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Testicular seminoma is characterized by a prominent lymphoid infiltrate and an excellent prognosis. Cytotoxic T-lymphocytes (CTLs) infiltrating seminoma tumour nests constitute a major subset of the lymphoid infiltrate. The objective of this study was to determine whether CTLs express markers of cytotoxic potential and activity and whether the number of activated CTLs correlates with the extent of apoptosis in testicular seminomas, as opposed to non-seminomatous testicular germ cell tumours (NSTGCTs). Twenty cases of pure seminoma as well as 20 cases of NSTGCTs including 16 mixed germ cell tumours (MGCTs) were studied. Immunohistochemistry for the cytotoxic markers TIA-1 (cytotoxic potential) and granzyme B (cytotoxic activity) and the T-cell markers CD3 and CD8 was performed on formalin-fixed, paraffin-embedded sections. The apoptotic index (AI) was determined by the TUNEL method. The number of CD3(+), CD8(+), TIA-1(+), and granzyme B(+) cells in tumour cell nests was markedly increased in testicular seminomas, compared with NSTGCTs (p<0.01). Activated granzyme B(+) cells numbered 25.6+/-5.2 per high power field in seminomas and 8.9+/-3.2, 8.1+/-3.9, and 0.4+/-0.2 for embryonal carcinomas, yolk sac tumours, and immature teratomas, respectively. Double immunohistochemical staining for granzyme B and CD8 revealed that 82.6+/-8.5% of granzyme B-expressing cells were CD8(+). The tumour cell AI was significantly increased in embryonal carcinoma, compared with the seminoma, yolk sac tumour, and immature teratoma subgroups (6.7+/-1.3, 2.3+/-0.3, 3.0+/-1.1, and 2.3+/-1.1, respectively, p<0.001). TUNEL/CD3 double immunostaining revealed that a significant proportion of the apoptotic seminomatous tumour cells were in direct contact with one or more CD3(+) lymphocytes (47.2+/-6.2%). The number of activated granzyme B(+) CTLs showed a strong linear correlation with the AI in the seminoma group (r=0.71, p<0.0001) but not in other subgroups. TUNEL/granzyme B double immunolabelling revealed that a proportion of activated granzyme B(+) lymphocytes (20%) were often seen in close contact with apoptotic tumour cells. The presence of increased numbers of activated cytotoxic lymphocytes in testicular seminomas suggests that apoptotic tumour cell death in this neoplasm may be triggered by cytotoxic granule effectors. This phenomenon may be one of the key host immune mechanisms leading to the excellent prognosis in this tumour.

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Electron microscopic examination still is the gold standard for classifying epidermolysis bullosa, although it is relatively expensive, time consuming, and not readily available. Immunoreagents have been developed recently to map antigens in the basement membrane on routinely processed specimens. The current study was performed to examine the diagnostic usefulness of immunohistochemistry, as compared with electron microscopic examination, for analyzing routine formalin-fixed paraffin-embedded sections of epidermolysis bullosa. This study investigated 39 consecutively diagnosed cases of epidermolysis bullosa in which both electron microscopic examination and immunohistochemistry were used. In each case, three monoclonal antibodies were used to stain for laminin 1, collagen IV, and keratin. The immunohistochemical patterns were defined as follows: epidermolysis bullosa simplex (laminin, collagen IV, or both at the dermal floor of the blister and keratin at both the dermal floor and the epidermal roof), junctional epidermolysis bullosa (laminin, collagen IV, or both at the dermal floor of the blister and keratin only at the epidermal roof), and dystrophic epidermolysis bullosa (collagen IV, laminin, or both, and keratin all at the epidermal roof). Altogether, electron microscopic examination subclassified epidermolysis bullosa into its three major forms in 37 of the 39 cases (95%), and immunohistochemistry in 33 of the 39 cases (85%). All of the classifiable cases were concordant. Specifically, immunohistochemistry was diagnostic in 10 of 14 (71%) epidermolysis bullosa simplex cases, 14 of 14 (100%) junctional epidermolysis bullosa cases, and 9 of 11 (82%) dystrophic epidermolysis bullosa cases. The most frequent cause for inconclusive immunohistochemical results was failure in staining of the basement membrane with the antibodies to both laminin and collagen IV. In conclusion, the use of immunohistochemistry on routinely processed specimens may be useful for subclassifying epidermolysis bullosa into its major forms in the majority of the cases, although it still cannot fully replace electron microscopic examination or immunofluorescence mapping in the diagnosis of epidermolysis bullosa.