepithelium, and squamous epithelium of mature teratoma (Fig. 4.4a) [65, 91, 92, 95]. Seminomas, YST (Fig. 4.4b), choriocarcinoma, and spermatocytic tumors are constantly negative [65, 91, 92, 95]. Accordingly, SOX2 expression differentiates seminomas from solid EC, while papillary structures in EC are clearly differentiated from Schiller -Duval bodies in YST [53]. Its negativity in the isolated foci of solid and hepatic areas of YST differentiates these YST variants from EC.
Fig. 4.4
(a) Nuclei of neuroectodermal and squamous epithelia of immature ovarian teratomas are stained by SOX2. (b) SOX2 expression is positive in embryonal carcinoma, while primitive endodermal elements of yolk sac tumor are negative
In mediastinal lesions, SOX2 is expressed in EC, isolated teratomatous glands, and foci of mature neural tissue and differentiates them from SOX2-negative lymphomas and epithelial tumors of the thymus [65].
Positive | Negative | |
|---|---|---|
Germ cell tumors | Embryonal carcinomas Immature teratoma (neuroectodermal and squamous component) Gliomatosis peritonei | Seminoma Yolk sac tumors Choriocarcinoma Teratomas Spermatocytic tumor |
Non-germ cell tumors | Squamous cell carcinoma, neuroendocrine carcinomas, primitive neuroectodermal tumors, melanomas, breast, pancreatic and biliary duct carcinomas, gliomas, etc. |
4.1.5 Lin28
4.1.5.1 Nomenclature
Protein lin-28 homolog, cell lineage abnormal 28, protein zinc finger CCHC domain-containing protein 1; ZCCHC1
4.1.5.2 Gene Function and Chromosomal Location
Lin28 was first identified in the nematode Caenorhabditis elegans , although their homologous genes exist in many species including mammals [96]. It is a monomeric cytoplasmic mRNA-binding protein that enhances the efficiency of protein synthesis. Additionally, Lin28 protein can be exported to the nucleus and nucleolus where it regulates the migration of mRNA to the cytoplasm [97].
Functionally, Lin28 plays an important role during embryonic development, particularly in skeletal muscle tissue, through upregulation of IGF2 mRNA, MYOD1, ARBP/36B4 ribosomal protein, and its own mRNA [98]. By contrast, Lin28 acts as a suppressor for the production of mature microRNA through its specific binding, followed by blocking and subsequent degradation of the let-7 precursor and thus contributing to the vital maintenance of embryonic stem cells [99]. Therefore, Lin28 may also promote protein synthesis through its direct association with the target mRNA, including that encoded by the transcription factor Oct4 [100]. For all these reasons, Lin28 is important for maintaining cell pluripotency during mammalian embryogenesis whereas, by contrast, it shows a very restricted expression in mature tissues. In fact, Lin28 together with OCT4, SOX2, and NANOG is used to reprogram human somatic cells into cells that exhibit the essential characteristics of ESC [101]. An experimental study has shown that Lin28 is essential in the development of mouse primordial germ cells and may be involved in their malignant transformation [102]. Likewise, this protein also plays an important role in the in vitro differentiation of germ cells from ESC and is expressed in the primordial germ cells up to the premeiotic stage [102, 103].
4.1.5.3 Recommended Clones and Practical Considerations
Antibody
Controls
For a positive control, seminoma or GCNIS can be used.
Staining pattern
Cytoplasmic with occasional membranous accentuation.
4.1.5.4 Expression
Recent immunohistochemical studies have demonstrated a 100 % positive staining of Lin28 in all precursor lesions (GCNIS and gonadoblastoma), seminoma (Fig. 4.5), EC, and YST, some primary and extragonadal teratomas (especially immature neuroepithelium), and choriocarcinoma (mononucleated trophoblastic cells). Isolated cases of spermatocytic tumor show weak staining [104–106]. In addition to its value in the diagnosis of GCT, Lin28 stains a higher proportion of YST cells as compared with SALL4 [106].
Fig. 4.5
Lin28 presents with a cytoplasmic and membrane staining pattern in a classic seminoma
Lin28 is variably expressed in isolated cases of breast, lung, ovary, colorectal, and liver carcinomas, where high expression is an indicator of poor prognosis [104, 106, 107]. The presence of Lin28 in colorectal cancer is significantly associated with lymph node metastasis [108]. In contrast with the expression of OCT4, which is only sporadically present in medulloblastoma and is significantly associated with a poor prognosis, Lin28 protein is more frequently detected but does not influence prognosis [109].
Positive | Negative | |
|---|---|---|
Germ cell tumors | Seminoma Embryonal carcinoma Yolk sac tumors Immature teratoma (neuroepithelium) Choriocarcinoma (cytotrophoblast) Spermatocytic tumor | Teratomas (epithelial elements) |
Non-germ cell tumors | Breast, lung, ovary, colorectal, or liver carcinomas |
4.1.6 NANOG
4.1.6.1 Nomenclature
NANOG is a homeobox transcription factor also known as FLJ12581.
4.1.6.2 Gene Function and Chromosomal Location
NANOG is another member of the network of pluripotency-maintaining factors, encoded by the human NANOG gene located on the chromosome region 12p13.31, a region frequently evoked in chromosomal studies of GCT. As compared with the murine model, where the Oct4, Nanog, and Sox2 genes co-regulate cohorts of downstream genes [110], in humans, they directly repress or activate small sets of downstream transcriptional regulators, controlling a broader range of cellular processes. Due to this uncoupled regulatory loop, these three genes do not work as pan-repressors of differentiation, but each one controls specific cell fates [111]. In human ESC, NANOG seems to have a lineage-restricted function to repress the neuroectodermal and neural crest commitment but, together with OCT4, it is required for their self-renewal. In a similar way to OCT4, NANOG is expressed by the cells of the inner cell mass of the blastocyst and in the pregastrulation epiblast but is not expressed by unfertilized oocytes, 2–16-cell embryos, or early morulae; it is only restricted to the proximal epiblast, while OCT4 has a diffuse expression [112]. NANOG has also been detected in the nuclei of the murine and human germline stem cells in the embryonic testis but is lost as the gonocytes mature to form spermatogonia in the adult testis [113].
4.1.6.3 Recommended Clones and Practical Considerations
Antibody
Controls
Similar to SALL4, GCNIS represents an optimal control.
Staining pattern
The antibody produces a nuclear staining that is by comparison less defined than SALL4.
4.1.6.4 Expression
In GCT, NANOG parallels OCT4 and stains the neoplastic cells of precursor lesions, seminoma, and EC, while YST, teratoma, and choriocarcinoma are consistently negative [65, 113]. Levels of NANOG mRNA are 2 to 12 times higher in seminoma compared with EC [113]; this is to be expected since seminoma represents a more primitive cell stage than EC.
NANOG is positive in other malignancies, including carcinomas of the prostate, lung adenocarcinomas, gliomas, rectal and gastric carcinomas, and oral squamous or nasopharyngeal carcinomas [28, 114]. An epithelial-mesenchymal transdifferentiation has been described in some of these cases, with the expression of NANOG and other stemness markers at the invasive front of the tumor. In these neoplasms, the expression is correlated with a worse prognosis, distant metastasis, and recurrence after conventional therapies [115].
A comparative study of NANOG expression between pre- and postmenopausal normal ovaries and those harboring ovarian epithelial malignancies demonstrated a high percentage of NANOG positive cells in the lining of ovarian inclusion cysts in patients with carcinomas, while the surface epithelium was consistently negative [116]. NANOG-positive cells were absent in the ovarian mucinous tumors, while in the serous carcinomas, there was a positive population that increased with tumor grade [117]. Nevertheless, other studies have demonstrated the presence of NANOG-positive stem cells on the surface epithelium of the ovary and also in the epithelium of the distal part of the fimbriae, results that may lend some support to recent theories on ovarian serous carcinomas pathogenesis [118].
Positive | Negative | |
|---|---|---|
Germ cell tumors | Seminoma Embryonal carcinoma | Yolk sac tumors Choriocarcinoma Teratomas Spermatocytic tumor |
Non-germ cell tumors | Carcinomas of the prostate, lung adenocarcinomas, gliomas, rectal cancer, gastric carcinomas, and oral squamous or nasopharyngeal carcinomas Epithelial-mesenchymal transdifferentiation areas |
4.1.7 UTF-1
4.1.7.1 Nomenclature
UTF-1 from undifferentiated embryonic cell transcription factor 1
UTF-1 is another transcription factor expressed in undifferentiated ESC and downregulated during their differentiation [119].
4.1.7.2 Gene Function and Chromosomal Location
UTF-1 is located on the chromosomal region 10q26.3 [120]. Its expression in ESC is regulated by a dimer composed of OCT4A and SOX2, and it is specifically attached to the chromatin, being excluded from the nucleoli during all phases of cell progression. Similar to OCT4 and NANOG, the UTF-1 is expressed by cells of the inner cell mass of the blastocyst and in the epiblast but is soon downregulated during development. It is maintained in primordial germ cells and, unlike OCT4 and SOX2 but similar to SALL4, is detected in the spermatogonia of the adult testis [121, 122].
4.1.7.3 Recommended Clones and Practical Considerations
Antibody
Most studies have used the mouse monoclonal antibody MAB4337 clone 5G10.2 which was raised against a recombinant GST fusion protein, human UTF1.
Controls
GCNIS or seminoma is optimal.
Staining patterns
With the clone 5G10.2, nuclear staining was obtained on various platforms. Alkaline pH is recommended for HIER.
4.1.7.4 Expression
The few available publications, mainly focused on GCT and their metastases, characterized UTF-1 as a marker for GCNIS and seminoma with variable staining intensity, while EC constantly shows strong and diffuse staining. A weak staining is detected in the YST epithelial component, while teratomas and choriocarcinomas are negative [122].
In other gonadal specific tumors, weak nuclear staining is also detected in a limited number of Sertoli and Leydig cell tumors. Similarly, carcinomas of the breast, stomach, and kidney and some soft tissue tumors such as alveolar soft part sarcoma, angiosarcoma, or epithelioid Ewing sarcoma may also be positive [122]. Semi-quantitative analysis of UTF-1 staining demonstrates a higher intensity in prostate and endometrial cancer, while lower levels are seen in colon and renal clear cell carcinomas [123].
Positive | Negative | |
|---|---|---|
Germ cell tumors | Seminoma Embryonal carcinoma Yolk sac tumors | Choriocarcinoma Teratomas Spermatocytic tumor |
Non-germ cell tumors | Sertoli and Leydig tumors Breast, stomach, and kidney carcinomas Soft tissue tumors like alveolar sarcoma, angiosarcoma, or epithelioid Ewing sarcoma, etc. |
4.1.8 TCL-1
4.1.8.1 Nomenclature
TCL-1 is also known as protein 1A of T-cell leukemia/lymphoma.
4.1.8.2 Gene Function and Chromosomal Location
TCL-1A is part of the TCL1 protein family and is encoded by a gene on chromosome region 14q32.13. This protein is involved in several chromosome translocations and gene inversions that characterize human prolymphocytic T-cell leukemia and some B-cell lymphomas, mainly through an increase in the phosphorylation and activation of the AKT1, AKT2, and AKT3 genes resulting in an antiapoptotic response. Also, after the induction of the nuclear translocation of AKT1, the TCL1A protein increases proliferation, stabilizes mitochondrial membrane potential, and promotes cell survival [124].
4.1.8.3 Recommended Clones and Practical Considerations
Antibody
Polyclonal antibodies and mouse monoclonal clone 27D6/20 have been used in the majority of the studies, both on lymphoid and GCT. In our limited experience, the rabbit clone EP105 was able to reproduce these results in all major types of GCT. An alkaline pH buffer should be used for HIER. Aside from the aforementioned clone EP105, mouse monoclonal antibodies have been developed against the whole TCL-1 human protein or just a fragment.
Controls
The strongest nuclear and cytoplasm staining of the T lymphocytes from tonsils with no background on the B lymphocytes should be used as a positive control.
Staining patterns
All developed antibodies exhibit nuclear and cytoplasmic staining.
4.1.8.4 Expression
Overexpression of human TCL1 gene has been implicated in the development of T-cell prolymphocytic leukemia. In the B-cell neoplasms, TCL1 gene acts as an oncogene, and the antibody is expressed in both the cytoplasmic and nuclear compartment of most B-cell lymphomas, including lymphoblastic lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma (60 %), and primary cutaneous B-cell lymphoma (55 %). TCL1 also has a constant and intense staining in Burkitt lymphomas. TCL1 is not expressed by Hodgkin/Reed-Sternberg cells or multiple myeloma, marginal cell lymphoma, anaplastic CD30-positive lymphoblastic T-cell lymphoma, peripheral T-cell lymphoma, or mycosis fungoides. These data indicate that TCL1 is expressed in more differentiated B cells of both neoplastic and reactive conditions [125].
The TCL1 protein is overexpressed in primary seminomas and its precursors (Fig. 4.6), suggesting a role in the pathogenesis of GCT [65, 126, 127]. Indeed, a relation between OCT4 and TCL1 has been demonstrated in mouse ESC as OCT4 binds to Tcl1 promoter and activates its transcription. Furthermore, a role in the regulation of proliferation, but not differentiation, has been demonstrated [128], which is in accordance with the idea of limited pluripotency in seminomas. A decrease of TCL1 expression, both in number of cells and intensity, was observed in metastatic seminomas after radiotherapy, while chemotherapy seems to have little effect on its expression [129]. Focal nuclear staining has been demonstrated in some EC while nuclear and cytoplasmic positivity is observed in isolated cells of spermatocytic tumors [129].
Positive | Negative | |
|---|---|---|
Germ cell tumors | Seminoma Focal in embryonal carcinoma and spermatocytic tumor | Yolk sac tumors Choriocarcinoma Teratomas |
Non-germ cell tumors | Most B-cell lymphomas Isolated cases of breast, esophagus, colon, gastric, urothelium, thyroid, biliary duct, and renal carcinomas | Hodgkin/Reed-Sternberg cells, multiple myeloma, marginal cell lymphoma, anaplastic CD30-positive lymphoblastic T-cell lymphoma, peripheral T-cell lymphoma, or mycosis fungoides |
Fig. 4.6
TCL1 nuclear and cytoplasmic staining in germ cell neoplasia in situ and intratubular seminoma
4.1.9 KLF4
4.1.9.1 Nomenclature
KLF4 from the Krüppel-like factor 4 is alternatively known as epithelial/endothelial zinc finger protein EZF or Gut-enriched Krüppel-like factor (GKLF).
4.1.9.2 Gene Function and Chromosomal Location
KLF4 is a member of the KLF family of transcription factors that acts as an upstream regulator of NANOG and represents a direct downstream of LIF-Stat3 signaling. Due to LIF signaling, KLF4 levels increase, bind to the NANOG promoter, and induce NANOG overexpression. OCT4 and SOX2 expression is not correlated with NANOG expression [130]. However, it seems that KLF4, together with NANOG, OCT4, and SOX2, represents the main transcription factors involved in the reprogramming of the fibroblast into a pluripotent stem cell stage [131]. It can be both an activator and a repressor, depending on with which genes it interacts, and it is aberrantly expressed in breast and colon cancer [132]. It can bind to the promoter region of its own gene located on chromosomal region 9q31.2 and can activate its own transcription. It plays an important role in terminal differentiation of some epithelial cells of the digestive tract but also lung, genital tract, and vascular endothelium [133].
4.1.9.3 Recommended Clones and Practical Considerations
Antibody
There is no standard regarding the clone to be used. Rabbit and mouse polyclonal antibodies are the most frequently employed, some of them requiring a 4 °C overnight incubation and thus have only limited use in routine diagnosis. Product H-180, a rabbit polyclonal raised against amino acids 1–180 of human KLF4, seems to provide a reasonable balance between nuclear staining and unspecific background.
Controls
As a marker of undifferentiated GCT, GCNIS or seminoma could both be used as positive controls.
Staining patterns
The antibody produces a nuclear staining similar to the other transcription factors.
4.1.9.4 Expression
Compared with NANOG, which highlights the nuclei of the spermatogonia in the human testis, KLF4 is strongly expressed in postmeiotic germ cells such as spermatids, where it plays an important role in spermiogenesis [133]. Limited data on KLF4 expression are available in GCT. Nuclear staining has been reported in GCNIS and seminoma [134].
In somatic tumors, KLF4 was proposed as yet another specific marker for monocytic differentiation in leukemia [135] and as a progression marker in nasopharyngeal [136] and prostate carcinomas [137].
Positive | Negative | |
|---|---|---|
Germ cell tumors | Seminoma | Embryonal carcinoma Yolk sac tumors Choriocarcinoma Teratomas Spermatocytic tumor |
Non-germ cell tumors | Monocytic leukemia, nasopharyngeal carcinomas, prostate adenocarcinomas | NA |
4.1.10 SOX17
4.1.10.1 Nomenclature
SOX17 gene might be alternatively referred to as SRY-related HMG-box gene 17.
4.1.10.2 Gene Function and Chromosomal Location
SOX17 is another member of the family of 20 SOX transcription factors with key roles in embryogenesis, being a Wnt signaling pathway antagonist and a critical specifier in pluripotent germ cell fate, as mentioned in Chap. 3. The protein is coded by the SOX17 gene located on the 8q11.23 chromosomal region, and its heterozygous mutations were detected in some congenital and sporadic urinary defects, generally grouped under the name of vesicoureteral reflux-3 (VUR-3). These are explained by the expression of SOX17 in the ureteric bud and metanephric mesenchyme of the developing kidney and urinary tract between 11.5–15.5 days after fertilization [138]. It is required also for both primitive mesoderm formation and normal looping of the embryonic heart tube as well as for the normal development of the definitive gut endoderm [139].
4.1.10.3 Recommended Clones and Practical Considerations
Antibody
From the limited data available, it seems that a goat polyclonal antibody, raised against the recombinant human SOX17, gives a clean nuclear staining. Other commercially available goat polyclonal antibodies against N-terminus of Sox-17 of mouse origin and a mouse monoclonal raised against E. coli-derived recombinant human SOX17 are both prone to nonspecific cytoplasmic staining [91]. For these reasons, a pH 6 buffer is recommended for HIER.
Controls
GCNIS and seminoma are used as positive controls.
Staining patterns
SOX17 produces a nuclear staining.
4.1.10.4 Expression
SOX17 is expressed in male and female fetal gonocytes, but in contrast with male spermatogenesis, no expression is detected in oogenesis beyond the stage of gonocyte [91]. In spermatogenesis, SOX17 is expressed in the stages of spermatogonium, spermatocyte, and spermatid, showing no staining in the other cells of the seminal epithelium [91].
Limited data are available on the expression of SOX17 in testicular GCT, but in contrast with SOX2, it stains cells of GCNIS and seminoma, while EC is negative [91, 95]. Thus, it is useful in the differentiation of seminoma from EC. Nuclear staining is present in all tumor components of YST, while the expression in teratoma is variable but always occurs in glandular elements, as expected of its role in endodermal development. Choriocarcinomas are negative [95].
Positive | Negative | |
|---|---|---|
Germ cell tumors | Seminoma Yolk sac tumors Teratomas (glands) | Embryonal carcinoma Choriocarcinoma Spermatocytic tumor |
Non-germ cell tumors | NA | NA |
4.1.11 AP-2γ
4.1.11.1 Nomenclature
AP-2γ, the activating enhancer-binding protein 2 gamma, is also called the transcription factor ERF-1 gene (estrogen receptor factor 1).
4.1.11.2 Gene Function and Chromosomal Location
The protein is encoded by the TFAP2C gene located on the 20q13.31 chromosome region. It is part of the AP2 family of transcription factors that interacts with various genes to control the ectoderm development, especially the skin and the neural crest [140]. In murine embryos, it has been shown to interact with transcription factors such as BLIMP1 and PRDM14 for the differentiation of epiblast-like cells into primordial germ cells [141]. It also cooperates with CDX2 in maintenance of the extraembryonic trophoblast. Together, they act in alternative pathways and are correlated with the repression of the pluripotency factor NANOG [142].
4.1.11.3 Recommended Clones and Practical Considerations
Antibody
AP-2γ clone 6E4/4 is a mouse monoclonal antibody raised against bacterially produced AP-2 protein and specifically recognizes the C-terminus of AP-2γ. It is the most frequently used clone, and at a pH 6 for HIER, it produces a sharp staining on various platforms.
Controls
GCNIS and seminomas can be used as positive control.
Staining patterns
Nuclear staining is the accepted pattern for this antibody.
4.1.11.4 Expression
In embryonal tissue, it is expressed by gonocytes, reaching its highest expression at 12 weeks of gestation, followed by a gradual downregulation, which correlates with testicular maturation. It is detected until the 37th week of gestation, being absent in the adult testis [143]. It is overexpressed by GCNIS, gonadoblastoma, and seminoma. Only focal expression was observed in EC, choriocarcinomas, skin, and skin adnexa of teratomas. YST and spermatocytic tumor are negative [47, 143].
AP-2γ also stains the granulosa cell layer of ovarian follicles and is weakly expressed in isolated cells of immature testicular granulosa cell tumors [47]. There is an AP-2γ progressive downregulation and loss of staining in primary melanomas as compared with nevi [144]. It is upregulated in advanced-stage ovarian carcinoma compared with early-stage carcinomas, borderline tumors, and ovarian surface epithelium [145], suggesting its possible relationship with tumor progression.
Positive | Negative | |
|---|---|---|
Germ cell tumors | Seminoma Focal in embryonal carcinoma, choriocarcinoma, and teratomas (epithelial elements) | Embryonal carcinoma Yolk sac tumors Choriocarcinoma Teratomas Spermatocytic tumor |
Non-germ cell tumors | Immature testicular granulosa cell tumors Nevi Advanced-stage ovarian carcinomas | Melanomas |
4.1.12 IMP3
4.1.12.1 Nomenclature
IMP3 or IGF-II mRNA-binding protein 3 is also known as KH domain containing protein overexpressed in cancer, KOC1, L523S, or IGF2BP3.
4.1.12.2 Gene Function and Chromosomal Location
IMP3 is the third member of IGF-II mRNA-binding protein family, which contains a combination of two RNA recognition motifs and four hnRNP K homology domains. They are coded by the IGF2BP3 gene located on the chromosomal region 7p15.3. They have a high affinity and multiple attachments to the IGF-II leader 3, an untranslated region of the insulin-like growth factor II, an important growth factor in embryogenesis [146].
In humans, IGF2 gene expression is controlled by the imprinting control region 1 (ICR1) located in chromosome 11p15.5. DNA methylation defects involving the ICR1 are responsible for two growth disorders: (1) the Beckwith-Wiedemann syndrome (maternal ICR1 hypermethylation) characterized by a disproportionate overgrowth of the fetus, malformations, and a high risk of developing renal tumors, rhabdomyosarcomas, or hepatoblastoma and (2) the Silver-Russell syndrome (paternal ICR1 loss of methylation) which is generally characterized by growth restriction [147].
4.1.12.3 Recommended Clones and Practical Considerations
Antibody
Clone 69.1 was raised against amino acids 2–580 of a recombinant protein similar to human IMP3 and has been used in the majority of the studies. Moreover, the IGF2BP3 (IMP3) mRNA is correlated with the status of protein expression by immunohistochemistry in ovarian clear cell carcinomas [148]. In our experience, the rabbit clone EP286 raised against a synthetic peptide corresponding to human IMP3 provides comparable results.
Controls
Cytoplasmic and nuclear staining is observed in a large panel of fetal tissues, while ganglionic and granular layers of the cerebellar cortex or germ cells of the adult ovary or testis (see below) can be used as a positive control.
Staining patterns
Cytoplasmic and nuclear staining is the accepted pattern.
4.1.12.4 Expression
IMP3 is expressed ubiquitously in fetal tissues, while in the gonads, it has been detected in resting and growing oocytes and granulosa cells of the ovary. In the adult testis, a cytoplasmic and nuclear staining is demonstrated in all spermatogonia, spermatocytes, and spermatozoa [149]. Extensive studies have demonstrated that IMP3 acts as an oncoprotein with no expression in benign tissues, whereas it is highly associated with aggressive and advanced cancer, promoting tumor cell proliferation, invasion, and metastasis [150, 151].
It has been suggested that IMP3 plays a role in GCT development, and its immunohistochemical expression has been demonstrated in the majority of these tumors, including spermatocytic tumor [149]. Its highest expression has been reported in EC, but what makes this antibody remarkable is the difference in its expression in male and female teratomas; it is positive in primary and metastatic testicular teratomas (Fig. 4.7) but negative in ovarian teratomas [151]. These data may further support reflect the malignant nature of testicular teratomas and the possibility of a different histogenesis from ovarian teratomas. This difference in expression might be explained by the parental imprinting of the Igf-II gene demonstrated in mice which shows that the difference in growth phenotype depends on the type of gamete contributing to the mutated allele. As the paternal allele is expressed in the majority of the tissues [152], the expression in only male teratomas might be simply sex related.
Fig. 4.7
Testicular teratoma and germ cell neoplasia in situ showing a nuclear and cytoplasmic expression for IMP3
For primary and metastatic GCT diagnosis, the antibody is of little use as it cannot be used either as a marker for GCNIS, due to its expression in spermatogonia, or to differentiate ovarian GCT from somatic carcinomas as it stains mucinous, clear cell, serous, and faintly endometrioid carcinomas of the ovary [148].
Positive | Negative | |
|---|---|---|
Germ cell tumors | Seminoma Embryonal carcinoma Yolk sac tumors Choriocarcinoma Spermatocytic tumor Testicular teratomas (epithelial elements) | Ovarian teratomas (epithelial elements) |
Non-germ cell tumors | Mucinous, clear cell, and endometrioid carcinomas of the ovary GISTs, angiomyolipoma, mastocytosis, thymic carcinomas, lung carcinomas, etc. | Solitary fibrous tumor, alveolar soft part sarcoma, desmoplastic small round cell tumor, glomus tumor, leiomyoma, etc. |
4.1.13 GATA3
4.1.13.1 Nomenclature
4.1.13.2 Gene Function and Chromosomal Location
GATA3 is an essential enhancer of T-cell receptors, binding to its alpha and delta receptor genes via the 2 GATA-type zinc fingers. The human GATA3 gene was mapped on the 10p15 chromosomal region, a critical region in the development of the parathyroid glands, inner ear, and kidneys [155]. Genetic mapping and subsequent functional studies indicate that deletion of chromosome 10 (del10p) or GATA3 gene mutations induce its functional haplo-insufficiency and causes human HDR syndrome, also known as Barakat syndrome, characterized by hypoparathyroidism, sensorineural deafness, and renal disease [156]. In embryonic life, besides its importance in development and differentiation of the luminal breast epithelium, parathyroid gland, adipose tissue, and other non-hematopoietic organs [157], GATA3 is required for the differentiation of CD4-positive T-helper 2 (Th2) cells, a process relevant in immune and inflammatory responses [158].
4.1.13.3 Recommended Clones and Practical Considerations
Antibody
With clone HG3-31 of GATA3, a mouse monoclonal antibody raised against the N-terminus of human recombinant GATA3, positivity in GCT has never been reported. However, the introduction of clone L50-823 raised against a peptide between trans-activation and DNA-binding domains of GATA3 has consistently demonstrated trophoblastic and endodermal expression [159]. Due to its reduced sensitivity, clone HG3-31 should not be used in routine diagnosis.
Controls
Urothelial and breast normal epithelium and carcinomas can be used as positive controls.
Staining patterns
Only nuclear pattern is considered positive.
4.1.13.4 Expression
Nuclear staining was detected in the epidermis, peritoneal mesothelium, and all trophoblastic cells of a 7-week-old embryo with some focal positivity in the caudal mesenchyme and endothelial cells [159]. Normal human secondary yolk sacs do not express GATA3, not even those from as early as the 5th and 6th weeks [160]. Meanwhile, the epidermis, epithelia of oral cavity, olfactory plate, periocular mesenchyme, skull base cartilage, isolated cells in the brain, and nerve glia as well as one-third of the tubular structures and glomerular mesangial cells were positive in a 10-week-old fetus [159].
In adult tissue, GATA3 staining is frequently encountered in many tissues, including the hematopoietic (blood, bone marrow, thymus, B, T, erythroid, and myeloid lineages), blood vessels (endothelial cells), adipocytes, adrenal gland, urothelium, mammary gland, brain, and hair follicles. In the kidney, the collecting ducts, distal tubules, and mesangial cells are positive. Variable staining in seminal vesicle and prostate basal cells is also observed. Terminal ducts of the parotid gland and the thymic cortex are also positive. In normal testis, no staining is observed. Decidual stromal change in the uterus and endometriosis may also be positive [159].
Initial immunohistochemical studies demonstrated a higher sensitivity of GATA3 nuclear staining for tumors of urothelial and breast origin, compared with classical markers [157]. An even lower expression is seen in triple negative, medullary, and metaplastic breast carcinomas [161]. Nevertheless, as more studies are published and clone L50-823 is used, GATA3 is seen to demonstrate an absence of specificity, being positive in many tissues such as parathyroid [162], salivary gland tumors [163]; mucinous and non-mucinous pancreatic adenocarcinomas; squamous cell carcinomas; basal cell carcinomas; chromophobe carcinomas of the kidney; mesotheliomas, poorly differentiated papillary, or mucinous adenocarcinomas of the lung; carcinomas of the stomach, colon, prostate, endometrium, thyroid, serous, and Brenner benign; and borderline ovarian tumors. Most extra-adrenal paragangliomas are positive, while the glandular component of synovial sarcomas may also be stained. Other types of sarcomas may also be stained [159]. It was also described as a sensitive marker of benign and malignant mesonephric lesions in the lower female genital tract [164].
Clone L50-823 also highlights the choriocarcinoma component of mixed GCT (both cyto- and syncytiotrophoblasts) (Fig. 4.8a) and the syncytiotrophoblastic cells of seminoma [159], which supports a GATA3 role in trophoblast differentiation [165]. Besides the staining in tumors with trophoblastic differentiation, endodermal elements are also strongly stained, while only focal elements of EC are identified [159]. This might be explained by cross-reactivity of the antibody with other GATA family members, as GATA-4, GATA-5, and GATA-6 are expressed in the endoderm in an overlapping manner [47, 158].
Fig. 4.8
(a) Area of trophoblastic differentiation expressing GATA3. (b) GATA3 nuclear staining highlights areas of primitive pattern of yolk sac tumor
A study on YST using clone L50-823 demonstrated that GATA3 is only expressed by the primitive areas of YST (reticular-microcystic, endodermal sinus, polyvesicular, polyembryoma) (Fig. 4.8b), while the less frequent somatic patterns (like glandular, hepatoid, and solid) are consistently negative. In routine diagnostic procedures, most YST are identified by the presence of primitive areas which express GATA3; consequently it is a good marker for classic types of YST. However, hepatoid, glandular, and solid YST, which often constitute a diagnostic challenge, do not consistently express GATA3 [160].
Positive | Negative | |
|---|---|---|
Germ cell tumors | Choriocarcinoma Syncytiotrophoblasts of seminomas Yolk sac tumors Squamous differentiation mature teratoma | Seminoma Embryonal carcinoma Teratomas Glandular and hepatoid YST Spermatocytic tumor |
Non-germ cell tumors | Urothelial carcinomas, breast carcinomas, parathyroid carcinomas, mucinous and non-mucinous pancreatic adenocarcinomas, squamous and basal cell carcinomas, etc. |
Table 4.1 summarizes pluripotentiality, transcription factors, and developmental stage-related markers used in immunohistochemical diagnosis of GCT, while Table 4.2 analyzes antibodies published in research papers but less frequently used in diagnostic histopathology. Figure 4.9 depicts neoplastic differentiation pathways and its corresponding pluripotentiality markers in both type II GCT (associated with GCNIS or gonadoblastoma) and parthenogenetically activated Type IV GCT.
Table 4.1
Pluripotentiality, developmental stage-related markers in GCT. The first five antibodies are the most useful ones in daily practice and recommended by the International Society of Urologic Pathology [3]
Antibody | EGC | GCNIS-GB | Sem | EC | YST | Choriocarcinoma | IT | MT | ST | Usefulness Availability |
|---|---|---|---|---|---|---|---|---|---|---|
SALL4 | + | + | + | + | + | ± | ± (glands and neuroepithelium) | − | ± | +++ |
OCT4 | + | + | + | + | − | − | − | − | − | +++ |
SOX2 | − | − | − | + | − | − | ± (neuroepithelium) | ± (squamous differentiation) | − | +++ |
CD117 | + | + | + | − | − | − | − | − | + | ++ |
SOX17 | + | + | + | − | + | − | − | ± (glands) | − | + |
GATA3 | − | − | − | + | + (syncytiotrophoblast) | − | + (squamous differentiation) | − | ± | |
Lin28 | + | + | + | + | + | + (cytotrophoblast) | ± (epithelial component) | − | − | − |
NANOG | + | + | + | + | − | − | − | − | − | − |
AP-2γ | + | + | + | ± | − | ± | − | ± (epithelial component) | − | − |
IMP3 | + | + | + | + | + | + | − | +M /− F | + | − |
UTF-1 | + | ND | + | + | + | − | − | − | − | − |
TCL-1 | ND | + | + | ± | − | − | − | − | ± | − |
KLF4 | ND | + | + | − | − | − | − | − | − | − |
Table 4.2
Antibodies published in research papers but less frequently used in diagnostic histopathology
Antibody | Pattern | Gene | Gene location | Expression in normal tissues | GCNIS-GB | Sem | EC | YST | ChoCa | T | ST | Somatic tumors |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
Nuclear | AURKB | 17p13.1 | Spermatogonia ± spermatocytes | + | + | − | − | ND | − | ND | Poor prognostic marker in different carcinomas | |
Nuclear | 4q27 | Spermatogonia | + | + | + | + | + | + | ND | Poor prognostic marker in stage I non-small cell lung cancer | ||
Nuclear | ESR2 | 14q23.2 | Spermatogonia, primary and secondary spermatocytes, and round spermatids Sertoli cells | ± | −/+ | −/+ | + | ± | + | ND | ND | |
Nuclear | GATA4 | Granulosa and theca cells in both preantral and antral follicles, Sertoli and Leydig cells through fetal and postnatal development, fetal germ cells and prepubertal spermatogonia | ND | + | ND | + | ND | ND | ND | Sertoli and Leydig cell tumors | ||
Cytoplasm | GDF3 | 12p13.1 | Cerebral cortex, hippocampus, cerebellum | + | + | + | + | + | − | ND | ND | |
GLUT3 [180] | Membrane | 12p13.31 | Vascular endothelium, spermatozoa | ND | + | + | + | − | + mature T | − | − | |
Cytoplasm | GPER1 | 7p22.3 | Sertoli cells, spermatogonia, and spermatocytes | + | + | + | − | − | −/+ | ND | High-risk endometrial carcinomas with low hormone receptors | |
Cytoplasm | HMGA1 | 6p21 | ND | + | + | + | − | ND | − | ND | Most malignant neoplasias | |
Cytoplasm and nuclear | MAGEA4 | Pre-spermatogonia, spermatogonia, and early spermatocytes | ± | + | − | − | − | − | + | Melanomas, certain carcinomas and sarcomas, uterine neoplasms, serous ovarian carcinomas | ||
Nuclear | MAGEC2 | NA | + | + | NA | NA | NA | NA | + | Melanoma, urothelial carcinomas, etc. | ||
NEK2 [192] | Nuclear | NEK2 | 1q32.3 | − | + | − | − | − | − | − | ND | ND |
Cytoplasm and nuclear | NUTM1 | 15q13 | NA | + | ± | ± | + hepatoid and intestinal/glandular differentiation | ND | ± immature hepatoid and intestinal/glandular differentiation | ND | Midline carcinomas | |
Cytoplasm | CTAG1B | Xq28 | Gonocytes (from18 months with maximum level at 40 months); spermatogonia and in primary spermatocytes of the adult testes | + | − | − | − | − | − | + | Melanomas, certain carcinomas and sarcomas, uterine neoplasms, serous ovarian carcinomas | |
PATZ1 [196] | Cytoplasm | PATZ1 | 22q12.2 | Sertoli, spermatogonia | + cyt | + cyt | + cyt | + cyt | + cyt | + cyt | NA | NA |
Nuclear | RNF4 | 4p16.3 | Spermatogonia, spermatocytes, spermatides | − | − | − | − | − | − | − | − | |
Oocytes | + | + | + | + | + | + | − | − | ||||
Nuclear | SOX9 | 17q24.3 | NA | + GCNIS but ± in gonadoblastoma | + | ND | ND | ND | ND | ND | Ductal pancreatic tumors | |
TSPY1 [202] | Nuclear | TSPY1 | Yp11.2 | NA | + | + | ND | ND | ND | ND | ND |
Fig. 4.9
Immunophenotypic expression of pluripotentiality factors in GCT developmental pathways. Type II tumors (postpubertal testicular and extragonadal), originated from germ cell neoplasia in situ (blue arrows and text), are different from ovarian parthenogenetically activated type IV (red arrows and text). Each tumor type exhibits a characteristic immunophenotype related to its degree of pluripotentiality, ranging from an unrestricted one in seminoma-dysgerminoma and embryonal carcinoma to absent in terminally differentiated tumors like choriocarcinoma and mature teratoma. Tumors of intermediate type of differentiation, such as yolk sac tumors, immature teratomas, and testicular postpubertal teratomas present only a partial, heterogeneous expression
4.2 Classic Markers
4.2.1 PLAP
4.2.1.1 Nomenclature
Placental alkaline phosphatase or PLAP; it is also known as Regan isoenzyme.
4.2.1.2 Gene Function and Chromosomal Location
Alkaline phosphatase encompasses a family of four different genes that encodes hydrolase enzymes. The genes that encode the placental, the placental-like, and the intestinal form are located on chromosome 2q37, while the fourth one which encodes the hepatic, osseous, and renal type is located on chromosome 1p36.12 [166–168].
PLAP is normally secreted by the placental syncytiotrophoblasts and physiologically is involved in cellular transport, proliferation, and differentiation, regulation of metabolism, and gene transcription [169]. Even if its serum levels might be separately identified [170], due to its expression in normal conditions and several malignancies [53, 170–174], it is not considered a reliable diagnostic tool, although high levels in GCT are associated with seminomas [175, 176].
4.2.1.3 Recommended Clones and Practical Considerations
Antibody
Mouse monoclonal antibodies such as 8A9 and NB-10 are the most frequently used. Clone SP15, a rabbit monoclonal antibody, can also be used with optimal results in the majority of the actual platforms. Alkaline buffer for the HIER is required.
Controls
Placenta is the optimal positive control. Normal testicular and ovarian parenchyma should be negative. A cross-reactivity with skeletal and smooth muscle fibers occurs with clone 8A9 and should be accepted, as stated on various vendors’ data sheets.
Staining patterns
Membranous and cytoplasmic are the expected patterns.
4.2.1.4 Expression
A membranous and rarely cytoplasmic positivity was identified in different normal tissues and various tumors other than germ cells [177, 178]. In the fetal testis, distinct positivity is observed in the primitive germ cells [179], but normal adult testicular tissue is negative [180]. In our experience, significant loss of immunoreactivity occurs in poorly fixed or autolyzed material.
PLAP is re-expressed not only in precursor neoplastic germ cell lesions (GCNIS and gonadoblastoma) [38, 180, 181] but also in infiltrative gonadal and extragonadal, primary, or metastatic GCT [182–184]. Seminomas account for the strongest reactivity in up to 100 % of the cases (Fig. 4.10a), followed by a lower intensity of the staining in 97 % of EC (Fig. 4.10b) and 85 % of YST. Staining is focal in cytotrophoblasts of choriocarcinoma [180, 181], and areas of immature elements in teratoma may also be positive [180]176.
Positive | Negative | |
|---|---|---|
Germ cell tumors | Seminoma Embryonal carcinoma Yolk sac tumors Choriocarcinoma Teratomas (epithelial elements) | Spermatocytic tumor |
Non-germ cell tumors | Gastrointestinal, gynecologic, lung, breast, and urologic tumors |
Fig. 4.10
(a) PLAP membranous expression in seminoma and germ cell neoplasia in situ while a similar pattern is seen in focal areas of embryonal carcinoma (b)
4.2.2 CD30
4.2.2.1 Nomenclature
CD30 is recognized under various names such as Ki-1 or TNFRSF8 (tumor necrosis factor receptor superfamily member 8) and is normally expressed by several embryonal tissues and consequently considered important in cell proliferation and development [185].
4.2.2.2 Gene Function and Chromosomal Location
4.2.2.3 Recommended Clones and Practical Considerations
Antibody
The monoclonal antibody generally used for its detection is Ber-H2, and it stains the cell membranes in alkaline HIER conditions.
Controls
Tonsil-activated inter- and perifollicular B and T cells should be used as low-expressing positive controls; they must show a distinct membranous staining and focally a dot-like reaction in the Golgi area of the cytoplasm. Positive classic Hodgkin’s lymphoma or EC could be used as positive controls. Staining should be as strong as possible and should not be accompanied by any unspecific staining or background.
Staining patterns
In lymphoid neoplasia, membrane and Golgi area patterns are frequently demonstrable. In EC, only membrane staining is noted. The presence of cytoplasmic staining could be related to tissue fixation and/or autolysis.
4.2.2.4 Expression
It was first described in various lymphoproliferative disorders, particularly expressed on the surface of Reed-Sternberg cells of Hodgkin’s lymphoma [190] and the cells of anaplastic large cell lymphoma (Ki-1 lymphoma) [191]. It is also overexpressed by the leukocytes of patients with chronic inflammatory diseases and has been suggested as a therapeutic target in autoimmune diseases [192].
In GCT, CD30 emerged as a highly specific antibody for EC (Fig. 4.11) [18, 43, 186, 193, 194]. Chemotherapy may modulate CD30 expression in EC, as it has been reported negative in a series of treated cases [195]. Additional EC markers may be necessary in these cases to reach a diagnosis [45]. As evidence of its close relationship with EC, minute foci of expression in classical seminoma would suggest early transformation into EC [196]. The accompanying undifferentiated GCNIS is constantly negative [17, 18, 43, 196], and, as expected, intratubular EC should stain positive. Generally, other GCT fail to express CD30 [17, 18, 43, 196, 197] and only isolated YST and mature teratoma may display focal positivity. This makes CD30 one of the most specific antibodies in a GCT diagnostic panel.
Positive | Negative | |
|---|---|---|
Germ cell tumors | Embryonal carcinoma | Seminoma Yolk sac tumor Choriocarcinoma Teratomas Spermatocytic tumor |
Non-germ cell tumors | Hodgkin’s lymphomas, anaplastic large cell lymphoma, anaplastic variant of diffuse large B-cell lymphoma, CD30-positive cutaneous lymphoproliferative disorders |
Fig. 4.11
CD30 specifically stains cell membranes of embryonal carcinoma
4.2.3 Alpha Fetoprotein
4.2.3.1 Gene Function and Chromosomal Location
Alpha-fetoprotein (AFP) is a member of the albuminoid gene superfamily, encoded by a gene localized on 4q11-q22 region [198]. This early protein is secreted by both primitive and secondary human yolk sac and is involved in the binding and transport of ligands. Among its many presumed functions, it has been proposed that it plays a role as growth regulator in fine-tuning the architectural interstitial growth patterns in developing organisms [199].
4.2.3.2 Recommended Clones and Practical Considerations
Antibody
Polyclonal antibodies have been used to demonstrate the presence of the protein in different tissues. A recently introduced rabbit monoclonal antibody, clone EP209, gives crisp staining on endodermal elements of mixed GCT. Its specificity and sensitivity are still to be determined in ample studies.
Controls
Well-fixed and non-autolysed embryonal liver is a reliable positive control.
Staining patterns
Both normal and pathologic positive tissues give an often granular cytoplasmic staining that is particularly strong in intracytoplasmic lumina, as it occurs in the human yolk sac [200]. As a protein secreted in the serum, a strong nonspecific background reaction and positivity in necrotic areas and cystic contents is regularly seen.
4.2.3.3 Expression
AFP remains a highly specific immunohistochemical marker for YST, although, in the same way as β-hCG, it is also secreted by many non-GCT not only in the female genital tract [201] but also by tumors of other organs, usually those of endodermal origin with an associated hepatoid component [202].
Diagnostically relevant serum AFP isoforms are produced in non-neoplastic liver disease (L1) and liver cancers (L3), while L2 isoform is characteristic of YST [203]. Interestingly, both L2 and L3 isoforms can be elevated in pediatric YST reflecting both yolk sac and hepatic differentiations [204]. In the secondary human yolk sac, AFP positivity is limited to the endodermal layer where intracellular vesicles and endodermal tubules are highlighted by strong membrane AFP expression [200]. In primitive YST patterns, AFP produces a strong granular cytoplasmic positivity (Fig. 4.12a), especially in intracellular lumina. It may also be present in the hyaline globules, although most of them are not immunoreactive for this antigen [205]. Classical patterns have a heterogeneous but consistent AFP expression. However, in somatic glandular patterns, staining is either only focal or absent, often restricted to cytoplasm and the apex of isolated columnar cells, and difficult to identify at low power [206]. AFP-positive areas in embryoids (Fig. 4.12b) and EC reflect differentiation into YST [207]. GCNIS, gonadoblastoma, seminoma, EC, choriocarcinoma, and spermatocytic tumor are negative.
Positive | Negative | |
|---|---|---|
Germ cell tumors | Yolk sac tumors Teratomas (some epithelial elements) | Seminoma Embryonal carcinoma Choriocarcinoma Teratomas Spermatocytic tumor
 Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
Get Clinical Tree app for offline access
|