Intratubular germ cell neoplasia of the human testis: heterogeneous protein expression and relation to invasive potential - PubMed (original) (raw)

. 2014 Sep;27(9):1255-1266.

doi: 10.1038/modpathol.2013.246. Epub 2014 Jan 24.

Maria Camacho-Moll 1, Joni Macdonald 1, Richard A Anderson 1, Christopher Jh Kelnar 2, Marie O'Donnell 3, Richard M Sharpe 1, Lee B Smith 1, Ken M Grigor 3, W Hamish B Wallace 2, Hans Stoop 4, Katja P Wolffenbuttel 4, Roland Donat 5, Philippa Tk Saunders # 1, Leendert Hj Looijenga # 4

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Intratubular germ cell neoplasia of the human testis: heterogeneous protein expression and relation to invasive potential

Rod T Mitchell et al. Mod Pathol. 2014 Sep.

Abstract

Testicular germ cell cancer develops from premalignant intratubular germ cell neoplasia, unclassified cells that are believed to arise from failure of normal maturation of fetal germ cells from gonocytes (OCT4(+)/MAGEA4(-)) into pre-spermatogonia (OCT4(-)/MAGEA4(+)). Intratubular germ cell neoplasia cell subpopulations based on stage of germ cell differentiation have been described, however the importance of these subpopulations in terms of invasive potential has not been reported. We hypothesized that cells expressing an immature (OCT4(+)/MAGEA4(-)) germ cell profile would exhibit an increased proliferation rate compared with those with a mature profile (OCT4(+)/MAGEA4(+)). Therefore, we performed triple immunofluorescence and stereology to quantify the different intratubular germ cell neoplasia cell subpopulations, based on expression of germ cell (OCT4, PLAP, AP2γ, MAGEA4, VASA) and proliferation (Ki67) markers, in testis sections from patients with preinvasive disease, seminoma, and non-seminoma. We compared these subpopulations with normal human fetal testis and with seminoma cells. Heterogeneity of protein expression was demonstrated in intratubular germ cell neoplasia cells with respect to gonocyte and spermatogonial markers. It included an embryonic/fetal germ cell subpopulation lacking expression of the definitive intratubular germ cell neoplasia marker OCT4, that did not correspond to a physiological (fetal) germ cell subpopulation. OCT4(+)/MAGEA4(-) cells showed a significantly increased rate of proliferation compared with the OCT4(+)/MAGEA4(+) population (12.8 versus 3.4%, P<0.0001) irrespective of histological tumor type, reflected in the predominance of OCT4(+)/MAGEA4(-) cells in the invasive tumor component. Surprisingly, OCT4(+)/MAGEA4(-) cells in patients with preinvasive disease showed significantly higher proliferation compared to those with seminoma or non-seminoma (18.1 versus 10.2 versus 7.2%, P<0.05, respectively). In conclusion, this study has demonstrated that OCT4(+)/MAGEA4(-) cells are the most frequent and most proliferative cell population in tubules containing intratubular germ cell neoplasia, which appears to be an important factor in determining invasive potential of intratubular germ cell neoplasia to seminomas.

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Figures

Figure 1

Figure 1

Expression of gonocyte markers (OCT4, AP2γ and PLAP; A-I) and spermatogonial markers (MAGEA4 and VASA; J-O) in human fetal testis, intratubular germ cell neoplasia containing tubules (Adult – intratubular germ cell neoplasia) and tubules from adult testis with active spermatogenesis (Adult – ‘Normal’). Gonocyte proteins are detected in human fetal germ cells and intratubular germ cell neoplasia cells, but are absent from the germ cells in tubules with apparently normal spermatogenesis; whilst spermatogonial proteins are expressed in germ cells in all tissue types. Human fetal samples are 14 (A,D), 16 (J) and 18 weeks (G,M) gestation. Scale bar = 50μm.

Figure 2

Figure 2

Representative image for expression of VASA (green) and AP2γ (red) in tubules from adult patients with testicular germ cell cancer. A) Tubule with apparently normal spermatogenesis: VASA is expressed in the germ cells with no expression of the intratubular germ cell neoplasia/gonocyte protein AP2γ. B) Tubule with abnormal spermatogenesis: VASA expression is seen in the presumptive spermatocytes towards the lumen (white arrowhead), however germ cells along the basement membrane express VASA (yellow arrowhead) or AP2γ (yellow arrow). A small proportion of cells co-express VASA and AP2γ (white arrow). C) Intratubular germ cell neoplasia tubule: The majority of cells express the intratubular germ cell neoplasia protein AP2γ (yellow arrow) with a small number of cells adjacent to the basement membrane expressing VASA (presumptive spermatogonia; pink arrow). A small number of germ cells expressing VASA are located towards the lumen (presumptive spermatocytes; white arrowhead).

Figure 3

Figure 3

Expression of OCT4 (red) and PLAP (green) in intratubular germ cell neoplasia containing tubules from men with testicular germ cell cancer (A,C) and in normal human fetal testis tissue (B). The majority of intratubular germ cell neoplasia cells co-express OCT4 and PLAP (A,C; yellow arrowheads). Occasional OCT4 positive intratubular germ cell neoplasia cells are negative for PLAP expression (A; white arrow). Similar sub-populations of OCT4+/PLAP− (B, white arrow) and OCT4+/PLAP+ (B, yellow arrowheads) cells are also identified in the human fetal testis. In tubules with intratubular germ cell neoplasia, occasional OCT4−/PLAP+ cells are identified (C; yellow arrows), however no similar population is seen in the human fetal testis. Counterstain (DAPI; blue).

Figure 4

Figure 4

Representative image for expression of OCT4 (red), VASA (A; blue), MAGEA4 (B; blue) and PLAP (green) in intratubular germ cell neoplasia containing tubules from patients with testicular germ cell cancer. A) VASA expression is demonstrated in putative ‘spermatogenic’ germ cells that are negative for intratubular germ cell neoplasia cell proteins PLAP and OCT4 (white arrow). A small proportion of the VASA+ cells that are negative for OCT4 express PLAP (white arrowhead). B) The majority of cells co-express OCT4 and PLAP without MAGEA4, other sub-populations are identified including PLAP+/OCT4+/MAGEA4+ (pink arrow) and PLAP+/OCT4−/MAGEA4− (pink arrowhead). Counterstain (DAPI; pale blue) in merged panels.

Figure 5

Figure 5

Quantification of putative intratubular germ cell neoplasia phenotypes. Expression (+) of OCT4, PLAP and MAGEA4 for intratubular germ cell neoplasia containing tubules (n=9; pre-invasive, seminoma and non-seminoma; n=3 each). Bars with different letters are significantly different from each other (p<0.05). Mean +/− SEM.

Figure 6

Figure 6

Proliferation in putative intratubular germ cell neoplasia cells. A) Overall proliferation in all intratubular germ cell neoplasia cells. B) Proliferation (Ki67+) of intratubular germ cell neoplasia (OCT4+) cells based on the co-expression with MAGEA4 in tubules from all patients (B), children with pre-invasive disease (C; Pre-Inv. Child; n=4), adults with pre-invasive disease (D; Pre-Inv. Adult; n=6), seminoma (n=7) and non-seminoma (n=8). Mean +/− SEM. * p<0.05, ** p<0.01, **** p<0.0001.

Figure 7

Figure 7

Proliferation of intratubular germ cell neoplasia cells based on diagnosis of pre-invasive disease (PRE INV; n=7), seminoma (SEM; n=7) or non-seminoma (NON-SEM; n=8). A) Proliferation of OCT4+ intratubular germ cell neoplasia cells. B) Proliferation of OCT4+/MAGEA4+ intratubular germ cell neoplasia cells. Mean +/− SEM. * p<0.05, ** p<0.01, in comparison with pre-invasive intratubular germ cell neoplasia.

Figure 8

Figure 8

Representative images for expression of OCT4 (red), PLAP (green) and MAGEA4 (blue) in testis sections from patients with A) Intra-tubular seminoma, B) Seminoma with surrounding ‘normal’ (*) tubules and C) Seminoma. Note the lack of expression of MAGEA4 in both intra-tubular seminoma and invasive seminoma. Counterstain (DAPI; pale blue) in merged panels (bottom right).

Figure 9

Figure 9

Schematic for germ cell maturation and proliferation in germ cells during transition from gonocyte to intratubular germ cell neoplasia and testicular germ cell cancer (bottom). Germ cell maturation from gonocyte to initiation of spermatogenesis is represented in the testis during the different stages of life (middle). For comparison, germ cell differentiation in the normal testis is also shown (top). Germ cells in the fetal testis may exhibit delayed maturation with persistence of gonocyte markers through childhood. A variety of germ cell protein profiles are present in the intratubular germ cell neoplasia tubule, however it is the cells expressing exclusively gonocyte proteins (with no spermatogonial proteins) that are more proliferative and contribute to the majority of the cells in intratubular seminoma and subsequently invasive seminoma. Cells expressing spermatogonial proteins are occasionally seen in the tubule or resultant tumour but exhibit low proliferation rates. Expression of OCT4 (red), PLAP (green) and MAGEA4 (blue) is shown for individual cells and cells with high rates of proliferation are indicated (yellow asterisk)

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References

    1. McGlynn KA, Devesa SS, Sigurdson AJ, et al. Trends in the incidence of testicular germ cell tumors in the United States. Cancer. 2003;97:63–70. - PubMed
    1. Richiardi L, Bellocco R, Adami HO, et al. Testicular cancer incidence in eight northern European countries: secular and recent trends. Cancer Epidemiol Biomarkers Prev. 2004;13:2157–2166. - PubMed
    1. Rajpert-De Meyts E. Developmental model for the pathogenesis of testicular carcinoma in situ: genetic and environmental aspects. Hum Reprod Update. 2006;12:303–323. - PubMed
    1. Skakkebaek NE. Possible carcinoma-in-situ of the testis. Lancet. 1972;2:516–517. - PubMed
    1. Waters BL, Trainer TD. Development of the human fetal testis. Pediatr Pathol Lab Med. 1996;16:9–23. - PubMed

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