Transcriptome and methylome analysis of CNS germ cell tumor finds its cell-of-origin in embryogenesis and reveals shared similarities with testicular counterparts.

BACKGROUND: CNS germ cell tumors (GCTs) predominantly develop in pediatric and young adult patients with variable responses to surgery, radiation, and chemotherapy. This study aimed to examine the complex and largely unknown pathogenesis of CNS GCTs. METHODS: We used a combined transcriptomic and methylomic approach in 84 cases and conducted an integrative analysis of the normal cells undergoing embryogenesis and testicular GCTs. RESULTS: Genome-wide transcriptome analysis in CNS GCTs indicated that germinoma had a transcriptomic profile representative of primitive cells during early embryogenesis with high meiosis/mitosis potentials, while nongerminomatous GCTs (NGGCTs) had differentiated phenotypes oriented toward tissue formation and organogenesis. Co-analysis with the transcriptome of human embryonic cells revealed that germinomas had expression profiles similar to those of primordial germ cells, while the expression profiles of NGGCTs were similar to those of embryonic stem cells. Some germinoma cases were characterized by extensive immune-cell infiltration and high expression of cancer-testis antigens. NGGCTs had significantly higher immune-cell infiltration, characterized by immune-suppression phenotype. CNS and testicular GCTs (TGCTs) had similar mutational profiles; TGCTs showed enhanced copy number alterations. Methylation analysis clustered germinoma/seminoma and nongerminoma/nonseminoma separately. Germinoma and seminoma were co-categorized based on the degree of the tumor microenvironment balance. CONCLUSIONS: These results suggested that the pathophysiology of GCTs was less dependent on their site of origin and more dependent on the state of differentiation as well as on the tumor microenvironment balance. This study revealed distinct biological properties of GCTs, which will hopefully lead to future treatment development.

Antibodies Against Hypothalamus and Pituitary Gland in Childhood-Onset Brain Tumors and Pituitary Dysfunction.

Purpose: To detect the presence of antipituitary (APA) and antihypothalamus antibodies (AHA) in subjects treated for brain cancers, and to evaluate their potential association with pituitary dysfunction. Methods: We evaluated 63 patients with craniopharyngioma, glioma, and germinoma treated with surgery and/or radiotherapy and/or chemotherapy at a median age of 13 years. Forty-one had multiple pituitary hormone deficiencies (MPHD), six had a single pituitary defect. GH was the most common defect (65.1%), followed by AVP (61.9%), TSH (57.1%), ACTH (49.2%), and gonadotropin (38.1%). APA and AHA were evaluated by simple indirect immunofluorescence method indirect immunofluorescence in patients and in 50 healthy controls. Results: Circulating APA and/or AHA were found in 31 subjects (49.2%) and in none of the healthy controls. In particular, 25 subjects out of 31 were APA (80.6%), 26 were AHA (83.90%), and 20 were both APA and AHA (64.5%). Nine patients APA and/or AHA have craniopharyngioma (29%), seven (22.6%) have glioma, and 15 (48.4%) have germinoma. Patients with craniopharyngioma were positive for at least one antibody in 39.1% compared to 33.3% of patients with glioma and to 78.9% of those with germinoma with an analogous distribution for APA and AHA between the three tumors. The presence of APA or AHA and of both APA and AHA was significantly increased in patients with germinoma. The presence of APA (P = 0.001) and their titers (P = 0.001) was significantly associated with the type of tumor in the following order: germinomas, craniopharyngiomas, and gliomas; an analogous distribution was observed for the presence of AHA (P = 0.002) and their titers (P = 0.012). In addition, we found a significant association between radiotherapy and APA (P = 0.03). Conclusions: Brain tumors especially germinoma are associated with the development of hypothalamic-pituitary antibodies and pituitary defects. The correct interpretation of APA/AHA antibodies is essential to avoid a misdiagnosis of an autoimmune infundibulo-neurohypophysitis or pituitary hypophysitis in patients with germinoma.

Intratumoural immune cell landscape in germinoma reveals multipotent lineages and exhibits prognostic significance.

AIMS: Alterations in microenvironments are a hallmark of cancer, and these alterations in germinomas are of particular significance. Germinoma, the most common subtype of central nervous system germ cell tumours, often exhibits massive immune cell infiltration intermingled with tumour cells. The role of these immune cells in germinoma, however, remains unknown. METHODS: We investigated the cellular constituents of immune microenvironments and their clinical impacts on prognosis in 100 germinoma cases. RESULTS: Patients with germinomas lower in tumour cell content (i.e. higher immune cell infiltration) had a significantly longer progression-free survival time than those with higher tumour cell contents (P = 0.03). Transcriptome analyses and RNA in-situ hybridization indicated that infiltrating immune cells comprised a wide variety of cell types, including lymphocytes and myelocyte-lineage cells. High expression of CD4 was significantly associated with good prognosis, whereas elevated nitric oxide synthase 2 was associated with poor prognosis. PD1 (PDCD1) was expressed by immune cells present in most germinomas (93.8%), and PD-L1 (CD274) expression was found in tumour cells in the majority of germinomas examined (73.5%). CONCLUSIONS: The collective data strongly suggest that infiltrating immune cells play an important role in predicting treatment response. Further investigation should lead to additional categorization of germinoma to safely reduce treatment intensity depending on tumour/immune cell balance and to develop possible future immunotherapies.

Tumor immune microenvironment is associated with the growth of intracranial germinomas.

INTRODUCTION: The role of immune checkpoint molecules and the tumor immune microenvironment in the development of intracranial germ cell tumors remains unclear. METHODS: We investigated the expression of programed cell death-1 (PD-1), programed cell death ligand-1 (PD-L1), and tumor-infiltrating lymphocytes (TILs) in 8 patients who had intracranial germinomas with sufficient tumor tissue by immunohistochemistry, to analyze the associations between their clinical courses and radiological features. The 8 patients were categorized based on the duration between symptom onset and pathological diagnosis into the long-term onset (LTO) group (> 1 year of symptoms) and the short-term onset (STO) group (< 1 year of symptoms). RESULTS: Three patients belonged to the LTO group and 5 patients to the STO group. Compared with STO tumors, LTO tumors were significantly associated with a lower ratio of PD-L1-positive tumor cells (p = 0.012), higher number of infiltrating CD3- and CD8-positive lymphocytes (p = 0.016, 0.003, respectively), and lower ratio of PD-1-positive cells per CD8-positive lymphocytes (p = 0.047). LTO germinomas were significantly smaller in size than STO tumors, not associated with hydrocephalus, and tended to be present in patients with older age at diagnosis and atypical tumor location. CONCLUSIONS: Our data suggest that the tumor immune microenvironment, including PD-1/PD-L1 signaling, is associated with the growth of intracranial germinomas.

PD-1/PD-L1 expression in a series of intracranial germinoma and its association with Foxp3+ and CD8+ infiltrating lymphocytes.

One histopathological characteristic of intracranial germinoma is abundant tumor-infiltrating lymphocytes (TILs) showing a two-cell pattern with large undifferentiated tumor cells. The programmed cell death 1 (PD-1)/programmed cell death 1 ligand (PD-L) axis has recently been recognized as an anti-tumor immune system. To evaluate intratumor immune status in intracranial germinoma, we examined expressions of PD-1 and PD-L1 (clone 28-8) and subtypes of TILs. Expressions of PD-1 and PD-L1 were detected immunohistochemically in 25 formalin-fixed, paraffin-embedded tumor specimens from 24 patients with intracranial germinoma consisting of 22 primary and 3 recurrent tumors. To evaluate subtypes of TILs, quantification of lymphocytes with CD3, CD8, CD4, and Foxp3 was performed. Statistical analyses were performed among PD-1, PD-L1 and subtypes of TILs. In 25 tumor tissue, expressions of PD-1 in TILs and PD-L1 in tumor cells were identified in 96% (24/25) and 92% (23/25), respectively. Expression of PD-1 was associated with CD3+ TIL density. Expression of PD-1 correlated with Foxp3+ TIL density and CD8+ TIL density, but not with CD4+ TIL density. Furthermore, expression of PD-1 correlated strongly with Foxp3+/CD4+ ratio. Taken together, increase of PD-1+ expression is associated with accumulation of Foxp3+ and CD8+ TILs. These findings intimate that PD-1/PD-L1 axis might shape the immune infiltration suggesting a modulation of the immune response and subsequent tumor growth in intracranial germinoma. Anti-PD-1 and anti-PD-L1 are potential immune therapeutic strategies in intracranial germinoma.

Type, Frequency, and Spatial Distribution of Immune Cell Infiltrates in CNS Germinomas: Evidence for Inflammatory and Immunosuppressive Mechanisms.

Central nervous system germinomas are characterized by a massive immune cell infiltrate. We systematically characterized these immune cells in 28 germinomas by immunophenotyping and image analysis. mRNA expression was analyzed by Nanostring technology and in situ RNA hybridization. Tumor infiltrating lymphocytes (TILs) were composed of 61.8% ± 3.1% (mean ± SE) CD3-positive T cells, including 45.2% ± 3.5% of CD4-positive T-helper cells, 23.4% ± 1.5% of CD8-positive cytotoxic T cells, 5.5% ± 0.9% of FoxP3-positive regulatory T cells, and 11.9% ±1.3% PD-1-positive TILs. B cells accounted for 35.8% ± 2.9% of TILs and plasma cells for 9.3% ± 1.6%. Tumor-associated macrophages consisted of clusters of activated PD-L1-positive macrophages and interspersed anti-inflammatory macrophages expressing CD163. Germinoma cells did not express PD-L1. Expression of genes encoding immune cell markers and cytokines was high and comparable to mRNA levels in lymph node tissue. IFNG and IL10 mRNA was detected in subfractions of TILs and in PD-L1-positive macrophages. Taken together, the strong immune reaction observed in germinomas involves inflammatory as well as various suppressive mechanisms. Expression of PD-1 and PD-L1 and infiltration of cytotoxic T cells are biomarkers predictive of response to anti-PD-1/PD-L1 therapies, constituting a rationale for possible novel treatment approaches.

Pituitary and systemic autoimmunity in a case of intrasellar germinoma.

Germinomas arising in the sella turcica are difficult to differentiate from autoimmune hypophysitis because of similar clinical and pathological features. This differentiation, nevertheless, is critical for patient care due to different treatments of the two diseases. We report the case of an 11-year-old girl who presented with diabetes insipidus and growth retardation, and was found to have an intra- and supra-sellar mass. Initial examination of the pituitary biopsy showed diffuse lymphocytic infiltration of the adenohypophysis and absent placental alkaline phosphatase expression, leading to a diagnosis of hypophysitis and glucocorticoid treatment. Because of the lack of clinical and radiological response, the pituitary specimen was re-examined, revealing this time the presence of scattered c-kit and Oct4 positive germinoma cells. The revised diagnosis prompted the initiation of radiotherapy, which induced disappearance of the pituitary mass. Immunological studies showed that the patient's serum recognized antigens expressed by the patient's own germinoma cells, as well as pituitary antigens like growth hormone and systemic antigens like the Sjögren syndrome antigen B and alpha-enolase. The study first reports the presence of pituitary and systemic antibodies in a patient with intrasellar germinoma, and reminds us that diffuse lymphocytic infiltration of the pituitary gland and pituitary antibodies does not always indicate a diagnosis of autoimmune hypophysitis.

The microenvironment of germ cell tumors harbors a prominent antigen-driven humoral response.

Germ cell tumors are a heterogeneous group of neoplasms derived from residual primordial tissue. These tumors are commonly found in the brain, testes, or ovaries, where they are termed germinomas, seminomas, or dysgerminomas, respectively. Like several other tumor types, germ cell tumors often harbor an immune cell infiltrate that can include substantial numbers of B cells. Yet little is known about whether the humoral immune response affects germ cell tumor biology. To gain a deeper understanding of the role B cells play in this tumor family, we characterized the immune cell infiltrate of all three germ cell tumor subtypes and defined the molecular characteristics of the B cell Ag receptor expressed by tumor-associated B cells. Immunohistochemistry revealed a prominent B cell infiltrate in the microenvironment of all tumors examined and clear evidence of extranodal lymphoid follicles with germinal center-like architecture in a subset of specimens. Molecular characterization of the Ig variable region from 320 sequences expressed by germ cell tumor-infiltrating B cells revealed clear evidence of Ag experience, in that the cardinal features of an Ag-driven B cell response were present: significant somatic mutation, isotype switching, and codon insertion/deletion. This characterization also revealed the presence of both B cell clonal expansion and variation, suggesting that local B cell maturation most likely occurs within the tumor microenvironment. In contrast, sequences from control tissues and peripheral blood displayed none of these characteristics. Collectively, these data strongly suggest that an adaptive and specific humoral immune response is occurring within the tumor microenvironment.

Renal cell carcinoma antigen is expressed by yolk sac tumors and yolk sac elements of embryonal carcinomas.

Renal cell carcinoma antigen is a rather specific marker for normal and neoplastic renal tissue. We investigated the expression of this antigen in 34 gonadal and extragonadal germ cell tumors, including 8 pure yolk sac carcinomas and 26 embryonal carcinomas, 15 of which were combined with teratomas, seminomas, and dysgerminomas. Renal cell carcinoma antigen was demonstrated in all 8 yolk sac tumors and 21 of 26 embryonal carcinomas (81%). In yolk sac tumors, renal cell carcinoma antigen reactivity was diffusely present throughout the tumors. In embryonal carcinomas, this marker was identified only in yolk sac components. Both intracytoplasmic and membranous staining patterns were present. No reactivity was noticed in embryonal carcinoma cells, seminoma, dysgerminoma, and other components of teratomas. The study suggests an antigenic similarity between renal tubules and yolk sac tumors. Furthermore, the renal cell carcinoma antigen may be used as an addition to the panel of immunocytochemical markers for yolk sac carcinomas.

Activated status of tumour-infiltrating lymphocytes and apoptosis in testicular seminoma.

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.

Radioimmunoscintigraphy in patients with ovarian cancer.

The use of radiolabeled monoclonal antibodies (MoAbs) has significantly improved the ability to detect tumor antigens, thus improving in vivo tumor diagnosis and treatment. The management of ovarian carcinoma still poses a challenging medical problem. Clinical trials using radioimmunoscintigraphy or a hand-held gamma detection probe intraoperatively were performed in patients with clinical evidence of primary or recurrent ovarian cancer. Immunoscintigraphy of ovarian cancer lesions has been performed mainly with 99mTc, 111In and 123I labeled with HMFG1, HMFG2, OC-125, B72.3, H17E2, OVTL3, MoAb170, Mov18 and other MoAbs. Antibody guided imaging using radioimmunoscintigraphy has demonstrated improved targeting of ovarian cancer, resulting in a highly sensitive and specific method. However, it is not yet known which type of MoAb is the most efficient for radioimmunoscintigraphy. Since these tumors represent a potentially curable disease, radioimmunoscintigraphy could contribute mainly to accurate staging as a supplementary to conventional diagnostic methods, as well as for the localization of active disease after chemotherapy and monitoring for the presence of recurrent disease. Nevertheless, prospective studies in a large number of patients should be undertaken in order to further evaluate the diagnostic contribution of this approach.

[Acute hepatitis B virus after chemotherapy for a case with germinoma].

A 28-year old man with HCG-producing germinoma had undergone chemotherapy and radiotherapy. On admission for the fifth session of maintenance chemotherapy, he was found to be positive for hepatitis B (HB)s antigen, but negative for HBs antibody. HBs antigen had been negative during previous admissions. Since liver function was normal, the patient underwent chemotherapy. During myelosuppression after chemotherapy, liver dysfunction developed and acute HB was diagnosed. He fortunately showed seroconversion 2 months after onset. Serum immunological examinations are required for patients receiving chemotherapy.

Expression patterns of cancer testis antigens in testicular germ cell tumors and adjacent testicular tissue.

PURPOSE: The human cancer-testis antigens (CTAs) are a group of tumor specific antigens recognized by cytotoxic T lymphocytes whose expression occurs in human malignancies as well as in normal testicular tissue. We studied a series of CTA gene transcripts in testicular germ cell tumors of various histological types to test the hypothesis that the expression of CTA in testicular germ cell tumors reflects developmental stages of tumorigenesis rather than constitutive tumor antigens recognized by cytotoxic T lymphocytes. MATERIALS AND METHODS: Total RNA was obtained from 31 primary and 3 metastatic testicular germ cell tumors, and 11 parenchymal tissues adjacent to the testicular germ cell tumors. We performed an expression study of the CTA genes MAGE-A, MAGE-B, GAGE, PAGE-1, HOM-MEL-40 (SSX2), NY-ESO-1, LAGE-1 and SCP-1 in these samples using reverse transcriptase-polymerase chain reaction. RESULTS: The results showed that expression patterns of CTA genes depended on the histological differentiation of the testicular germ cell tumors. Overall CTA expression was more common in seminomas than in nonseminomatous germ cell tumors. Specifically all 13 seminomas (100%) demonstrated the positive expression of MAGE-B1 and MAGE-B2, while 3 of 17 nonseminomatous germ cell tumor samples (18%) showed positive expression of these genes. All 5 teratomatous elements (100%) had homogenous null expression with regard to all CTA genes examined. In addition, we detected deficiencies in CTA expression in 7 of 11 parenchymal tissues adjacent to the testicular germ cell tumors (64%). CONCLUSIONS: These data support the idea that CTA transcripts in testicular germ cell tumors serve as developmental footprints of testicular germ cell tumors rather than as constitutive tumor antigens recognized by cytotoxic T lymphocytes.

Persistence of clonal T-cell expansions following high-dose chemotherapy and autologous peripheral blood progenitor cell rescue.

Analysing the regeneration of T lymphocytes after high-dose chemotherapy with autologous peripheral blood progenitor cell rescue (PBPCR) may help elucidate the mechanisms of immune recovery. The T-cell receptor variable beta chain (TCRBV) repertoire of adult patients undergoing high-dose chemotherapy was analysed by flow cytometry, before and after treatment. Four patients were found to have a stable expansion present (TCRBV3, 17, 21 and 22) ranging from 8% to 42% of the CD4(+) or CD8(+) repertoire. We demonstrated that, in these patients, following high-dose chemotherapy and autologous stem cell transplantation, the clonal expansions reappeared in peripheral blood and returned to pretransplant levels. Three expansions (CD3(+)CD8(+)TCRBV3(+), CD3(+)CD4(+)TCRBV21(+) and CD3(+)CD8(+)TCRBV22(+)) were further defined by sequence analysis of the complementarity-determining region (CDR)3 portion within the TCR rearrangements. These were shown to be predominantly clonal, with the same sequences being identified in peripheral blood before and after PBPCR, providing evidence that the overwhelming majority of T cells in these expansions arise from mature lymphocytes. This study demonstrated that patients undergoing autologous PBPCR for high-dose chemotherapy regenerate clonal expansions, consistent with pretreatment levels. They also regenerate T-cell repertoires with each TCRBV family represented to a similar level as that prior to high-dose chemotherapy.

A new highly specific monoclonal antibody against placental alkaline phosphatase: a potential marker for the early detection of testis tumour.

OBJECTIVE: To develop specific monoclonal antibodies (mAbs) against human germ cell tumours. MATERIALS AND METHODS: A single-cell suspension obtained from tumour tissue fragments (consisting of both tumour and normal compartments) from a patient with seminoma was used as an immunogen. Spleen cells from immunized mice were used to develop mAbs. Tissue specificity, biochemical characteristics and competitive studies were analysed using immunocytochemical staining, dot blots and a Western blot analysis, to identify target antigen(s). RESULTS: The immunization protocol led to the development of 107 hybridomas, 90 of which were negative against the original tissue biopsies. The remaining 17 showed positivity against various tissue compartments. One selected mAb (ATC2) showed specific staining on germ cell tumours but not on normal tissues, and positive staining with some human tumour cell lines. The target antigen for ATC2 was confirmed to be placental alkaline phosphatase (PLAP) based on: Western blot analysis compared with commercially available PLAP; comparison of the data with another well-known anti-PLAP mAb (H17E2, although the two mAbs recognized different antigenic epitopes); heat resistance characteristics; high-performance liquid chromatography of the ATC2 target antigen and purified PLAP. CONCLUSION: The selected mAb ATC2 has high specificity for human germ cell tumours, the target antigen for ATC2 being PLAP, although the antigenic epitope(s) differ from those recognized by H17E2. Thus ATC2 may be useful for monitoring serum levels of PLAP in patients with testis cancer and may be relevant for detecting cancer cells in the semen of individuals with suspected testis cancer, particularly in those with equivocal findings on ultrasonography.

Human endogenous retrovirus protein cORF supports cell transformation and associates with the promyelocytic leukemia zinc finger protein.

Human endogenous retrovirus sequences (HERVs) reside in the genomes of primates and humans for several million years. The majority of HERVs is non-coding but a limited set is intact and can express proteins. We have recently identified an almost intact HERV-K(HML-2) provirus on chromosome 7 and have documented that most patients with germ cell tumors (GCTs) display antibodies directed against proteins of HERV-K(HML-2). To address whether these proteins merely represent tumor markers or contribute to neoplastic transformation, we examined the transforming potential of various HERV sequences and studied physical interactions between HERV and cellular proteins by yeast two-hybrid and biochemical assays. cORF, a protein encoded by the C-terminal open reading frame within the env gene, supports tumor growth in nude mice and associates with the promyelocytic leukemia zinc finger protein (PLZF). The interaction domains map between amino acid residues 21 and 87 of cORF, and between residues 245 and 543 of PLZF. PLZF is critical for spermatogenesis in mice. Abnormal spermatogenesis or maturation of gonocytes is thought to predispose humans to the development of germ cell tumors. Thus, cORF of human endogenous retroviruses may contribute to tumor development by interfering with processes during spermatogenesis that involve PLZF.

Transient increase in mitogen-induced lymphoproliferative responses in patients with testicular cancer after BEP chemotherapy.

OBJECTIVES: To investigate the impact of polychemotherapy on cellular immunity in patients with testicular cancer. METHODS: Lymphocyte subpopulations, lymphoproliferative responses to mitogenic stimulation, and mitogen-induced release of soluble interleukin-2 receptor from peripheral blood mononuclear cells were investigated in 15 patients with testicular germ cell tumors a median of 61 months (range 7 to 73) after polychemotherapy with bleomycin, etoposide, and cisplatin (BEP). RESULTS: The numbers of peripheral blood T cells (CD3+), CD4+ and CD8+ subsets, and lymphoproliferative responses to pokeweed mitogen, phytohemagglutinin, and concanavalin A in patients were comparable to those of healthy control subjects. When two groups of patients were formed according to elapsed time from BEP polychemotherapy and study onset (group A, 12 months and group B, 69 months after termination of BEP), a significant increase in lymphoproliferative response to concanavalin A (P <0.05) was found in group A 1 year after chemotherapy. CONCLUSIONS: BEP chemotherapy administered to patients with testicular cancer does not result in impairment of cellular immunity but rather leads to a significant increase in the capacity of patients' lymphocytes to respond to mitogenic stimulation up to 1 year after polychemotherapy. Moreover, the increased T-cell activity found after BEP therapy may contribute to the high rate of long-term complete remission.

Diagnostic value of anti-alpha FP antibody levels in a metastatic germ cell tumor of unknown primary site.

BACKGROUND: A 21 year old man with a metastatic germ cell tumor of unknown primary not responding to chemotherapy was scheduled to have a blind bilateral orchiectomy to eradicate the possible primary site although palpation and ultrasonography of the testicles had always been normal. METHOD: The patient underwent a radioimmunoscintigraphy with Anti-alpha FP antibody scan (AFP-Scan). RESULTS: On the basis of the scintigraphic results the patient underwent a left orchiectomy and additionally removal of the lymph node metastases. Histology revealed the presence of an in situ carcinoma in the left testis and a mixed tumor present in the abdominal lymph node metastases. Fluorescent in situ hybridization on tumor cells did not show any abnormalities related to chromosome 12, a finding connected with the somatic type of germ cell tumors. CONCLUSION: Anti-alpha FP antibody scan was helpful in detecting the primary site and saving the life of the patient without resulting in hypogonadism.

Characterization and distribution of an oncofetal antigen (M2A antigen) expressed on testicular germ cell tumours.

M2A antigen is an oncofetal antigen associated with germ cell neoplasia, present in testis on fetal gonocytes and re-expressed on carcinoma in situ (CIS) and germ cell tumours. We developed a panel of monoclonal antibodies (mAb), M2A (IgG2a), D1-26 (IgG2b) and D2-40 (IgG1), to this antigen in order to characterize its structure and study its distribution among germ cell tumours. M2A antigen was purified by sequential lectin and antibody affinity chromatography and characterized as a monomeric M, 40 000 surface sialoglycoprotein, extensively glycosylated with O-linked carbohydrate structures, but devoid of N-linked sugars. Terminal sialic acid residues were required for reactivity with mAb M2A and D1-26, but not D2-40. Sections of 69 testicular germ cell tumours, fixed in formalin and embedded in paraffin, were stained with mAb D2-40 to examine the distribution of M2A antigen. Uniform membrane staining was observed in seminomas, and focal staining in 69% of embryonal carcinomas, 29% of teratomas and 25% of yolk sac tumours. CIS in the vicinity of all germ cell tumours also displayed uniform membrane staining. The characterization of M2A antigen, and the development of mAb which react with it in conventionally preserved archival specimens, provide important initiatives to study the origin and progression of germ cell neoplasia.