23 research outputs found
Towards Optimal Diagnosis of Type II Germ Cell Tumors
The aim of the work described in this thesis is to improve the understanding of the pathobiology of testicular cancer (typ
Molecular determinants of treatment response in human germ cell tumors
PURPOSE: Germ cell tumors (GCTs) are highly sensitive to cisplatin-based
chemotherapy. This feature is unexplained, as is the intrinsic
chemotherapy resistance of mature teratomas and the resistant phenotype of
a minority of refractory GCTs. Various cellular pathways may influence the
efficacy of chemotherapy. Their impact has not been investigated in a
comprehensive study of tumor samples from clinically defined subgroups of
GCT patients. EXPERIMENTAL DESIGN: We investigated proteins involved in
regulation of apoptosis (p53, BAX, BCL-2, and BCL-X(L)), cell cycle
control [p21 and retinoblastoma protein (RB)], and drug export and
inactivation [P-glycoprotein, multidrug resistance-associated protein
(MRP) 1, MRP2, breast cancer resistance protein, lung resistance protein,
metallothionein, and glutathione S-transferase pi] immunohistochemically
in samples of unselected GCT patients (n = 20), patients with advanced
metastatic disease in continuous remission after first-line chemotherapy
(n = 12), and chemotherapy-refractory patients (n = 24). Mature teratoma
components (n = 10) within tumor samples from all groups were analyzed
separately. The apoptotic index was studied by terminal deoxynucleotidyl
transferase-mediated nick end labeling assay. RESULTS: Invasive GCTs of
all groups showed a correlation between wild-type p53 and apoptotic index
(r(s) = 0.66; P < 0.001). The levels of the antiapoptotic proteins BCL-2
and BCL-X(L) were generally low. p21 was hardly detectable and did not
correlate with p53 (r(s) = 0.29; P = 0.07). No significant differences
among the three patient groups were identified regarding any of the
investigated parameters (all Ps were >0.08), even though only individual
samples from chemotherapy-resistant cases showed a strong staining for
MRP2 and GSTpi. In contrast to other components, mature teratomas showed
an intense p21 and RB staining and were mostly positive for MRP2, lung
resistance protein, and GSTpi. CONCLUSIONS: Our results indicate a
multifactorial basis for the chemosensitivity of GCTs with lack of
transporters for cisplatin, of antiapoptotic BCL-2 family members, of p21
induction by p53, and of RB and an intact apoptotic cascade downstream of
p53. These findings suggest a preference for apoptosis over cell cycle
arrest after up-regulation of p53. None of the examined parameters offers
a general explanation for the chemotherapy-resistant phenotype of
refractory tumors. The up-regulation of various factors interfering with
chemotherapy efficacy and ability for a p21-induced cell cycle arrest may
explain the intrinsic chemotherapy resistance of mature teratomas
DICER1 RNase IIIb domain mutations are infrequent in testicular germ cell tumours
Background: Testicular Germ Cell Tumours (TGCT) are the most frequently occurring malignancy in males from 15-45 years of age. They are derived from germ cells unable to undergo physiological maturation, although the genetic basis for this is poorly understood. A recent report showed that mutations in the RNase IIIb domain of DICER1, a micro-RNA (miRNA) processing enzyme, are common in non-epithelial ovarian cancers. DICER1 mutations were found in 60% of Sertoli-Leydig cell tumours, clustering in four codons encoding metal-binding sites. Additional analysis of 14 TGCT DNA samples identified one case that also contained a mutation at one of these sites. Findings. A number of previous studies have shown that DICER1 mutations are found in Q) within the RNase IIIb domain in one TGCT sample, which was predicted to disturb DICER1 function. Conclusion: Overall our findings suggest a mutation frequency in TGCTs of ∼1%. We conclude therefore that hot-spot mutations, frequently seen in Sertoli-Leydig cell tumours, are not common in TGCTs
Cripto: Expression, epigenetic regulation and potential diagnostic use in testicular germ cell tumors
Type II germ cell tumors arise after puberty from a germ cell that was incorrectly programmed during fetal life. Failure of testicular germ cells to properly differentiate can lead to the formation of germ cell neoplasia in situ of the testis; this precursor cell invariably gives rise to germ cell cancer after puberty. The Nodal co-receptor Cripto is expressed transiently during normal germ cell development and is ectopically expressed in non-seminomas that arise from germ cell neoplasia in situ, suggesting that its aberrant expression may underlie germ cell dysregulation and hence germ cell cancer. Here we investigated methylation of the Cripto promoter in mouse germ cells and human germ cell cancer and correlated this with the level of CRIPTO protein expression. We found hypomethylation of the CRIPTO promoter in undifferentiated fetal germ cells, embryonal carcinoma and seminomas, but hypermethylation in differentiated fetal germ cells and the differentiated types of non-seminomas. CRIPTO protein was strongly expressed in germ cell neoplasia in situ along with embryonal carcinoma, yolk sac tumor and seminomas. Further, cleaved CRIPTO was detected in media from seminoma and embryonal carcinoma cell lines, suggesting that cleaved CRIPTO may provide diagnostic indication of germ cell cancer. Accordingly, CRIPTO was detectable in serum from 6/15 patients with embryonal carcinoma, 5/15 patients with seminoma, 4/5 patients with germ cell neoplasia in situ cells only and in 1/15 control patients. These findings suggest that CRIPTO expression may be a useful serological marker for diagnostic and/or prognostic purposes during germ cell cancer management
Coamplification of DAD-R, SOX5, and EKI1 in human testicular seminomas, with specific overexpression of DAD-R, correlates with reduced levels of apoptosis and earlier clinical manifestation
Seminomas and nonseminomas represent the invasive stages of testicular
(TGCTs) of adolescents and adults. Although TGCTs are characterized by
extra copies of the short arm of chromosome 12, the genetic basis for gain
of 12p in the pathogenesis of this cancer is not yet understood. We have
demonstrated that gain of 12p is related to invasive growth and that
amplification of specific 12p sequences, i.e., 12p11.2-p12.1, correlates
with reduced apoptosis in the tumors. Here we show that three known genes
map within the newly determined shortest region of overlap of
amplification (SROA): DAD-R, SOX5, and EKI1. Whereas EKI1 maps close to
the telomeric region of the SROA, DAD-R is the first gene at the
centromeric region within the 12p amplicon. Although all three genes are
amplified to the same level within the SROA, expression of DAD-R is
significantly up-regulated in seminomas with the restricted 12p
amplification compared with seminomas without this amplicon. DAD-R is also
highly expressed in nonseminomas of various histologies and derived cell
lines, both lacking such amplification. This finding is of particular
interest because seminomas with the restricted 12p amplification and
nonseminomas are manifested clinically in the third decade of life and
show a low degree of apoptosis. In contrast, seminomas lacking a
restricted 12p amplification, showing significantly lower levels of DAD-R
with pronounced apoptosis, manifest clinically in the fourth decade of
life. A low level of DAD-R expression is also observed in normal
testicular parenchyma and in parenchyma containing the precursor cells of
this cancer, i.e., carcinoma in situ. Therefore, elevated DAD-R expression
in seminomas and nonseminomas correlates with invasive growth and a
reduced level of apoptosis associated with an earlier clinical
presentation. These data implicate DAD-R as a candidate gene responsible
in part for the pathological effects resulting from gain of 12p sequences
in TGCTs. In addition, our results also imply differences in expression
regulation of DAD-R between seminomas and nonseminomas
Restricted 12p amplification and RAS mutation in human germ cell tumors of the adult testis
Human testicular germ-cell tumors of young adults (TGCTs), both seminomas
and nonseminomas, are characterized by 12p overrepresentation, mostly as
isochromosomes, of which the biological and clinical significance is still
unclear. A limited number of TGCTs has been identified with an additional
high-level amplification of a restricted region of 12p including the K-RAS
proto-oncogene. Here we show that the incidence of these restricted 12p
amplifications is approximately 8% in primary TGCTs. Within a single cell
formation of i(12p) and restricted 12p amplification is mutually
exclusive. The borders of the amplicons cluster in short regions, and the
amplicon was never found in the adjacent carcinoma in situ cells.
Seminomas with the restricted 12p amplification virtually lacked apoptosis
and the tumor cells showed prolonged in vitro survival like seminoma cells
with a mutated RAS gene. However, no differences in proliferation index
between these different groups of seminomas were found. Although patients
with a seminoma containing a homogeneous restricted 12p amplification
presented at a significantly younger age than those lacking it, the
presence of a restricted 12p amplification/RAS mutation did not predict
the stage of the disease at clinical presentation and the treatment
response of primary seminomas. In 55 primary and metastatic tumors from 44
different patients who failed cisplatinum-based chemotherapy, the
restricted 12p amplification and RAS mutations had the same incidence a
Prevalence of c-KIT Mutations in Gonadoblastoma and Dysgerminomas of Patients with Disorders of Sex Development (DSD) and Ovarian Dysgerminomas
Activating c-KIT mutations (exons 11 and 17) are found in 10-40% of testicular seminomas, the majority being missense point mutations (codon 816). Malignant ovarian dysgerminomas represent ~3% of all ovarian cancers in Western countries, resembling testicular seminomas, regarding chromosomal aberrations and c-KIT mutations. DSD patients with specific Y-sequences have an increased risk for Type II Germ Cell Tumor/Cancer, with gonadoblastoma as precursor progressing to dysgerminoma. Here we present analysis of c-KIT exon 8, 9, 11, 13 and 17, and PDGFRA exon 12, 14 and 18 by conventional sequencing together with mutational analysis of c-KIT codon 816 by a sensitive and specific LightCycler melting curve analysis, confirmed by sequencing. The results are combined with data on TSPY and OCT3/4 expression in a series of 16 DSD patients presenting with gonadoblastoma and dysgerminoma and 15 patients presenting pure ovarian dysgerminomas without DSD. c-KIT codon 816 mutations were detected in five out of the total of 31 cases (all found in pure ovarian dysgerminomas). A synonymous SNP (rs 5578615) was detected in two patients, one DSD patient (with bilateral disease) and one patient with dysgerminoma. Next to these, three codon N822K mutations were detected in the group of 15 pure ovarian dysgerminomas. In total activating c-KIT mutations were found in 53% of ovarian dysgerminomas without DSD. In the group of 16 DSD cases a N505I and D820E mutation was found in a single tumor of a patient with gonadoblastoma and dysgerminoma. No PDGFRA mutations were found. Positive OCT3/4 staining was present in all gonadoblastomas and dysgerminomas investigated, TSPY expression was only seen in the gonadoblastoma/dysgerminoma lesions of the 16 DSD patients. This data supports the existence of two distinct but parallel pathways in the development of dysgerminoma, in which mutational status of c-KIT might parallel the presence of TSPY
Characterization of Endothelial Cells Associated with Hematopoietic Niche Formation in Humans Identifies IL-33 As an Anabolic Factor
Bone marrow formation requires an orchestrated interplay between osteogenesis, angiogenesis, and hematopoiesis that is thought to be mediated by endothelial cells. The nature of the endothelial cells and the molecular mechanisms underlying these events remain unclear in humans. Here, we identify a subset of endoglin-expressing endothelial cells enriched in human bone marrow during fetal ontogeny and upon regeneration after chemotherapeutic injury. Comprehensive transcriptional characterization by massive parallel RNA sequencing of these cells reveals a phenotypic and molecular similarity to murine type H endothelium and activation of angiocrine factors implicated in hematopoiesis, osteogenesis, and angiogenesis. Interleukin-33 (IL-33) was significantly overexpressed in these endothelial cells and promoted the expansion of distinct subsets of h
A 46,XY female DSD patient with bilateral gonadoblastoma, a novel SRY missense mutation combined with a WT1 KTS splice-site mutation
Patients with Disorders of Sex Development (DSD), especially those with gonadal dysgenesis and hypovirilization are at risk of developing malignant type II germ cell tumors/cancer (GCC) (seminoma/dysgerminoma and nonseminoma), with either carcinoma in situ (CIS) or gonadoblastoma (GB) as precursor lesion. In 10-15% of 46,XY g
Stem cell factor receptor (c-KIT) codon 816 mutations predict development of bilateral testicular germ-cell tumors
Testicular germ-cell tumors (TGCTs) of adolescents and adults originate
from intratubular germ cell neoplasia (ITGCN), which is composed of the
malignant counterparts of embryonal germ cells. ITGCN cells are
characterized, among others, by the presence of stem cell factor receptor
c-KIT. Once established, ITGCN will always progress to invasiveness.
Approximately 2.5-5% of patients with a TGCT will develop bilateral
disease and require complete castration, resulting in infertility, a need
for lifelong androgen replacement, and psychological stress. To date, the
only way to predict a contralateral tumor is surgical biopsy of the
contralateral testis to demonstrate ITGCN. We did a retrospective study of
224 unilateral and 61 proven bilateral TGCTs (from 46 patients, in three
independently collected series in Europe) for the presence of activating
c-KIT codon 816 mutations. A c-KIT codon 816 mutation was found in three
unilateral TGCT (1.3%), and in 57 bilateral TGCTs (93%; P < 0.0001). In
the two wild-type bilateral tumors for which ITGCN was available, the
preinvasive cells contained the mutation. The mutations were somatic in
origin and identical in both tumors. We conclude that somatic activating
codon 816 c-KIT mutations are associated with development of bilateral
TGCT. Detection of c-KIT codon 816 mutations in unilateral TGCT identifies
patients at risk for bilateral disease. These patients may undergo
tailored treatment to prevent the development of bilateral disease, with
retention of testicular hormonal function