Inhibition of bromodomain and extra‐terminal proteins increases sensitivity to venetoclax in chronic lymphocytic leukaemia

The development of drugs able to target BTK, PI3k‐delta and BCL2 has dramatically improved chronic lymphocytic leukaemia (CLL) therapies. However, drug resistance to these therapies has already been reported due to non‐recurrent changes in oncogenic pathways and genes expression signatures. In this study, we investigated the cooperative role of the BCL2 inhibitor venetoclax and the BRD4 inhibitor JQ1. In particular, we found that JQ1 shows additional activity with venetoclax, in CLL cell lines and in ex vivo isolated primary CD19⁺ lymphocytes, arguing in favour of combination strategies. Lastly, JQ1 is also effective in venetoclax‐resistant CLL cell lines. Together, our findings indicated that the BET inhibitor JQ1 could be a promising therapy in CLL, both as first‐line therapy in combination with venetoclax and as second‐line therapy, after the emergence of venetoclax‐resistant clones.     

Reduced Proficiency in Homologous Recombination Underlies the High Sensitivity of Embryonal Carcinoma Testicular Germ Cell Tumors to Cisplatin and Poly (ADP-Ribose) Polymerase Inhibition

Testicular Germ Cell Tumors (TGCT) and patient-derived cell lines are extremely sensitive to cisplatin and other interstrand cross-link (ICL) inducing agents. Nevertheless, a subset of TGCTs are either innately resistant or acquire resistance to cisplatin during treatment. Understanding the mechanisms underlying TGCT sensitivity/resistance to cisplatin as well as the identification of novel strategies to target cisplatin-resistant TGCTs have major clinical implications. Herein, we have examined the proficiency of five embryonal carcinoma (EC) cell lines to repair cisplatin-induced ICLs. Using γH2AX staining as a marker of double strand break formation, we found that EC cell lines were either incapable of or had a reduced ability to repair ICL-induced damage. The defect correlated with reduced Homologous Recombination (HR) repair, as demonstrated by the reduction of RAD51 foci formation and by direct evaluation of HR efficiency using a GFP-reporter substrate. HR-defective tumors cells are known to be sensitive to the treatment with poly(ADP-ribose) polymerase (PARP) inhibitor. In line with this observation, we found that EC cell lines were also sensitive to PARP inhibitor monotherapy. The magnitude of sensitivity correlated with HR-repair reduced proficiency and with the expression levels and activity of PARP1 protein. In addition, we found that PARP inhibition strongly enhanced the response of the most resistant EC cells to cisplatin, by reducing their ability to overcome the damage. These results point to a reduced proficiency of HR repair as a source of sensitivity of ECs to ICL-inducing agents and PARP inhibitor monotherapy, and suggest that pharmacological inhibition of PARP can be exploited to target the stem cell component of the TGCTs (namely ECs) and to enhance the sensitivity of cisplatin-resistant TGCTs to standard treatments.     

Molecular biomarkers as potential targets for therapeutic strategies in human testicular germ cell tumors: An overview

Testicular germ cell tumors (TGCTs), the most common malignancy in males between 15 and 34 years of age and the most frequent cause of death from solid tumors in this age group. TGCTs can be subdivided into seminoma and non‐seminoma germ cell tumors (NSGCTs), including embryonal cell carcinoma, choriocarcinoma, yolk sac tumor, and teratoma. Seminomas and NSGCTs do not only present distinctive clinical features, but they also show significant differences as far as therapy and prognosis are concerned. Seminomas are highly sensitive to both radiation and chemotherapy, with a good prognosis, non‐seminomas are sensitive to platinum‐based combination chemotherapy and are less susceptible to radiation, with the exception of teratomas. The different therapeutic outcome might be explained by inherent properties of the cells from which testicular neoplasia originate. The unique treatment sensitivity of TGCTs is unexplained so far, but it is likely to be related to intrinsic molecular characteristics of the PGCs/gonocytes, from which these tumors originate. Many discovered bio‐markers including OCT3/4, SOX2, SOX17, HMGA1, HMGA2, PATZ1, GPR30, Aurora B, estrogen receptor β, and others have given further advantages to discriminate between histological subgroups. In addition, therapeutic approaches for the treatment of TGCTs have been proposed: humanized antibodies against receptors/surface molecules on cancer cells, inhibitors of serine–threonine, and tyrosine kinases, and others. The mini‐review will be an overview on the molecular alterations identified in TGCTs and on novel targeted antineoplastic strategies that might help to treat chemotherapy resistant TGCTs. J. Cell. Physiol. 228: 1641–1646, 2013. © 2013 Wiley Periodicals, Inc.     

Testicular germ cell tumours: the paradigm of chemo-sensitive solid tumours

Testicular germ cell tumours (TGCTs) are the most frequent solid malignant tumour in men 20–40 years of age and the most frequent cause of death from solid tumours in this age group. Up to 50% of the patients suffer from metastatic disease at diagnosis. The majority of metastatic testicular cancer patients, in contrast to most other metastatic solid tumours, can be cured with highly effective cisplatin-based chemotherapy. From a genetic point of view, almost all TGCTs in contrast to solid tumours are characterised by the presence of wild type p53. High p53 expression levels are associated with elevated Mdm2 levels and a loss of p21^Waf1/Cip1 expression suggesting a changed functionality of p53. Expression levels of other proteins involved in the regulation of cell cycle progression indicate a deregulated G1–S phase checkpoint in TGCTs. After cisplatin-induced DNA damage, the increasing levels of p53 lead to the trans-activation of a number of genes but not of p21^Waf1/Cip1, preferentially directing TGCT cells into apoptosis or programmed cell death, both via the mitochondrial and the death receptor apoptosis pathways. The sensitivity of TGCTs to chemotherapeutic drugs may lay in the susceptibility of germ cells to apoptosis. Taken together, this provides TGCT as a tumour type model to investigate and understand the molecular determinants of chemotherapy sensitivity of solid tumours. This review aims to summarise the current knowledge on the biological basis of cisplatin-induced apoptosis and response to chemotherapy in TGCTs.     

Tumor-suppressive Function of Protein-tyrosine Phosphatase Non-receptor Type 23 in Testicular Germ Cell Tumors Is Lost upon Overexpression of miR142–3p microRNA

Protein-tyrosine phosphatase non-receptor type 23 (PTPN23) is a candidate tumor suppressor involved in the tumorigenesis of various organs. However, its physiological role(s) and detailed expression profile(s) have not yet been elucidated. We investigated the function and regulation of PTPN23 in the formation of testicular germ cell tumors (TGCTs). Expression of PTPN23 in human TGCT cell lines was significantly lower than that in spermatogonial stem cells in mice. Overexpression of PTPN23 in NEC8, a human TGCT cell line, suppressed soft agar colony formation in vitro and tumor formation in nude mice in vivo. These data indicate that PTPN23 functions as a tumor suppressor in TGCTs. Multiple computational algorithms predicted that the 3′ UTR of human PTPN23 is a target for miR-142-3p. A luciferase reporter assay confirmed that miR-142-3p bound directly to the 3′ UTR of PTPN23. Introduction of pre-miR-142 in the PTPN23 transfectant of NEC8 led to suppressed expression of PTPN23 and increased soft agar colony formation. Quantitative RT-PCR data revealed a significantly higher expression of miR-142-3p in human seminomas compared with normal testes. No difference in mRNA expression between seminoma and non-seminoma samples was detected by in situ hybridization. Both quantitative RT-PCR and immunohistochemical analyses revealed that PTPN23 expression was significantly lower in TGCTs than in normal testicular tissues. Finally, a lack of PTPN23 protein expression in human TGCTs correlated with a relatively higher miR-142-3p expression. These data suggest that PTPN23 is a tumor suppressor and that repression of PTPN23 expression by miR-142-3p plays an important role in the pathogenesis of TGCTs.     

JQ1, a BET‐bromodomain inhibitor,inhibits human cancer growth and suppresses PD‐L1 expression

Most traditional cytotoxic chemotherapeutic agents have poor aqueous solubility and significant toxicity. Hence, there is a need to develop molecule‐targeted drugs. Programmed death‐ligand 1 (PD‐L1) is associated with the prognosis of several cancer types, and blockade of PD‐1/PD‐L1 signaling increases the amplitude of anti‐tumor immunity. In the present study, we investigated the effects of JQ1, a bromodomain and extraterminal‐bromodomain inhibitor, on cell growth, and messenger RNA (mRNA) and protein levels of PD‐L1 in renal cell carcinoma primary culture cells, and prostate, liver, and lung cancer cell lines. The results of the cell counting kit‐8 assay suggested that JQ1 inhibits cell growth in a dose‐dependent manner. The mRNA and protein levels of PD‐L1 decreased in the primary culture of JQ1‐treated renal carcinoma, prostate cancer, liver cancer, and lung cancer cell lines. In addition, the mRNA level of PD‐L2 also decreased in the JQ1‐treated cells. Overall, JQ1 might be a potential anti‐tumor agent.     

Deciphering the molecular effects of romidepsin on germ cell tumours: DHRS2 is involved in cell cycle arrest but not apoptosis or induction of romidepsin effectors

Testicular germ cell tumours (GCTs) mostly affect young men at age 17‐40. Although high cure rates can be achieved by orchiectomy and chemotherapy, GCTs can still be a lethal threat to young patients with metastases or therapy resistance. Thus, alternative treatment options are needed. Based on studies utilising GCT cell lines, the histone deacetylase inhibitor romidepsin is a promising therapeutic option, showing high toxicity at very low doses towards cisplatin‐resistant GCT cells, but not fibroblasts or Sertoli cells. In this study, we extended our analysis of the molecular effects of romidepsin to deepen our understanding of the underlying mechanisms. Patients will benefit from these analyses, since detailed knowledge of the romidepsin effects allows for a better risk and side‐effect assessment. We screened for changes in histone acetylation of specific lysine residues and analysed changes in the DNA methylation landscape after romidepsin treatment of the GCT cell lines TCam‐2, 2102EP, NCCIT and JAR, while human fibroblasts were used as controls. In addition, we focused on the role of the dehydrogenase/reductase DHRS2, which was strongly up‐regulated in romidepsin treated cells, by generating DHRS2‐deficient TCam‐2 cells using CRISPR/Cas9 gene editing. We show that DHRS2 is dispensable for up‐regulation of romidepsin effectors (GADD45B, DUSP1, ZFP36, ATF3, FOS, CDKN1A, ID2) but contributes to induction of cell cycle arrest. Finally, we show that a combinatory treatment of romidepsin plus the gluccocorticoid dexamethasone further boosts expression of the romidepsin effectors and reduces viability of GCT cells more strongly than under single agent treatment. Thus, romidepsin and dexamethasone might represent a new combinatorial approach for treatment of GCT.     

SERPINE2 is a possible candidate promotor for lymph node metastasis in testicular cancer

Testicular germ cell tumors (TGCTs) commonly metastasize to the lymph node or lung. However, it remains unclear which genes are associated with TGCT metastasis. The aim of this study was to identify gene(s) that promoted human TGCT metastasis. We intraperitoneally administered conditioned medium (CM) from JKT-1, a cell-line from a human testicular seminoma, or JKT-HM, a JKT-1 cell sub-line with high metastatic potential, into mice with JKT-1 xenografts. Administration of CM from JKT-HM significantly promoted lymph node metastasis. A cDNA microarray analysis showed that JKT-HM cells highly expressed the Serpine peptidase inhibitor, clade E, member 2 (SERPINE2), which encodes a secreted protein. Administration of CM from SERPINE2-silenced JKT-HM cells inhibited lymph node metastasis in the xenograft model, compared with administration of CM from JKT-HM cells. There was no significant difference in xenograft volume. Moreover, administration of CM from SERPINE2-over-expressing JKT-1 was likely to promote lymph node metastasis in the xenograft model. There was no difference in the in vitro proliferation or migration of JKT-1 cells cultured with CM from JKT-HM cells, compared to that with CM from JKT-1. There was no promotion of proliferation or lymphangiogenesis in the xenografts, as measured by Ki-67 and LYVE-1 immunohistochemistry, respectively. Although we could not clarify how SERPINE2 promoted lymph node metastasis, it may be a promoter in the development of lymph node metastasis in the human seminoma cells in a mouse xenograft model.     

p53 inactivation by MDM2 and MDMX negative feedback loops in testicular germ cell tumors

Testicular germ cell tumors (TGCT) are unique in their excellent response to DNA-damaging chemotherapy. Mutation of p53 is rare in both untreated and relapsed TGCTs, suggesting that p53 fails to respond effectively against malignant transformation in germ cells. Previous studies implicated the presence of a poorly defined TGCT-specific mechanism of p53 inactivation. Here we show that disruption of p53-MDM2 binding using the MDM2-specific inhibitor Nutlin activates p53 in TGCT cells and is sufficient to induce strong apoptosis. Knockdown of MDMX cooperates with Nutlin to activate p53. Surprisingly, we found that p53 activation induced a two-fold increase in MDMX mRNA and protein expression in TGCT cells. A p53-responsive promoter is identified in MDMX intron 1 that contains a functional p53-binding site, suggesting that MDMX also functions as a negative feedback regulator of p53 in a cell line-dependent fashion. These findings suggest that MDM2 and MDMX are responsible for the functional inactivation of p53 in TGCT. Furthermore, TGCT cells are unique in having a strong apoptosis response to p53. Direct activation of p53 by targeting MDM2 and MDMX may provide a backup approach for the treatment of TGCTs resistant to DNA-damaging drugs.     

Y chromosome instability in testicular cancer

Approximately 15-25% of male infertility cases carry extensive azoospermic factor (AZF) deletions. Moreover, about 80% of Finnish testicular germ cell tumors (TGCT) and about 23-25% of TGCTs from other geographic regions carry short and interstitial AZF deletions. In infertility cases the AZF deficiency occurs in the germ cells of the proband father giving rise to mosaic sperm populations comprising non-deleted and deleted sperms. Fertilization of an oocyte by a Y deleted sperm will give rise to an AZF-deleted and infertile F1 male. In TGCTs the AZF deletions take place in the initial stages of embryogenesis producing individuals that are a mosaic of Y deleted and non-deleted cell lineages. Carcinoma in situ (CIS) is a premalignant lesion that some believe may develop in gonads of male embryos before the ninth week of age due to transformation of a totipotent primordial germ cell. If the transformed cell carries AZF deletions the resultant CIS will also have Y deletions. CIS will differentiate into seminoma or into embryonal carcinoma and non-seminomas in about 1 x 10(-3) of the young adults carrying premalignant CIS outgrowths; if the CIS lesion has AZF deletions the derived forms of testicular cancer will also exhibit these deletions. AZF deletions play no role in the development of testicular cancers. On the other hand, they are a marker of Y chromosome instability and eventually of a more generalized pattern of genome instability associated with the appearance of TGCT. Genetic factors such as malfunction of metabolizing genes, DNA repairing genes, Y-linked or X-linked genes have been considered as possible causes of AZF deletions in testicular cancer. Yet, the exact identification of the genes involved remains elusive. AZF deletions have also been identified in non-Hodgkin lymphomas and in colorectal cancers, two forms of malignancy that have been found to be associated with TGCTs.     

Enhancers and suppressors of testicular cancer susceptibility in single- and double-mutant mice

Susceptibility to spontaneous testicular germ cell tumors (TGCTs), a common cancer affecting young men, shows unusual genetic complexity. Despite remarkable progress in the genetics analysis of susceptibility to many cancers, TGCT susceptibility genes have not yet been identified. Various mutations that are inherited as Mendelian traits in laboratory mice affect susceptibility to spontaneous TGCTs on the 129/Sv inbred genetic background. We compared the frequency of spontaneous TGCTs in single- and double-mutant mice to identify combinations that show evidence of enhancer or suppressor effects. The lower-than-expected TGCT frequencies in mice with partial deficiencies of TRP53 and MGF-SLJ and in 129.MOLF-Chr19 (M19) consomic mice that were heterozygous for the A(y) mutation suggest that either these genes complement each other to restore normal functionality in TGCT stem cells or together these genes activate mechanisms that suppress incipient TGCTs. By contrast, the higher-than-expected TGCT frequencies in Mgf(Sl-J)-M19 compound heterozygous mice suggest that these mutations exacerbate each other's effects. Together, these results provide clues to the genetic and molecular basis for susceptibility to TGCTs in mice and perhaps in humans.     

A molecular cytogenetic approach to studying platinum resistance

The technique of comparative genomic hybridisation (CGH) has until recently been used to screen for common genomic abnormalities in fresh tumour material; it has identified previously unrecognised regions of amplification associated with poor prognosis subtypes of breast cancer and lymphoma. Our group has applied this technique to resistant cell lines and their sensitive counterparts in order to define chromosomal abnormalities associated with acquired drug resistance. We have demonstrated the applicability of this technique to the study of drug resistance using cell lines with known mechanisms of resistance. The ability to detect novel genomic alterations in cell lines with novel mechanisms of resistance was also demonstrated. We subsequently examined the CGH profiles of seven different cell lines made resistant to three platinum analogues and showed the most consistent abnormalities to involve over-representation of regions 4q and 6q. More recently, we have applied the CGH technique to a series of testicular germ cell tumours (TGCTs) collected as formalin-fixed paraffin-embedded biopsy specimens from patients, both pre- and post-therapy using a platinum-based regimen (POMB/ACE). Previous reports have shown over-representation of X, 7q, 8q and 12p and loss of 13q to occur in 25% of primary TGCTs. Over-representation of 12p was confirmed in the majority of these biopsy samples; deletion of 13q was noted in the initial biopsies of several patients. We also demonstrated alterations of 4p, 4q, 5q and 6q in this series of patients. Newly acquired deletions of 2q and 18q and amplifications of 8q were frequently observed in post-chemotherapy samples from resistant tumours. The CGH studies on these patients with TGCT will not only enable us to correlate our observations on clinical material with those from long-term cell lines, but should also identify sites of key genes involved in clinical platinum resistance.     

Romidepsin and tamoxifen cooperatively induce senescence of pancreatic cancer cells through downregulation of FOXM1 expression and induction of reactive oxygen species/lipid peroxidation

Background

Although improvement has been made in therapeutic strategies against pancreatic carcinoma, overall survival has not significantly enhanced over the past decade. Thus, the establishment of better therapeutic regimens remains a high priority.

Methods

Pancreatic cancer cell lines were incubated with romidepsin, an inhibitor of histone deacetylase, and tamoxifen, and their effects on cell growth, signaling and gene expression were analyzed. Xenografts of human pancreatic cancer CFPAC1 cells were medicated with romidepsin and tamoxifen to evaluate their effects on tumor growth.

Results

The inhibition of the growth of pancreatic cancer cells induced by romidepsin and tamoxifen was effectively reduced by N-acetyl cysteine and α-tocopherol, respectively. The combined treatment greatly induced reactive oxygen species production and mitochondrial lipid peroxidation, and these effects were prevented by N-acetyl cysteine and α-tocopherol. Tamoxifen enhanced romidepsin-induced cell senescence. FOXM1 expression was markedly downregulated in pancreatic cancer cells treated with romidepsin, and tamoxifen further reduced FOXM1 expression in cells treated with romidepsin. Siomycin A, an inhibitor of FOXM1, induced senescence in pancreatic cancer cells. Similar results were obtained in knockdown of FOXM1 expression by siRNA.

Conclusion

Since FOXM1 is used as a prognostic marker and therapeutic target for pancreatic cancer, a combination of the clinically available drugs romidepsin and tamoxifen might be considered for the treatment of patients with pancreatic cancer.

     

Elevated H3K27ac in aged skeletal muscle leads to increase in extracellular matrix and fibrogenic conversion of muscle satellite cells

Epigenetic alterations occur in various cells and tissues during aging, but it is not known if such alterations are also associated with aging in skeletal muscle. Here, we examined the changes of a panel of histone modifications and found H3K27ac (an active enhancer mark) is markedly increased in aged human skeletal muscle tissues. Further analyses uncovered that the H3K27ac increase and enhancer activation are associated with the up‐regulation of extracellular matrix (ECM) genes; this may result in alteration of the niche environment for skeletal muscle stem cells, also called satellite cells (SCs), which causes decreased myogenic potential and fibrogenic conversion of SCs. In mice, treatment of aging muscles with JQ1, an inhibitor of enhancer activation, inhibited the ECM up‐regulation and fibrogenic conversion of SCs and restored their myogenic differentiation potential. Altogether, our findings not only uncovered a novel aspect of skeletal muscle aging that is associated with enhancer remodeling but also implicated JQ1 as a potential treatment approach for restoring SC function in aging muscle.     

Loss of drug-induced activation of the CD95 apoptotic pathway in a cisplatin-resistant testicular germ cell tumor cell line

Testicular germ cell tumors (TGCTs) are unusually sensitive to cisplatin. In the present study the role of the CD95 death pathway in cisplatin sensitivity of TGCT cells was studied in Tera and its in vitro acquired cisplatin-resistant subclone Tera-CP. Cisplatin induced an increase in CD95 membrane expression, which preceded the onset of apoptosis. Cisplatin-induced apoptosis was efficiently blocked by caspase-8 inhibitor zIETD-fmk in Tera cells, but only partially in Tera-CP cells. In addition, cisplatin induced FADD and caspase-8 recruitment to the CD95 receptor in Tera cells, which was not noticed in Tera-CP cells. Moreover, overexpression of vFLIP reduced apoptosis induction by cisplatin in Tera cells. CD95L-blocking experiments revealed the involvement of CD95/CD95L interactions in cisplatin-induced apoptosis of Tera cells as well as cisplatin-sensitive 833KE TGCT cells. Tera and 833KE cells, treated with low doses of cisplatin, were sensitive for an apoptosis-inducing anti-CD95 antibody. In contrast, CD95L blocking had no effect on cisplatin-induced apoptosis in Tera-CP or Scha, an intrinsic resistant TGCT cell line, nor did anti-CD95 antibody induce additional apoptosis in cisplatin-treated Tera-CP or Scha cells. Taken together, these results show that (1) cisplatin sensitivity of TGCT cells is dependent on the activation of the CD95 death pathway and (2) loss of cisplatin-induced activation of this CD95 signaling pathway may result in resistance to cisplatin.     

EMMPRIN/CD147-encriched membrane vesicles released from malignant human testicular germ cells increase MMP production through tumor–stroma interaction

Background

Elevated levels of EMMPRIN/CD147 in cancer tissues have been correlated with tumor progression but the regulation of its expression is not yet understood. Here, the regulation of EMMPRIN expression was investigated in testicular germ cell tumor (TGCTs) cell lines.

Methods

EMMPRIN expression in seminoma JKT-1 and embryonal carcinoma NT2/D1 cell lines was determined by Western blot, immunofluorescence and qRT-PCR. Membrane vesicles (MVs) secreted from these cells, treated or not with EMMPRIN siRNA, were isolated by differential centrifugations of their conditioned medium. MMP-2 was analyzed by zymography and qRT-PCR.

Results

The more aggressive embryonic carcinoma NT2/D1 cells expressed more EMMPRIN mRNA than the seminoma JKT-1 cells, but surprisingly contained less EMMPRIN protein, as determined by immunoblotting and immunostaining. The protein/mRNA discrepancy was not due to accelerated protein degradation in NT2/D1 cells, but by the secretion of EMMPRIN within MVs, as the vesicles released from NT2/D1 contained considerably more EMMPRIN than those released from JKT-1. EMMPRIN-containing MVs obtained from NT2/D1, but not from EMMPRIN-siRNA treated NT2/D1, increased MMP-2 production in fibroblasts to a greater extent than those from JKT-1 cells.

Conclusion and general significance

The data presented show that the more aggressive embryonic carcinoma cells synthesize more EMMPRIN than seminoma cells, but which they preferentially target to secreted MVs, unlike seminoma cells which retain EMMPRIN within the cell membrane. This cellular event points to a mechanism by which EMMPRIN expressed by malignant testicular cells can exert its MMP inducing effect on distant cells within the tumor microenvironment to promote tumor invasion. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Germinal tumor invasion and the role of the testicular stroma

Testicular germ cell tumors (TGCTs) are the most frequent neoplasia among young people and their incidence has grown very quickly during recent decades in North America and Europe. Many studies have been carried out in order to elucidate the factors involved in the appearance and progression of these tumors. Little is known about the role of cancer cell-stroma crosstalk in TGCT invasive processes. Here, we review several factors which may be implicated in germ cell tumor progression, such as matrix metalloproteinases, insulin-like growth factor, transforming growth factor beta, the cadherin/catenin complex and integrins. Paradoxically, some of these molecules are also involved in the regulation of normal testicular function. Finally, we discuss prospects for future research on the role of the stroma in the progression and differentiation of male germ cell tumors.     

Serum lactate dehydrogenase (S-LDH) and S-LDH isoenzymes in patients with testicular germ cell tumors

The activities of serum lactate dehydrogenase (S-LDH) and S-LDH isoenzymes were determined in 252 patients with a history of testicular germ cell tumors (TGCT). Fifteen of 37 patients with TGCT lesions and seven of 215 without had raised levels of S-LDH (above 8.0 mukat/l (480 U/l)). Of the patients with TGCT lesions, four had only raised S-LDH-1 levels, one only raised S-LDH-2 (and normal S-LDH), two only raised S-LDH-3 (one with normal S-LDH), and 10 had five combinations of raised levels of S-LDH isoenzymes with a predominance of S-LDH-1. S-LDH and S-LDH-1 correlated significantly with the total tumor volume in the patients with TGCT lesions, especially pronounced in those with lesions from seminoma. Of 34 patients with TGCT metastases, 13 with raised S-LDH levels lived significantly shorter lengths of time than 21 with normal S-LDH. Similarly, 11 with raised S-LDH-1 (above 3.0 mukat/l (180 U/l) lived significantly shorter times than 23 with normal S-LDH-1. S-LDH is a valuable tumor marker in patients with TGCT, especially in those with seminoma. Routine determination of S-LDH isoenzymes in addition to S-LDH in patients with TGCT is not recommended. In patients with a history of TGCT and an unexplained elevation of S-LDH levels, a raised S-LDH-1 level indicates the presence of TGCT lesions.