Comparison of Hepatitis E Virus Sequences from Humans and Swine, the Netherlands, 1998-2015.

Pigs are suspected to be a major source of zoonotic hepatitis E virus (HEV) infection in industrialized countries, but the transmission route(s) from pigs to humans are ill-defined. Sequence comparison of HEV isolates from pigs with those from blood donors and patients in 372 samples collected in the Netherlands in 1998 and 1999 and between 2008 and 2015 showed that all sequences were genotype 3 except for six patients (with travel history). Subgenotype 3c (gt3c) was the most common subtype. While the proportion of gt3c increased significantly between 1998 and 2008, it remained constant between 2008 and 2015. Among the few circulating HEV subtypes, there was no difference observed between the human and the pig isolates. Hepatitis E viruses in humans are very likely to originate from pigs, but it is unclear why HEV gt3c has become the predominant subtype in the Netherlands.

Low SARS-CoV-2 seroprevalence in blood donors in the early COVID-19 epidemic in the Netherlands.

The world is combating an ongoing COVID-19 pandemic with health-care systems, society and economies impacted in an unprecedented way. It is unclear how many people have contracted the causative coronavirus (SARS-CoV-2) unknowingly and are asymptomatic. Therefore, reported COVID-19 cases do not reflect the true scale of outbreak. Here we present the prevalence and distribution of antibodies to SARS-CoV-2 in a healthy adult population of the Netherlands, which is a highly affected country, using a high-performance immunoassay. Our results indicate that one month into the outbreak (i) the seroprevalence in the Netherlands was 2.7% with substantial regional variation, (ii) the hardest-hit areas showed a seroprevalence of up to 9.5%, (iii) the seroprevalence was sex-independent throughout age groups (18-72 years), and (iv) antibodies were significantly more often present in younger people (18-30 years). Our study provides vital information on the extent of exposure to SARS-CoV-2 in a country where social distancing is in place.

Usutu virus infection in Dutch blood donors.

BACKGROUND: The screening of Dutch blood donations for West Nile virus (WNV) may be imminent, as WNV emerges in nearby countries and more donors travel to WNV-affected regions. Since 2016 the related, mosquito-borne Usutu virus (USUV) causes seasonal mortality in Dutch birds. To what extent will human USUV infections affect Dutch WNV donor screening? STUDY DESIGN AND METHODS: From April through September 2018, plasma samples from blood donations in blackbird-rich regions were stored. When increased bird mortality was reported in August, samples from July, August, and September were tested for USUV-RNA in pools of eight, using a home-brew combined WNV/USUV-PCR assay. Reactive pools were deconstructed. Original plasma units and samples of previous and follow-up donations of reactive donors were tested for USUV- and WNV-RNA, and for antibody responses. RESULTS: The number of USUV RNA-positive, WNV RNA-negative donations was 0 of 2688 donations in July, 6 of 4416 in August (1:736), and 1 of 4936 in September. The seven infected donors tested negative for USUV-RNA in preceding and follow-up donations. For 6 donors, seroconversion for USUV-antibodies was demonstrated. All index donations tested positive in a commonly used PCR-assay for WNV donor screening. Three exposed recipients did not show signs of infection. Screening a random subset of 1092 donations from September for USUV-IgG antibodies showed that 22 donors tested reactive; for three donors retrospective testing identified an USUV PCR-positive pre-seroconversion donation. CONCLUSION: Seasonal USUV infection in Dutch blood donors is common. Cross-reactivity in molecular assays for WNV-screening occurs, but can be resolved using USUV- and WNV-specific PCR-primers and sequencing of viral RNA.

Risk factors for hepatitis E virus seropositivity in Dutch blood donors.

BACKGROUND: A marked increase of hepatitis E cases has recently been observed in the Netherlands. Causes of the (re-)emergence of hepatitis E virus (HEV) and exact sources and routes of transmission of HEV infection are currently unknown. We aimed to identify risk factors for HEV seropositivity. METHODS: Using the Wantai EIA, 2100 plasma samples of blood donors from all over the Netherlands aged 18-70 years were tested for anti-HEV IgG antibodies. A questionnaire on socio-demographic characteristics, health, and potential risk factors for HEV exposure was sent to these participants. RESULTS: The overall IgG-seroprevalence was 31% (648/2100) and increased with age. Several food products were independently associated with IgG-seropositivity in a multivariate analysis adjusting for age and gender among 1562 participants who completed the questionnaire: traditional Dutch dry raw sausages called "cervelaat", "fijnkost", "salami" and "salametti" which are generally made from raw pork and beef (aOR 1.5; 95%CI 1.2-1.9), frequent consumption of bovine steak (aOR 1.3; 95%CI 1.0-1.7), and frequent consumption of smoked beef (aOR 1.3 95%CI 1.0-1.7). Although not frequently reported, contact with contaminated water was also a risk factor for seropositivity (aOR 2.5; 95%CI 1.5-4.4). Lower seroprevalence was associated with eating raspberries, going out for dinner, and contact with wild animals and dogs. CONCLUSION: Several pork food products, mainly dry raw sausages, and contact with contaminated water were associated with past HEV infection in the Netherlands. Further investigation is needed into the prevalence and infectivity of HEV in these risk factor food products, as well as investigation of the production methods and possible origin of HEV-contamination within these sausages, e.g. very small amounts of pork liver, pig-derived blood products as food additive, or the pork muscle tissue.

Meat consumption is a major risk factor for hepatitis E virus infection.

BACKGROUND: The incidence of autochthonous hepatitis E virus genotype 3 (HEV gt3) infections in Western Europe is high. Although pigs are a major reservoir of the virus, the exact sources and transmission route(s) of HEV gt3 to humans remain unclear. METHODS: To determine the role of meat consumption at a population level, the seroprevalence of anti-HEV IgG antibodies was compared between Dutch blood donors with a vegetarian lifestyle and donors who consume meat on a daily basis. RESULTS: The age-weighted anti-HEV IgG seroprevalence among donors not eating meat was significantly lower than among meat-eating donors (12.4% vs 20.5%, p = 0.002). For both groups the prevalence strongly increased with age and the difference in prevalence was apparent for all age groups. CONCLUSIONS: Compared with meat-eating donors, the incidence of HEV infection is significantly lower among donors not eating meat, indicating that meat consumption is a major risk factor for HEV infection.

Risk Factors and Screening for Trypanosoma cruzi Infection of Dutch Blood Donors.

BACKGROUND: Blood donors unaware of Trypanosoma cruzi infection may donate infectious blood. Risk factors and the presence of T. cruzi antibodies in at-risk Dutch blood donors were studied to assess whether specific blood safety measures are warranted in the Netherlands. METHODOLOGY: Birth in a country endemic for Chagas disease (CEC), having a mother born in a CEC, or having resided for at least six continuous months in a CEC were considered risk factors for T. cruzi infection. From March through September 2013, risk factor questions were asked to all donors who volunteered to donate blood or blood components. Serum samples were collected from donors reporting one or more risk factors, and screened for IgG antibodies to T. cruzi by EIA. RESULTS: Risk factors for T. cruzi infection were reported by 1,426 of 227,278 donors (0.6%). Testing 1,333 at-risk donors, none (0.0%; 95%, CI 0.0-0.4%) was seroreactive for IgG antibodies to T. cruzi. A total of 472 donors were born in a CEC; 553 donors reported their mother being born in a CEC; and 1,121 donors reported a long-term stay in a CEC. The vast majority of reported risk factors were related to Suriname and Brazil. Overall, the participants resided for 7,694 years in CECs, which equals 2.8 million overnight stays. Of those, 1.9 million nights were spent in Suriname. CONCLUSIONS/SIGNIFICANCE: Asymptomatic T. cruzi infection appears to be extremely rare among Dutch blood donors. Blood safety interventions to mitigate the risk of T. cruzi transmission by transfusion would be highly cost-ineffective in the Netherlands, and are thus not required.

Incidence and duration of hepatitis E virus infection in Dutch blood donors.

BACKGROUND: The incidence of hepatitis E virus (HEV) infection in the Netherlands is high. Blood donors are not routinely screened for HEV infection, but since January 2013, donations used for the production of solvent/detergent (S/D)-treated plasma have been screened for HEV RNA. STUDY DESIGN AND METHODS: Donations were screened for HEV RNA in pools of 96 and 192 donations. In addition, all donations made between 60 days before and after each HEV RNA-positive donation were tested individually for HEV RNA and anti-HEV immunoglobulin G. RESULTS: The screening of 59,474 donations between January 2013 and December 2014 resulted in identification of 45 HEV RNA-positive donations (0.076%) from 41 donors. HEV RNA loads ranged from 80 to 2.3 × 10(6) IU/mL. The number of positive donations increased significantly over time (p = 0.03). Thirty-three of 90 donations made up to 60 days before or after HEV RNA-positive donations were positive when tested individually, while they had not been detected in the pool screening. The mean duration of HEV viremia in the healthy blood donor is estimated to be 68 days. CONCLUSION: The incidence of HEV infection in the Netherlands is high and increased during the study period. In 2013 and 2014, HEV RNA was detected in 1 per 762 donations intended for production of S/D plasma.

Screening of blood donors for chronic Coxiella burnetii infection after large Q fever outbreaks.

BACKGROUND: The Netherlands experienced major Q fever outbreaks from 2007 through 2009. An increasing number of human chronic Q fever cases has been reported in the affected area. Blood donors unaware of chronic Coxiella burnetii infection might be infectious for transfusion recipients. Local blood donations were screened for serologic signs of chronic Coxiella infection. STUDY DESIGN AND METHODS: From August 2012 through January 2013, a total of 2490 serum samples were collected from all consenting blood donors in the most affected Q fever outbreak area and screened for Phase II anti-Coxiella immunoglobulin G antibodies using an enzyme-linked immunosorbent assay (ELISA). (Phase II antibodies are considered indicative for resolved or ongoing Coxiella infection.) Reactive samples were confirmed by quantitative immunofluorescent Phase I and II antibody testing. A Phase I antibody titer of at least 1024 was considered indicative for chronic Coxiella infection. For 179 donors archived samples from 2009 and 2010 were available to study the long-term course of Coxiella antibodies. RESULTS: A total of 110 of 2490 donors were confirmed positive for Phase II Coxiella antibodies (4.4%; 95% confidence interval, 3.7%-5.3%), of which 79 were reactive for Phase I antibodies, with a maximum titer of 256. In 15 of 24 donors (62.5%), testing positive for Phase II antibodies in 2009 and 2010, ELISA reactivity had declined to negativity in 2012 and 2013. CONCLUSION: After large Q fever outbreaks in the Netherlands, no sign of potentially infectious chronic Coxiella infection was found among blood donors in the most affected area. Using an ELISA for detection, Coxiella antibodies in previously exposed donors waned quickly.

Past and present of hepatitis E in the Netherlands.

BACKGROUND: Recent studies show that endemic hepatitis E virus (HEV) infection occurs frequently in some developed countries. In the Netherlands in 2013, the routine screening of 35,220 plasma donations for HEV RNA showed 20 donors to be viremic (1:1761), which seems to contradict reports of declining HEV seroprevalence in the recent past. STUDY DESIGN AND METHODS: To asses HEV infection pressure changes over time, archived samples from Dutch blood donations collected in 1988 and 2000 were tested for anti-HEV immunoglobulin (Ig)G. The findings were compared to the HEV seroprevalence among donors in 2011. RESULTS: The age-adjusted prevalence of anti-HEV IgG for Dutch donors aged 18 to 64 declined from 46.6% in 1988 to 27.3% in 2000 and to 20.9% in 2011. The reduction of seroprevalence was apparent for all age groups between 1988 and 2000, and for donors older than 40 between 2000 and 2011, but the seroprevalence among donors aged 18 to 29 increased between 2000 and 2011. Recent changes in HEV infection pressure are more apparent in the youngest donors, who to a lesser extent reflect cumulative exposure to HEV in the past. Donors aged 18 to 21 showed decreasing HEV seroprevalence from 19.8% in 1988 to 7.0% in 1995 and to 4.3% in 2000, followed by an increase to 12.7% in 2011. CONCLUSION: HEV antibody patterns in young and old Dutch donors, in 1988 to 2011, suggest that decades ago, HEV was ubiquitous and most persons acquired infection. Subsequently HEV incidence was low during a prolonged period, to increase again in recent years.

Coxiella burnetii infection among blood donors during the 2009 Q-fever outbreak in The Netherlands.

BACKGROUND: In 2007, 2008, and 2009 outbreaks of Q-fever occurred in The Netherlands with increasing magnitude. The 2009 outbreak with 2354 reported cases is the largest human Q-fever outbreak ever recorded. To assess the extent of infection and the safety of donated blood, we tested local blood donations for presence of Coxiella burnetii antibodies and DNA. STUDY DESIGN AND METHODS: Starting May 2009, more than 40,000 serum samples were collected from all consenting blood donors in the areas with high Q-fever incidence. The 1004 samples from the areas with the highest number of reported cases were tested for C. burnetii DNA by polymerase chain reaction; seroprevalence and incidence were determined using enzyme-linked immunosorbent assay and immunofluorescence assays (IFAs) in the subset of 543 donors of whom a follow-up sample was available. RESULTS: A total of 6 of 1004 donor samples tested reactive for C. burnetii DNA. Confirmatory testing (IFA) on the index and follow-up samples demonstrated seroconversion in two donors, high-level preexisting antibodies in one donor, and no seroconversion in three donors. Immunoglobulin (Ig)G testing of the 543 serum pairs showed that 66 were reactive in the latest sample, of which 10 represented seroconversions. CONCLUSION: In the area with highest incidence during a large Q-fever outbreak, 3 of 1004 blood donations contained C. burnetii DNA (0.3%; 95% confidence interval, 0.1%-1.0%). A total of 66 of 543 (12.2%) donors tested positive for anti-Coxiella IgG. Ten seroconversions were detected, resulting in an incidence of 5.7% per year, which is more than 10-fold higher than the local number of reported clinical cases (0.47% per year).

Inhibition of androgen receptor functions by gelsolin FxxFF peptide delivered by transfection, cell-penetrating peptides, and lentiviral infection.

BACKGROUND: Prostate cancer (PC) growth is dependent on the androgen-androgen receptor (AR) axis. Because current androgen ablation therapies of PC lead to resistance, novel approaches to block AR activity are urgently needed. METHODS: We inhibited AR function beyond the level of hormone binding by blockade of the coactivator groove in the ligand-binding domain (LBD) using a high-affinity gelsolin FxxFF peptide. Following peptide selection, the effect of the gelsolin FxxFF peptide on AR functions was determined in Hep3B cells that were transiently transfected with pM-peptide expression vectors or were incubated with synthetic gelsolin FxxFF peptide coupled to the TAT cell-penetrating peptide. Lentiviruses expressing the gelsolin FxxFF peptide were used to study endogenous AR target gene expression in LNCaP cells. RESULTS: pM-Gelsolin FxxFF efficiently interfered with AR N/C interaction and specifically inhibited AR-regulated reporter gene activity. The peptide did not inhibit progesterone receptor (PR) and glucocorticoid receptor (GR) activity, nor constitutively active gene promoters. The peptide also specifically blocked in vitro interactions of AR LBD with peptides. Like the gelsolin FxxFF peptide expressed by an expression vector, synthetic TAT-gelsolin FxxFF peptide efficiently blocked AR N/C interaction and inhibited full-length AR-regulated reporter gene activity. It hardly affected PR and GR activity, but the effect on constitutively active promoters was variable. Lentiviral gelsolin FxxFF peptide inhibited expression of KLK2 and NDRG1, but hardly affected PSA and TMPRSS2. CONCLUSIONS: Our results show that the AR coactivator groove may function as a target to overcome therapeutic failure that arises during current androgen ablation therapies.

Systematic structure-function analysis of androgen receptor Leu701 mutants explains the properties of the prostate cancer mutant L701H.

One mechanism of prostate tumors for escape from androgen ablation therapies is mutation of the androgen receptor (AR). We investigated the unique properties of the AR L701H mutant, which is strongly stimulated by cortisol, by a systematic structure-function analysis. Most amino acid substitutions at position 701 did not affect AR activation by 5alpha-dihydrotestosterone. Further analysis of the AR Leu(701) variants showed that AR L701M and AR L701Q, like AR L701H, had changed ligand responsiveness. AR L701M was strongly activated by progesterone but not by cortisol, whereas the opposite was observed for AR L701Q and AR L701H. Next, we analyzed a panel of structurally related steroids to study which of the OH groups at positions 11beta, 17alpha, and 21, which discriminate cortisol from progesterone, underlie the differential responses to both hormones. The results showed that the 17alpha-OH group was essential for activation of AR L701H and AR L701Q, whereas its absence was important for activation of AR L701M. Modeling indicated a conserved H-bonding network involving the steroidal 17alpha-OH group, His(701) or Gln(701), and the backbone of Ser(778). This network is absent in Leu(701) and in other mutants. A hydrophobic leucine or methionine at position 701 is unfavorable for the 17alpha-OH group. Our results indicate that the specific amino acid residue at position 701, its interaction with the backbone of Ser(778), and the steroidal 17alpha-hydroxyl group of the ligand are all important for the distinct transcriptional responses to progesterone and cortisol of AR mutants, including the prostate cancer mutant L701H.

Functional screening of FxxLF-like peptide motifs identifies SMARCD1/BAF60a as an androgen receptor cofactor that modulates TMPRSS2 expression.

Androgen receptor (AR) transcriptional activity is tightly regulated by interacting cofactors and cofactor complexes. The best described cofactor interaction site in the AR is the hormone-induced coactivator binding groove in the ligand-binding domain, which serves as a high-affinity docking site for FxxLF-like motifs. This study aimed at identifying novel AR cofactors by in silico selection and functional screening of FxxLF-like peptide motifs. Candidate interacting motifs were selected from a proteome-wide screening and from a supervised screening focusing on components of protein complexes involved in transcriptional regulation. Of the 104 peptides tested, 12 displayed moderate to strong in vivo hormone-dependent interactions with AR. For three of these, ZBTB16/PLZF, SMARCA4/BRG1, and SMARCD1/BAF60a, the full-length protein was tested for interaction with AR. Of these, BAF60a, a subunit of the SWI/SNF chromatin remodeling complex, displayed hormone-dependent interactions with AR through its FxxFF motif. Vice versa, recruitment of BAF60a by the AR required an intact coactivator groove. BAF60a depletion by small interfering RNA in LNCaP cells demonstrated differential effects on expression of endogenous AR target genes. AR-driven expression of TMPRSS2 was almost completely blocked by BAF60a small interfering RNA. In summary, our data demonstrate that BAF60a directly interacts with the coactivator groove in the AR ligand-binding domain via its FxxFF motif, thereby selectively activating specific AR-driven promoters.

A novel mutation F826L in the human androgen receptor in partial androgen insensitivity syndrome; increased NH2-/COOH-terminal domain interaction and TIF2 co-activation.

A novel mutation F826L located within the ligand binding domain (LBD) of the human androgen receptor (AR) was investigated. This mutation was found in a boy with severe penoscrotal hypospadias (classified as 46,XY DSD). The AR mutant F826L appeared to be indistinguishable from the wild-type AR, with respect to ligand binding affinity, transcriptional activation of MMTV-luciferase and ARE2-TATA-luciferase reporter genes, protein level in genital skin fibroblasts (GSFs), and sub-cellular distribution in transfected cells. However, an at least two-fold higher NH2-/COOH-terminal domain interaction was found in luciferase and GST pull-down assays. A two-fold increase was also observed for TIF2 (transcription intermediary factor 2) co-activation of the AR F826L COOH-terminal domain. This increase could not be explained by a higher stability of the mutant protein, which was within wild-type range. Repression of transactivation by the nuclear receptor co-repressor (N-CoR) was not affected by the AR F826L mutation. The observed properties of AR F826L would be in agreement with an increased activity rather than with a partial defective AR transcriptional activation. It is concluded that the penoscrotal hypospadias in the present case is caused by an as yet unknown mechanism, which still may involve the mutant AR.

Androgen receptor ligand-binding domain interaction and nuclear receptor specificity of FXXLF and LXXLL motifs as determined by L/F swapping.

The androgen receptor (AR) ligand-binding domain (LBD) binds FXXLF motifs, present in the AR N-terminal domain and AR-specific cofactors, and some LXXLL motifs of nuclear receptor coactivators. We demonstrated that in the context of the AR FXXLF motif many different amino acid residues at positions +2 and +3 are compatible with strong AR LBD interaction, although a preference for E at +2 and K or R at +3 was found. Pairwise systematic analysis of F/L swaps at +1 and +5 in FXXLF and LXXLL motifs showed: 1) F to L substitutions in natural FXXLF motifs abolished AR LBD interaction; 2) binding of interacting LXXLL motifs was unchanged or increased upon L to F substitutions; 3) certain noninteracting LXXLL motifs became strongly AR-interacting FXXLF motifs; whereas 4) other nonbinders remained unaffected by L to F substitutions. All FXXLF motifs, but not the corresponding LXXLL motifs, displayed a strong preference for AR LBD. Progesterone receptor LBD interacted with some FXXLF motifs, albeit always less efficiently than corresponding LXXLL motifs. AR LBD interaction of most FXXLF and LXXLL peptides depended on classical charge clamp residue K720, whereas E897 was less important. Other charged residues lining the AR coactivator-binding groove, K717 and R726, modulated optimal peptide binding. Interestingly, these four charged residues affected binding of individual peptides independent of an F or L at +1 and +5 in swap experiments. In conclusion, F residues determine strong and selective peptide interactions with AR. Sequences flanking the core motif determine the specific mode of FXXLF and LXXLL interactions.

Identification of germ cells at risk for neoplastic transformation in gonadoblastoma: an immunohistochemical study for OCT3/4 and TSPY.

Carcinoma in situ (CIS) is the precursor of malignant testicular germ cell tumors (GCTs) of adolescents and young adults, being the neoplastic counterpart of primordial germ cells/gonocytes. Carcinoma in situ cells will develop into invasive seminoma/nonseminoma. Gonadoblastoma (GB) is the precursor of invasive GCTs in dysgenetic gonads, predominantly dysgerminoma (DG). In this process, part of the Y chromosome (GBY region) is involved, for which TSPY is a candidate gene. A detailed immunohistochemical survey was performed for the known diagnostic markers, germ cell/placental alkaline phosphatase (PLAP), c-KIT, and OCT3/4, as well as testis-specific protein on the Y chromosome (TSPY) on a series of GBs, and adjacent invasive DGs. All 5 patients were XY individuals (4 females and 1 male). In contrast to c-KIT, PLAP was positive in all cases. The immature germ cells of GBs were positive for OCT3/4, whereas the mature germ cells were negative for this marker, but positive for TSPY. In every GB, a minor population of germ cells positive for both markers could be identified, similar to most CIS cells and early invasive DG. On progression to an invasive tumor, TSPY can be lost, a process that is also detectable in invasive testicular GCTs compared to CIS. These results indicate that GB is a heterogeneous mix of germ cells, in which the OCT3/4-positive cells have the potential to undergo progression to an invasive tumor. These early invasive stages are initially also positive for TSPY (like CIS), supporting a positive selection mechanism. Therefore, OCT3/4 in combination with TSPY is valuable to identify malignant germ cells in dysgenetic gonads. This could allow better prediction of the risk of progression to a GCT. In addition, the data support the model that GB represents the earliest accessible developmental stage of malignant GCTs.

Involvement of E-cadherin and beta-catenin in germ cell tumours and in normal male fetal germ cell development.

Intercellular contacts, mediated by E-cadherin, are essential for germ cell migration and maturation. Furthermore, it has been suggested that decrease or loss of E-cadherin correlates with tumour progression and invasive behaviour. beta-catenin is involved in a number of different processes, including cell--cell interaction when bound to cadherins, and determination of cell fate in pluripotent cells when activated via the Wnt signal-transduction pathway. To shed more light on the role of these factors in normal fetal germ cell development and the pathogenesis of germ cell tumours (GCTs), the present study investigated the presence and localization of E-cadherin and beta-catenin by immunohistochemistry. E-cadherin was only weakly expressed in or absent from fetal germ cells of the second and third trimesters, and was not expressed in carcinoma in situ/intratubular germ cell neoplasia unclassified (CIS/ITGCNU) and gonadoblastoma, the precursor of an invasive GCT in dysgenetic gonads. In GCTs, it was generally not expressed in seminoma and dysgerminoma, but was found in the vast majority of non-seminoma cells. beta-catenin was found in the cytoplasm of fetal germ cells at all gestational ages and in spermatogenesis in post-pubertal testes. It was also present in CIS/ITGCNU and gonadoblastoma. Whereas seminomas and dysgerminoma were negative, non-seminoma cells were frequently found to express beta-catenin. Expression of both factors therefore reflects the degree of differentiation of these tumours. No differences for either E-cadherin or beta-catenin were observed between samples of tumours resistant or sensitive to chemotherapy, and E-cadherin expression did not correlate with vascular invasion. E-cadherin and beta-catenin therefore play a role in both normal and malignant germ cell development and differentiation that warrants further investigation, but they seem to be of limited value as predictive or prognostic factors in GCTs.

Role of P53 and MDM2 in treatment response of human germ cell tumors.

PURPOSE: Testicular germ cell tumors (TGCTs) of adolescents and adults are very sensitive to systemic treatment. The exquisite chemosensitivity of these cancers has been attributed to a high level of wild-type P53. MATERIALS AND METHODS: To clarify the role of P53 in treatment sensitivity and resistance of TGCTs, we performed immunohistochemistry and Western blotting analysis on a series of 39 fresh-frozen primary TGCTs before therapy (unselected series). In a series of formalin-fixed paraffin-embedded TGCTs of patients with fully documented clinical course, including treatment-sensitive (n = 17) and -resistant (n = 18) tumors, P53 status was assessed by immunohistochemistry and mutation analysis. In addition, the involvement of MDM2, a P53 antagonist, was investigated by immunohistochemistry, reverse transcriptase polymerase chain reaction, and in situ hybridization. RESULTS: Immunohistochemistry demonstrated absence of staining for P53 in 36%, 41%, and 17% of the unselected, responding, and nonresponding TGCTs, respectively. Of the positive TGCTs, most tumors, ie, 49%, 41%, and 33%, showed 1% to 10% positive nuclei. This overall low level of P53 was confirmed by Western blotting. Mutation analysis revealed only one silent P53 mutation in one of the responding patients. All embryonal carcinomas were homogeneously positive for MDM2, encoded by the full length mRNA, while a heterogeneous pattern was found for the other histologic components. Amplification of MDM2 was detected in one out of 12 embryonal carcinomas. CONCLUSION: Although our results are in line with previous findings of the presence of wild-type P53 in TGCTs, they show that a high level of P53 does not relate directly to treatment sensitivity of these tumors, and inactivation of P53 is not a common event in the development of cisplatin resistance.