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SALL4 Is a Novel Diagnostic Marker forTesticular Germ Cell Tumors

Dengfeng Cao, MD, PhD,* Jianping Li, BS,* Charles C. Guo, MD,wRobert W. Allan, MD,z and Peter A. Humphrey, MD, PhD*

Abstract: The diagnosis of testicular germ cell tumors (GCTs)

sometimes can be challenging without ancillary markers. Here

we performed an immunohistochemical study of a novel stem

cell marker SALL4 in a large series of 110 primary testicular

GCTs (65 pure and 45 mixed) containing the following types of

tumors and/or tumor components: 50 intratubular germ cell

neoplasias (ITGCNs), 62 classic seminomas, 2 spermatocytic

seminomas, 39 embryonal carcinomas (EC), 5 pediatric and 26

postpubertal yolk sac tumors (YST), 7 pediatric and 25

postpubertal teratomas, and 5 choriocarcinomas. We compared

SALL4 with OCT4 in all GCTs, and SALL4 to a-fetoprotein(AFP) and glypican-3 in all YSTs. To test SALL4 specificity,

23 testicular non-GCTs (10 Leydig cell tumors, 4 Sertoli

cell tumors, 3 adenomatoid tumors, 3 paratesticular rhabdo-

myosarcomas, 2 diffuse large B-cell lymphomas, and 1 rete

testis papillary cystadenoma) and 275 nontesticular tumors

(158 metastatic carcinomas, 12 metastatic melanomas, 11

primary and 2 metastatic mesotheliomas, and 72 primary and

20 metastatic sarcomas) were also stained for SALL4. All

ITGCNs, classic seminomas, and ECs demonstrated strong

SALL4 and OCT4 staining in more than 90% tumor cells. All 31

YSTs (5 pediatric and 26 postpubertal) showed strong positive

SALL4 staining in more than 90% tumor cells but had negative

OCT4 staining. Both spermatocytic seminomas showed positive

SALL4 staining in 80% to 95% tumor cells in all 3 types of

tumor cells with weak-to–moderate staining intensity. Mono-

nucleated trophoblastic cells were variably positive for SALL4

staining in all 5 choriocarcinomas. Focal SALL4 staining was

seen in 4 of 7 pediatric and 23 of 27 postpubertal teratomas.

OCT4 staining was not seen in any spermatocytic seminoma,

choriocarcinoma, or teratoma. No SALL4 staining was seen in

all 23 testicular non-GCTs. Of 275 nontesticular tumors, only 10

carcinomas and 1 sarcoma showed focal (<25% tumor cells)

weak SALL4 staining. The only non-neoplastic cells within the

testis stained with SALL4 were spermatogonia and few primary

spermatocytes. AFP staining was seen in 29 of 31 YST but it was

often focal and patchy. Although all 31 YSTs showed glypican-3

staining, 14 (45%) show staining in less than 30% tumor cells.

Our findings indicate that SALL4 is a novel sensitive and

relatively specific marker for testicular GCTs. SALL4 is a more

sensitive marker than AFP and glypican-3 for YST.

Key Words: SALL4, testis, germ cell tumor, yolk sac tumor,

diagnostic marker

(Am J Surg Pathol 2009;33:1065–1077)

More than 90% testicular tumors are of germ cellorigin.44 Although most are malignant, testicular

GCTs are curable with modern therapy.11 TesticularGCTs of different histologic types differ in their clinicalbehavior and prognosis, and therefore are manageddifferently. For example, seminoma is generally treatedwith low-dose radiation, whereas nonseminomatousGCTs are not treated with radiation therapy.3,11 There-fore, accurate pathologic diagnosis and proper classifica-tion of testicular GCTs is critical for selecting appropriatetreatment and determining prognosis. The diagnosisof testicular GCT tumor is usually straightforward basedon examination of hematoxylin and eosin sections, butsometimes it can be challenging because about half of theadult testicular GCTs contain more than one type oftumor and different types of testicular GCTs often showoverlapping morphologic features and can mimic oneanother and mimic non-GCTs.47,48 In these difficult cases,ancillary diagnostic markers are often needed.

Several diagnostic markers have been used tofacilitate the diagnosis of testicular GCTs. Earlier markersincluded placental-like alkaline phosphatase, CD30, CD117(C-KIT), and a-fetoprotein (AFP). Although these markersare useful for testicular GCTs, they show only moderatesensitivity and/or specificity.5,12,14,15,19,20,26,29,34,46,51 Inaddition, their expression may be lost in metastatic foci27

or after treatment.4 Recently, glypican-3 has beenproposed as a new marker for yolk sac tumor (YST),but its staining is typically focal.31,57 For these reasons,novel markers are urgently needed. Recently, novelembryonic stem cell markers such as OCT4, NANOG,and SOX2 have emerged as more sensitive and specificmarkers for testicular GCTs. For example, OCT4 andCopyright r 2009 by Lippincott Williams & Wilkins

From the *The Lauren V. Ackerman Laboratory of Surgical Pathology,Division of Anatomic and Molecular Pathology, Department of Patho-logy and Immunology, Washington University School of Medicine,Saint Louis, MO; wDepartment of Pathology, The University of TexasMD Anderson Cancer Center, Houston, TX; and zDepartment ofPathology, University of Florida, Gainesville, FL.

Correspondence: Dengfeng Cao, MD, PhD, The Lauren V. AckermanLaboratory of Surgical Pathology, Division of Anatomic andMolecular Pathology, Department of Pathology and Immunology,Washington University in Saint Louis, 660 S Euclid Avenue, CampusBox 8118, Saint Louis, MO 63110 (e-mail: [email protected]).

ORIGINAL ARTICLE

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NANOG label seminoma and embryonal carcinoma(EC).13,16,40 SOX2 labels EC.8,40 OCT4, NANOG, andSOX2 are sensitive and specific markers for testicularGCTs, but they are only expressed in a subset of testicularGCTs: intratubular germ cell neoplasia (ITGCN), semi-noma, and EC. None of them is expressed in other typesof testicular GCTs such as spermatocytic seminoma,YST, and choriocarcinoma, and thus in certain circum-stances their diagnostic utility is limited.

OCT4, NANOG, and SOX2 function to maintainthe pluripotency and self-renewal of embryonic stem cells.Recent studies have shown that the maintenance of thepluripotency and self-renewal of embryonic stem cells isregulated by a group of genes not only including OCT4,NANOG, SOX2 but also SALL4.23,49,52,53 Within thisnetwork, SALL4 regulates transcription of OCT4,55,56

indicating that SALL4 acts upstream of OCT4. Therelationship between SALL4 and OCT4, NANOG andSOX2 suggests that SALL4 might also be a marker fortesticular GCTs. The aim of this study is to investigate thepotential diagnostic utility of SALL4 in a large series of110 primary testicular GCTs (65 pure and 45 mixed). Totest SALL4 specificity for testicular GCTs, 23 testicularnon-GCTs and 275 nontesticular non-GCTs (158 meta-static carcinomas, 12 metastatic melanomas, 11 primaryand 2 metastatic mesotheliomas, and 72 primary and 20metastatic sarcomas) of different origins were also stainedfor SALL4. The SALL4 result on carcinomas andmelanomas has been also reported in a previous study.6

We compared SALL4 with OCT4 in all testicular GCTs.We also compared SALL4 with 2 current diagnosticmarkers AFP and glypican-3 for YSTs.

MATERIALS AND METHODS

Case SelectionApproval to perform this study was obtained

from Institutional Review Boards of each institution.The surgical pathology files of Washington UniversityMedical Center, Shands Hospital at The University ofFlorida, and the University of Texas MD AndersonCancer Center were searched for primary testicular GCTsand non-GCTs. One hundred and ten primary testicularGCTs were retrieved for this study including 65 puretumors (44 classic seminomas, 5 pure ECs, 5 pure YSTs,9 pure teratomas, and 2 spermatocytic seminomas) and45 mixed GCTs (with classic seminoma component in18 cases, YST component in 26, EC component in 34,teratoma component in 25, and choriocarcinoma compo-nent in 5 cases). Eleven of these 110 patients are children(6 with pure mature teratomas, 4 with pure YSTs, 1 withmixed GCT consisting of mature teratoma and YST) andthe remaining 100 tumors are from postpubertal patients.The diagnosis of all cases and classification of eachcomponent were established using the most recent WorldHealth Organization criteria.9

To test SALL4 specificity, 23 primary testicularnon-GCTs including 10 Leydig cell tumors, 4 Sertoli celltumors, 3 adenomatoid tumors, 3 paratesticular rhabdo-

myosarcomas, 2 diffuse large B-cell lymphomas, and 1rete testis papillary cystadenoma were also stained forSALL4. In addition, 275 nontesticular tumors fromvarious organs and sites were also stained for SALL4:158 metastatic carcinomas (6 head and neck squamouscell carcinomas, 8 thyroid carcinomas, 12 lung carcino-mas, 8 breast carcinomas, 7 hepatocellular carcinomas,3 cholangiocarcinoma, 2 ampullary adenocarcinomas,10 pancreatic adenocarcinomas, 18 gastric adenocarcino-mas, 15 esophageal carcinomas, 10 renal-cell carcinomas,10 urothelial carcinomas, 12 prostatic adenocarcinomas,18 ovarian carcinomas, 6 uterine carcinomas, 13 colo-nic adenocarcinomas), 12 metastatic melanomas, 11primary and 2 metastatic mesotheliomas (5 abdominal,8 pleural), and 72 primary and 20 metastatic sarcomas(3 primary and 4 metastatic alveolar soft part sarcomas,6 primary and 1 metastatic angiosarcomas, 7 chondro-sarcomas, 2 clear-cell sarcomas of soft tissue, 3 primaryand 2 metastatic epithelioid sarcomas, 7 fibrosarcomas,6 primary and 2 metastatic leiomyosarcomas, 5 primaryand 1 metastatic liposarcomas, 9 malignant fibroushistiocytomas, 7 primary and 1 metastatic malignantperipheral nerve sheath tumors, 6 primary and 1 meta-static osteosarcomas, 2 primary and 2 metastatic primitiveneuroectodermal tumors/Ewing’s sarcomas, 8 primary and1 metastatic rhabdomyosarcomas, and 1 primary and 5metastatic synovial sarcomas). The immunohistochemicalstatus of SALL4 in these carcinomas and melanomas wasalso reported in a previous study.6

Immunohistochemical Staining and EvaluationFor each case, 1 to 3 formalin-fixed paraffin-

embedded tissue blocks were retrieved to generate 4 mmunstained slides for immunohistochemical staining witha SALL4 monoclonal antibody (clone 6E3, dilution1:100, Abnova Corporation, Taiwan, Republic of China).For GCTs, OCT4 immunostain was also performed witha monoclonal antibody (SC5629, dilution 1:100, SantaCruz Technologies Inc, Santa Cruz, CA). Appropriatepositive and negative controls were included for eachrun of immunostains. For both SALL4 and OCT4, onlynuclear staining was considered positive. The staining wasscored as weak, moderate, or strong. The percentage oftumor cells labeled with SALL4 or OCT4 was semiquan-titatively scored as 0 (no tumor cells staining), 1r30%tumor cells), 2+(31% to 60%), 3+(61% to 90%), and4+(>90%).

For YSTs, we also performed AFP (predilutedantibody, Ventana Medical systems, Tuscon, AZ) andglypican-3 (prediluted antibody, Biomosaic, Burlington,VT) immunostain. The percentage of cells stained withAFP or glypican-3 (cytoplasmic and/or membranous)was scored using the same criteria as described above.

Statistical AnalysisFisher exact test was used to compare the staining

results of SALL4 to those of OCT4 in GCTs. Chi-Squaretest was used to compare SALL4 staining to that of

Cao et al Am J Surg Pathol � Volume 33, Number 7, July 2009

1066 | www.ajsp.com r 2009 Lippincott Williams & Wilkins

AFP and glypican-3 in YSTs. A P<0.05 is consideredstatistically significant.

RESULTS

SALL4 in ITGCNs (N = 50)Among the 110 testicular GCTs, 50 have associated

ITGCN on sections used for SALL4 immunohistochem-ical staining. All 50 ITGCNs showed strongly 4+ SALL4staining (Table 1, Fig. 1). All OCT4-positive ITGCNtumor cells were strongly positive for SALL4 (Fig. 1).

SALL4 in Pure Classic Seminomas and ClassicSeminoma Components (N = 62)

All 62 classic seminomas (44 pure seminomas and18 as a component in mixed GCTs) showed strong 4+SALL4 staining in tumor cells (Table 1, Fig. 2).

SALL4 in Spermatocytic Seminomas (N = 2)Two spermatocytic seminomas (patient age 52 and

64 years old, respectively) were stained for SALL4. Thepercentage of tumor cells stained with SALL4 was 80% to85% (3+) and 90% to 95% (4+), respectively (Table 1).The staining intensity in both cases was weak-to–mode-rate, similar to that in spermatogonia but weaker thanthat (strong) in ITGCN, classic seminoma, EC, and YST.All 3 types of cells (intermediate cells, small cells, andlarger mononuclear or multinucleated cells) were positivefor SALL4 (Fig. 3).

SALL4 in Pure EC and EC Components (N = 39)All 39 ECs (5 pure and 34 as a component in mixed

GCTs) showed strong 4+ SALL4 staining in tumor cells(Table 1, Fig. 4).

TABLE 1. Immunohistochemical Staining of SALL4 in Testicular Germ Cell Tumors

SALL4 Staining

Tumor Type (Pure and Component) 0 1+ 2+ 3+ 4+

Intratubular germ cell neoplasia (N=50) 0 0 0 0 50 (100%)Classic seminoma (N=62) 0 0 0 0 62 (100%)Spermatocytic seminoma (N=2) 0 0 0 1 (50%) 1 (50%)Embryonal carcinoma (N=39) 0 0 0 0 39 (100%)Pediatric yolk sac tumor (N=5) 0 0 0 0 5 (100%)Postpubertal yolk sac tumor (N=26) 0 0 0 0 26 (100%)Pediatric teratoma (N=7) 3 4 (57%) 0 0 0Postpubertal teratoma (N=27) 4 23 (85%) 0 0 0Choriocarcinoma (N=5) 0 1 (20%) 0 3 (60%) 1 (20%)

FIGURE 1. Immunohistochemical staining of SALL4 in ITGCN. ITGCN cells are characterized by large nuclei with irregular contourand prominent nucleoli, and clear cytoplasm (A1, B1) and they are strongly positive for SALL4 (A2, B2). These ITGCNs cells areconfirmed by their strong OCT4 staining (A3, B3). ITGCN indicates intratubular germ cell neoplasia.

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SALL4 in Pediatric Pure Yolk Sac Tumors andYolk Sac Tumor Components (N = 5)

Among the 31 YSTs included in this study, 5occurred in pediatric patients (mean age 17mo, age 6 to25mo). Four YSTs were pure and 1 occurred in associ-ation with a mature teratoma component. The histologicpatterns displayed by these 5 YSTs included solid in 2,microcystic in 5, macrocystic in 1, glandular in 5,polyvitelline vesicular in 4, and endodermal sinus patternin 1. In all 5 YSTs, the tumor cells showed 4+ SALL4staining in all histologic patterns (Table 1, Fig. 5). Thestaining intensity was strong in all histologic patternsexcept the glandular area in 2 of 5 cases showed mixedstrong and focally weak-to–moderate staining (cells withweak-to–moderate staining accounting for less than5% SALL4-positive tumor cells).

SALL4 in Postpubertal Pure Yolk Sac Tumors andYolk Sac Tumor Components (N = 26)

Twenty-six of 31 YSTs in this study were inpostpubertal patients including 1 pure tumor (in a 19-year-old male) and 25 as a component in mixed GCTs(mean age 28.5 y, range: 15 to 52 y). The histologicpatterns exhibited by these 26 YSTs included solid in 15,microcystic in 22, macrocystic in 1, glandular 3, papillaryin 2, endodermal sinus in 3, parietal in 7, myxoid in 5,polyvesicular vitelline in 1, and hepatoid in 1. In all 26cases the tumor cells showed 4+ (>90% tumor cells)SALL4 staining (Table 1, Fig. 5). All histologic patternswere strongly positive for SALL4 staining, but focal(<5% positive cells) weak-to–moderate staining wasseen in the myxoid pattern in 2 of 5 cases and in theglandular pattern in 1 of 3 cases, respectively.

FIGURE 2. Immunohistochemical staining of SALL4 in classic seminoma. Seminoma can grow as solid (A1, A2), intertubular(B1, B2), and intratubular (C1, C2) patterns. It may also invade rete testis (D1, D2). Strong SALL4 staining is seen in more than95% tumor cells (A3–D3). These tumor cells are also strongly positive for OCT4 (A4–D4).



SALL4 in Pediatric Pure Teratomas andTeratomatous Components (N = 7)

We included 7 pediatric teratomas including 6 pureand 1 as a component in a mixed GCT (mature teratomaand YST) (Table 1). The mean age for these patients was2.8 years (age range: 4mo to 8 y). Two of these 7 tumorscontained immature elements (primitive neuroepithelialtissue). The immature elements in 1 of these 2 casesshowed 1+ moderate SALL4 staining (10% immatureelements stained) (Fig 6) and the mature elements in bothcases were negative for SALL4. In the 5 tumors with onlymature elements, the enteric type glands showed weak 1+staining (1%, 1%, and 10% cells in enteric glands stained,respectively) in 3 cases including the one in associationwith YST (Fig. 6). Other mature elements were negativefor SALL4 staining in all these 5 cases.

SALL4 in Postpubertal Pure Teratomas andTeratomatous Components (N = 27)

This study included 27 teratomas in postpubertalpatients including 3 pure and 24 as a component in mixedGCTs. All 3 pure teratomas were morphologicallymature. Thirteen of the latter 24 teratomas containedonly mature elements (13/24); eleven contained bothmature and immature elements (11/24).

Of the 3 pure mature teratomas, only one showed1+ weak SALL4 staining in the enteric type glands

(1% cells stained in these glands). Among the teratomasoccurring as a component of a mixed GCT, the onlymature elements showing SALL4 staining were theteratomatous glands (most were enteric-type glands,few were nonmucinous glands) in 22 of 24 (92%) cases(Fig. 6). The percentage of total teratomatous glandularepithelial cells stained with SALL4 ranged from 1% to20% (mean 7%). Most of the SALL4-positive glandswere focally stained with SALL4 in the lining epi-thelial cells but occasionally as many as 70% to 80%lining cells in a gland were stained with SALL4. Thestaining intensity was usually weak except in 1 casethe nonmucinous glands showed focal strong staining(Fig. 6).

As mentioned above, immature elements wereidentified in 11/24 teratomas occurring as a componentof mixed GCTs. They contained 2 types of immaturetissue: primitive neuroepithelial tissue with blastema-likemesenchyme (n=5) and low-grade immature mesen-chyme (n=11). The primitive neuroepithelial tissue andblastema-like stroma showed 1+ weak to focally strongSALL4 staining in 4 of 5 cases (percentage of SALL4positive cells in immature elements is approximately 20%in 3 cases and 1% in 1 case) (Fig. 6). The low-grademesenchyme showed 1+ weak SALL4 staining in 7 ofthese 11 cases (percentage of cells stained with SALL4ranges from 1% to 5%, mean 4%).

FIGURE 3. Immunohistochemical staining of SALL4 in spermatocytic seminoma (A1, B1). The staining intensity of SALL4 inspermatocytic seminoma (A2, B2) is similar to that of adjacent normal spermatogonia (B2). Few primary spermatocytes also showdot-like staining (B2). All 3 types of tumor cells (small, intermediate, and large) are stained with SALL4 (A2, B2). In contrast, alltumor cells are negative for OCT4 (A3, B3).



FIGURE 4. Immunohistochemical staining of SALL4 in embryonal carcinoma (A1 papillary, B1 glandular, C1 solid, and D1intratubular growth pattern). Strong SALL4 staining is seen in more than 95% tumor cells (A2–D2). These tumor cells are alsostrongly positive for OCT4 (A3–D3). Rarely embryonal carcinoma grows intimately with yolk sac tumor in a double-layeredpattern (E1), in which yolk sac tumor grows beneath the embryonal carcinoma. Both embryonal carcinoma and yolk sac tumorcomponents are strongly positive for SALL4 (E2) but only embryonal carcinoma is positive for OCT4, whereas yolk sac tumor isnot (E3).



SALL4 in Choriocarcinomatous Components(N = 5) and Scattered Syncytiotrophoblastin Other Types of GCTs (N = 13)

Choriocarcinoma was identified as a component in 5mixed GCTs. Mononucleated trophoblastic cells werepositive for SALL4 in all 5 cases: 1+ (1% to 2% tumorcells) in 1, 3+ in 3, and 4+ in 1 (Table 1, Fig. 7). Thestaining intensity was moderate in 2 of 5 cases, moderatewith focally strong in 1, and weak in the remaining 2 cases(Fig. 7). Syncytiotrophoblastic cells were negative for SALL4in all 5 cases (Fig. 7). In addition, scattered syncytiotropho-blastic cells were identified in 13 nonchoriocarcinomatousGCTs and all of them were negative for SALL4 staining(Fig. 7).

SALL4 in Primary Testicular Non-GCTsAll 10 Leydig cell tumors, 4 Sertoli cell tumors, 3

adenomatoid tumors, 3 paratesticular rhabdomyosarco-mas, 2 diffuse large B-cell lymphomas, and 1 rete papail-lary cystadenoma were negative for SALL4 staining.

SALL4 in Nontesticular TumorsTwo hundred seventy-five nontesticular tumors

from various organs and sites including 158 metastaticcarcinomas, 12 metastatic melanomas, 13 mesotheliomas(11 primary and 2 metastatic) and 92 sarcomas (72primary and 20 metastatic) were also stained withSALL4. SALL4 result on the carcinoma and melanomastaining has been previously reported.6 Of all these

FIGURE 5. Immunohistochemical stainings of SALL4, AFP, and glypican-3 in postpubertal (A, B) and pediatric (C, D) YSTs (A1microcystic, B1 solid and microcystic, C1 polyvesicular vitelline, and D1 papillary). Although AFP is often positive in YSTs, the stainis often focal and patchy (A2–D2). All YSTs are positive for glypican-3 staining, but the staining is focal and patchy in more than50% cases (A3–C3) and in some cases some pattern can be entirely negative (D3). In contrast, strong SALL4 staining is seen inmore than 90% tumor cells in all YSTs (A4–D4). OCT4 staining is negative in all YSTs (not shown). AFP indicates a-fetoprotein,YSTs, yolk sac tumors.



tumors, only 10 (6 esophageal, 3 gastric, and 1 colonic)metastatic carcinomas and 1 primary sarcoma (malignantfibrous histiocytoma) showed 1+ weak nuclear SALL4staining. The mean percentage of tumor cells positivefor SALL4 was 10% (range: 0.5% to 25%) in these10 carcinomas and 5% to 10% in the sarcoma.

SALL4 in Non-neoplastic Testicular TissueThe non-neoplastic seminiferous tubules were pre-

sent on the sections for SALL4 staining in 50 casesincluding 9 pediatric testes and 41 postpubertal testes.Positive SALL4 staining was observed in spermatogoniain all 50 testes (both pediatric and postpubertal) (Fig. 8).The staining intensity in most of spermatogonia wasweak-to–moderate (Fig. 8) but rarely spermatogonia

showed moderate-to–strong SALL4 staining (still weakerthan that in neoplastic cells). In addition, a few (<5%)primary spermatocytes showed dot-like weak SALL4staining (Fig. 3). Secondary spermatocytes, spermatids,spermatozoa, and Sertoli cells were negative for SALL4.Leydig cells, rete testis, epididymis, spermatic cord,fibroblasts, blood vessels, and hematopoietic cells showedno staining for SALL4. OCT4 was negative in allcomponents of non-neoplastic testicular tissue.

Comparison of SALL4 Stain to That of OCT4in Testicular GCTs

To compare SALL4 with OCT4, we performedimmunostain for OCT4 in all testicular GCTs. Table 2

FIGURE 6. Immunohistochemical staining of SALL4 in pediatric (A–H) and postpubertal (I–P) testicular teratomas. In thispediatric-mixed germ cell tumor consisting of mature teratoma and yolk sac tumor (A, C, E), the teratomatous enteric type glandsshow weak SALL4 staining (B, D), whereas the adjacent yolk sac tumor component is strongly positive for SALL4 andspermatogonia show weak-to–moderate SALL4 staining (B, F). In another pediatric pure teratoma, the immature primitiveneuroepithelial tissue (G) shows focal SALL4 staining with moderate-to–strong staining intensity (H). In postpubertal patients, theteratomatous glands (I) are usually weakly positive for SALL4 (J) but sometimes the less mature-appearing glands (K) can showstrong SALL4 staining (L). Immature elements such as blastema-like stroma (K) and primitive neuroepithelial tissue (M, O) can alsoshow variable staining intensity from weak-to–strong SALL4 staining (L, N, P).



summarized the comparison of SALL4 result to that ofOCT4. OCT4 was also 4+ strongly positive in allITGCNs, classic seminomas and ECs. No OCT4 stainingwas observed in any spermatocytic seminoma, YST,teratoma, or choriocarcinoma.

Comparison of SALL4 to a-fetoprotein andGlypican-3 in Yolk Sac Tumors

To compare SALL4 with currently used YSTmarkers AFP and glypican-3, we performed AFP andglypican-3 immunostains for all 31 YSTs. Twenty-nine of31 (94%) YSTs were positive for AFP including 1+ in 14cases, 2+ in 7 cases, and 3+ in 8 cases (Table 3, Fig. 5).No YST showed 4+ AFP staining (>90% tumor cells).Two of 31 cases (6%) were negative for AFP. There wasoften a high background staining in AFP staining. All 31YSTs (100%) showed glypican-3 staining including 1+ in14 (45%), 2+ in 5 (16%), 3+ in 7 (23%), and 4+ in 5(16%) (Table 3, Fig. 5). Of the 14 (45%) YSTs showingonly 1+ glypican-3 staining, 4 (2 pure, 2 as a componentin mixed GCTs) showed staining in less than 5% tumorcells. In contrast to AFP and glypican-3, SALL4 staining

in more than 90% tumor cells was seen in all 31 YSTs(100% sensitivity) (Table 3, P<0.0001 for both SALL4vs. AFP and SALL4 vs. glypican-3).

DISCUSSIONSALL4 is a zinc finger transcription factor that

shares homology to the Drosophila spalt (sal) gene.38 InDrosophila sal acts as a region-specific homeotic genewith an important role in the specification of head and tailregions during embryonic development.17 In the mouse,SALL4 is essential to early embryogenesis and homo-zygous mutant mice exhibit early embryonic lethality.39,50

In human beings SALL4 is located on chromosome20q13.13–13.2.18 As in other species, SALL4 is alsoessential to human development and mutations in SALL4lead to acro-renal-ocular and Okihiro syndromes.18

SALL4 forms a regulatory circuit with OCT4, NANOG,and SOX2 to maintain embryonic stem cell pluripotencyand self-renewal.21,23,49,52,53,55,56 In this self-stabilizingnetwork SALL4 regulates OCT4 transcription, suggestingaction upstream of OCT4.55 In this study, we have shownthat by immunohistochemical staining SALL4 proteinwas detected in all types of testicular GCTs. In contrast,OCT4 only labeled ITGCNs, classic seminoma, and EC,as observed by us and others.16,40 Therefore SALL4 had abroader expression than OCT4 in testicular GCTs. Inembryonic stem cells SALL4 acts upstream of OCT4,55 itis unknown whether similar relationship exists in testi-cular GCTs.

Our findings have clearly demonstrated diagnosticutility of SALL4 for testicular GCTs. In the currentstudy, SALL4 stained more than 90% tumor cells in allITGCNs, classic seminomas, ECs, and YSTs (bothpediatric and postpubertal). In 2 spermatocytic semino-mas, approximately 80% to 85% and 90% to 95% tumorcells were stained with SALL4, respectively. ThereforeSALL4 demonstrated 100% sensitivity for ITGCNs,seminomas (classic and spermatocytic), ECs and YSTs.In addition, SALL4 also stained some (27 of 34)teratomas and the mononucleated trophoblastic cells in all5 choriocarcinomas. In contrast, all 23 testicular non-GCTsshowed no SALL4 staining. Among 275 nontesticulartumors (158 metastatic carcinomas, 12 metastatic melano-mas, 11 primary and 2 metastatic mesotheliomas, 72primary and 20 metastatic sarcomas) from various organsand sites, only 10 carcinomas6 and 1 sarcoma showed weakSALL4 staining in less than 25% of tumor cells. In theliterature, the only other types of tumors that have beenreported to show positive SALL4 staining were precursor B-cell lymphoblastic lymphoma and acute myeloid leukemia,whereas other types of hematopoietic tumors were negativefor SALL4.7 All these indicate that SALL4 is a novelsensitive diagnostic marker for testicular GCTs especiallyfor ITGCN, classic and spermatocytic seminomas, EC, andYST with relatively high specificity (96.3% if using anytumor cells stained with SALL4 as the cutoff, 100% if usingat least 25% tumor cells stained with SALL4 as the cutoff inour study). In a previous study,6 we have demonstrated that

FIGURE 7. Immunohistochemical staining of SALL4 in testicularchoriocarcinoma (A1, B1) and isolated syncytiotrophoblast (C1).SALL4 staining is positive in mononucleated trophoblastic cellswith weak-to–strong intensity, whereas syncytiotrophoblasticcells in choriocarcinomas are negative for SALL4 staining (A2,B2). Syncytiotrophoblastic cells in isolated forms in other typesof testicular germ cell tumors are negative for SALL4 staining,whereas the adjacent embryonal carcinoma cells in this case arestrongly positive for SALL4 (C2).



all 22 metastatic seminomas, 20 metastatic ECs and 7 of 8metastatic YSTs from testis showed strong SALL4 stainingin more than 90% tumor cells. The remaining 1 metastaticYST showed strong SALL4 staining in 80% tumor cells.

Therefore the high (100%) sensitivity of SALL4 forseminoma, EC and YST not only holds for their primaryforms within testis but also for their metastatic forms inextragonadal sites.6

FIGURE 8. Immunohistochemical staining of SALL4 in non-neoplastic testicular tissue. Spermatogonia both in children (A1) andpostpubertal males (B1) are weakly to moderately positive for SALL4 stain (A2, B2). The SALL4 staining intensity in spermatogonia isless intense or weaker than that in adjacent intratubular germ cell neoplasia cells (C1 H&E; C2 SALL4 stain) and infiltrating seminomacells (D1 H&E; D2 SALL4 stain). Spermatogonia are negative for OCT4 stain (A3–D3). H&E indicates hematoxylin and eosin.



Besides its role in distinguishing a GCT from a non-GCT, SALL4 is also useful for classifying difficult GCTs.For example, it is sometimes difficult to distinguishglandular EC from glandular YST and glandular YSTfrom glandular teratoma. A panel including SALL4,OCT4, and epithelial membrane antigen (EMA) will helpease the difficulty. The glands in EC will be SALL4+/OCT4+/EMA� , whereas those in YST will beSALL4+/OCT4� /EMA� . Teratomatous glands aretypically positive for EMA but negative for OCT4.SALL4 staining in teratomatous glands is typically weakif present. Another example is that morphologically‘‘atypical’’ seminoma might mimic the solid variant ofEC and YST. In this scenario SALL4 clearly alsodemonstrates its utility in distinguishing them when it isused in conjunction with other markers such as OCT4and CD30. Seminoma has a profile characterized bySALL4+/OCT4+/CD30� , whereas EC is positive forall these 3 markers. In contrast, YST is positive forSALL4 but negative for OCT4 and CD30.

Compared with other types of testicular GCTs, YSTis notoriously for displaying multiple histologic patternsmimicking many other types of tumors, and thus pose thegreatest diagnostic challenges.45,47 Current markers forYST include AFP and glypican-3, but both are lack ofadequate sensitivity.10,28,29,31,57 Here, we comparedSALL4 with AFP and glypican-3 in all 31 YSTs. PositiveAFP staining was seen in 94% (29 of 31) of YSTs,in keeping with that (range: 50% to 100%) reportedin the literature.10,28,29,31 However, as observed byothers,10,28,29,31 we also found that AFP staining in YSTswas typically focal and patchy: 1+ (<30% tumor cells)staining in 14 (45%) YSTs including 7 with no more than10% tumor cells stained. In our study, all 31 YSTsshowed positive glypican-3 staining, similar to thatreported in 2 previous studies.31,57 However, 1+

(<30% tumor cells) glypcan-3 staining was seen in 14of our 31 (45%) YSTs. In the 2 previous studies, positiveglypican-3 staining in less than 50% tumor cells was seenin 50%31 and 12%57 YSTs, respectively. These findingsindicate that although AFP and glypican-3 are goodmarkers for YST, the fact that their staining was seen inless than 30% tumor cells in nearly half of YSTs limittheir diagnostic utility in certain conditions such aslimited biopsy material in metastatic sites. Furthermore,AFP and glypican-3 are not specific for YST, either. AFPwas often positive in teratomatous enteric type glands.Glypican-3 also labeled EC, choriocarcinoma, andimmature teratoma.31,57 In contrast to AFP and glypi-can-3, SALL4 strongly stained more than 90% tumorcells in all 31 YSTs. Even in metastatic sites, such highsensitivity of SALL4 with staining in more than 80% to90% tumor cells was still maintained for metastatictesticular YST.6 Although SALL4 is not entirely specificfor YST; our findings do indicate that SALL4 is a moresensitive marker than AFP and glypican-3 for YST.

Besides demonstrating diagnostic utility of SALL4in testicular GCTs, our findings that SALL4 wasexpressed in all types of GCTs, both in pediatric andpostpubertal patients, might help shed some light on thepathogenesis of testicular GCTs. Testicular GCTs can bedivided into 3 genetically distinct groups: teratomaand YST in neonates and infants, classic seminoma andnonseminomatous GCTs in postpubertal patients, andspermatocytic seminoma in old patients.24 These 3 groupsof tumors are though to arise from germ cells at differentstages of development.25,41,43 Gene imprinting study hassuggested that the neoplastic transformation of pediatricGCTs (teratoma and YST) initiates during germ cellmigration, whereas this occurs at a later stage in adulttesticular GCTs.41 Spermatocytic seminoma is thought tooriginate in primary spermatocyte.25,43 In postpubertal

TABLE 2. Comparison of SALL4 Immunohistochemical Staining to That of OCT4 in Testicular Germ Cell Tumors

Tumor Type SALL4 Positive OCT4 Positive P

Intratubular germ cell neoplasia (N=50) 50 (100%) 50 (100%) 1.00Classic seminoma (N=62) 62 (100%) 62 (100%) 1.00Spermatocytic seminoma (N=2) 2 (100%) 0 0.33 (but only 2 cases)Embryonal carcinoma (N=39) 39 (100%) 39 (100%) 1.00Pediatric yolk sac tumor (N=5) 5 (100%) 0 <0.01Postpubertal yolk sac tumor (N=26) 26 (100%) 0 <0.01Pediatric teratoma (N=7) 4 (57%) 0 0.02Postpubertal teratoma (N=27) 23 (85%) 0 <0.01Choriocarcinoma (N=5) 5 (100%) 0 <0.01

TABLE 3. Comparison of Immunohistochemical Staining of SALL4 to Those of AFP and Glypican-3 in 31 Testicular Yolk SacTumors

SALL4 Staining

Marker 0 1+ 2+ 3+ 4+ P

SALL4 0 0 0 0 31 (100%) <0.0001 (SALL4 vs. AFP)AFP 2 (6%) 14 (45%) 7 (23%) 8 (25%) 0Glypican-3 0 14 (45%) 5 (16%) 7 (23%) 5 (16%) <0.001 (SALL4 vs. glypican-3)



patients ITGCN is considered the precursor to classicseminoma and nonseminomatous GCTs, whereas pedia-tric YST and teratoma are thought not to originate fromITGCN but from either embryonic stem cells or earlymigrating primordial germ cells.1,2,42 One of the keyevents in the malignant transformation of germ cells is theblock in germ cell differentiation. It has been proposedthat dysregulation of embryonic pluripotency genes mightplay some role in this process.35,36 In this study, we havedemonstrated SALL4 protein in all 3 types of GCTs thatarise from different mechanisms at different stages ofgerm cell development, suggesting that dysregulation ofSALL4 might be a common factor in the pathogenesis ofall 3 types of testicular GCTs. Of interest, the proteinexpression level of SALL4 (evidenced by semiquantativeimmunohistochemical staining) varied in different typesof GCTs and seemed to correlate with the degree oftumor differentiation. For example, the staining inten-sity of SALL4 in the more poorly differentiated tumorssuch as ITGCN, classic seminoma, EC, and YST (bothpediatric and postpubertal patients) was diffuse andstrong (stronger than that in normal spermatogonia),whereas in the more differentiated tumors such asteratomas and spermatocytic seminomas the stainingwas focal and/or weak (weaker than that in normalspermatogonia, stronger in immature elements thanmature elements). These observations suggest thatSALL4 might be important for these tumors to maintaintheir poorly differentiated status. Previous studies haveshown that depletion of SALL4 targets in embryonic stemcells and extraembryonic endoderm cells results indifferentiation.22 In addition, it is also possible thatSALL4 might be also involved in the apoptotic pathwaysby preventing apoptosis in testicular GCTs to maintaintumor growth as its does in acute leukemia.54 Furtherstudies to investigate SALL4 in testicular GCTs will behelpful for us to better understand its possible role inthese tumors.

Finally, besides having a broader expression profilein testicular GCTs than OCT4, SALL4 also differs fromOCT4 in that SALL4 is expressed in normal spermato-gonia in both in children and postpubertal males and afew primary spermatogonia in postpubertal testis. Incontrast, OCT4 is not expressed in any germ cells after 3to 4 months of postnatal age (the final differentiation ofgonocytes into infantile spermatogonia).37 It is unknown,what mechanism turns off SALL4 expression as sperma-togonia differentiate into more mature germ cells.However, our finding does raise an interesting question:does SALL4 play a role in self-renewal of spermatogonia,both in children and adults, as it does in embryonic stemcells?49,52,53,55,56 In human testis, type A spermatogoniaare thought to be stem cells in renewal of spermatogoniaand in spermatogenesis.30,32,33 Our study is the first one todemonstrate SALL4 protein in normal spermatogoniaand testicular GCTs, so further studies to explore thisaspect of SALL4 function would be interesting.

In summary, we investigated the expression of anovel stem cell marker SALL4 by immunohistochemistry

in a large series of 110 primary testicular GCTs, 23testicular non-GCTs, and 275 non-testicular tumors. Ourresults indicate that SALL4 is a novel sensitive andrelatively specific diagnostic marker for testicular GCTs.

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