Hans Stoop

A Genetic Screen Implicates miRNA-372 and miRNA-373 As Oncogenes in Testicular Germ Cell Tumors

Endogenous small RNAs (miRNAs) regulate gene expression by mechanisms conserved across metazoans. While the number of verified human miRNAs is still expanding, only few have been functionally annotated. To perform genetic screens for novel functions of miRNAs, we developed a library of vectors expressing the majority of cloned human miRNAs and created corresponding DNA barcode arrays. In a screen for miRNAs that cooperate with oncogenes in cellular transformation, we identified miR-372 and miR-373, each permitting proliferation and tumorigenesis of primary human cells that harbor both oncogenic RAS and active wild-type p53. These miRNAs neutralize p53-mediated CDK inhibition, possibly through direct inhibition of the expression of the tumorsuppressor LATS2. We provide evidence that these miRNAs are potential novel oncogenes participating in the development of human testicular germ cell tumors by numbing the p53 pathway, thus allowing tumorigenic growth in the presence of wild-type p53.

Aberrant p53 protein expression is associated with an increased risk of neoplastic progression in patients with Barrett's oesophagus

Objective The value of surveillance for patients with Barretts oesophagus (BO) is under discussion given the overall low incidence of neoplastic progression and lack of discriminative tests for risk stratification. Histological diagnosis of low-grade dysplasia (LGD) is the only accepted predictor for progression to date, but has a low predictive value. The aim of this study was therefore to evaluate the value of p53 immunohistochemistry for predicting neoplastic progression in patients with BO. Design We conducted a case–control study within a prospective cohort of 720 patients with BO. Patients who developed high-grade dysplasia (HGD) or oesophageal adenocarcinoma (OAC) were classified as cases and patients without neoplastic progression were classified as controls. P53 protein expression was determined by immunohistochemistry in more than 12u2005000 biopsies from 635 patients and was scored independently by two expert pathologists who were blinded to long-term outcome. Results During follow-up, 49 (8%) patients developed HGD or OAC. P53 overexpression was associated with an increased risk of neoplastic progression in patients with BO after adjusting for age, gender, Barrett length and oesophagitis (adjusted relative risks (RRa) 5.6; 95% CI 3.1 to 10.3), but the risk was even higher with loss of p53 expression (RRa 14.0; 95% CI 5.3 to 37.2). The positive predictive value for neoplastic progression increased from 15% with histological diagnosis of LGD to 33% with LGD and concurrent aberrant p53 expression. Conclusions Aberrant p53 protein expression is associated with an increased risk of neoplastic progression in patients with BO and appears to be a more powerful predictor of neoplastic progression than histological diagnosis of LGD.

Stem cell factor as a novel diagnostic marker for early malignant germ cells

Carcinoma in situ (CIS) of the testis is the pre‐invasive stage of type II testicular germ cell tumours (TGCTs) of adolescents and adults. These tumours are the most frequently diagnosed cancer in Caucasian adolescents and young adults. In dysgenetic gonads, the precursor of type II GCTs can be either CIS or a lesion known as gonadoblastoma (GB). CIS/GB originates from a primordial germ cell (PGC)/gonocyte, ie an embryonic cell. CIS can be cured by local low‐dose irradiation, with limited side effects on hormonal function. Therefore, strategies for early diagnosis of CIS are essential. Various markers are informative to diagnose CIS in adult testis by immunohistochemistry, including c‐KIT, PLAP, AP‐2γ, NANOG, and POU5F1 (OCT3/4). OCT3/4 is the most informative and consistent in presence and expression level, resulting in intense nuclear staining. In the case of maturational delay of germ cells, frequently present in gonads of individuals at risk for type II (T)GCTs, use of these markers can result in overdiagnosis of malignant germ cells. This demonstrates the need for a more specific diagnostic marker to distinguish malignant germ cells from germ cells showing maturation delay. Here we report the novel finding that immunohistochemical detection of stem cell factor (SCF), the c‐KIT ligand, is informative in this context. This was demonstrated in over 400 cases of normal (fetal, neonatal, infantile, and adult) and pathological gonads, as well as TGCT‐derived cell lines, specifically in cases of CIS and GB. Both membrane‐bound and soluble SCF were expressed, suggestive of an autocrine loop. SCF immunohistochemistry can be a valuable diagnostic tool, in addition to OCT3/4, to screen for precursor lesions of TGCTs, especially in patients with germ cell maturation delay. Copyright

Global DNA methylation in fetal human germ cells and germ cell tumours: association with differentiation and cisplatin resistance

Differences in the global methylation pattern, ie hyper‐ as well as hypo‐methylation, are observed in cancers including germ cell tumours (GCTs). Related to their precursor cells, GCT methylation status differs according to histology. We investigated the methylation pattern of normal fetal, infantile, and adult germ cells (n = 103) and GCTs (n = 251) by immunohistochemical staining for 5‐

12p-Amplicon structure analysis in testicular germ cell tumors of adolescents and adults by array CGH

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Role of epigenetics in the etiology of germ cell cancer

cytidine. The global methylation pattern of male germ cells changes from hypomethylation to hypermethylation, whereas female germ cells remain unmethylated at all stages. Undifferentiated GCTs (seminomas, intratubular germ cell neoplasia unclassified, and gonadoblastomas) are hypomethylated, whereas more differentiated GCTs (teratomas, yolk sac tumours, and choriocarcinomas) show a higher degree of methylation. Embryonal carcinomas show an intermediate pattern. Resistance to cisplatin was assessed in the seminomatous cell line TCam‐2 before and after demethylation using 5‐azacytidine. Exposure to 5‐azacytidine resulted in decreased resistance to cisplatin. Furthermore, after demethylation, the stem cell markers NANOG and POU5F1 (OCT3/4), as well as the germ cell‐specific marker VASA, showed increased expression. Following treatment with 5‐azacytidine, TCam‐2 cells were analysed using a high‐throughput methylation screen for changes in the methylation sites of 14 000 genes. Among the genes revealing changes, interesting targets were identified: ie demethylation of KLF11, a putative tumour suppressor gene, and hypermethylation of CFLAR, a gene previously described in treatment resistance in GCTs. Copyright

SOX2 as a novel marker to predict neoplastic progression in Barrett's esophagus

All invasive testicular germ cell tumors of adolescents and adults (TGCTs), that is, seminomas and nonseminomas, show gain of 12p sequences, mostly as isochromosomes. Although several candidate genes have been suggested, the relevant gene(s) have not been identified yet. About 10% of testicular seminomas, however, show a more restricted amplification of the 12p11.2–p12.1 region, in which the various amplicons show an apparent overlap, allowing for the shortest region of amplification overlap approach, aiming at the identification of pathogenetically relevant sequences residing in this region. Here we report on a high-resolution 12p-amplicon architecture analysis using microarray-based comparative genomic hybridization, the results of which were subsequently confirmed by fluorescent in situ hybridization studies. The 12p-specific microarray contained 63 positionally selected BAC clones, which are more or less evenly distributed over the short arm of chromosome 12 (average spacing: less than 500u2009Kb), including 20 clones within the region of amplification. Out of a series of 17 seminomas, seven seminomas showed amplification of the whole amplicon region, of which three showed a dip in T/R value in the center of the amplified area. A more complex amplification pattern was found in the other 10 seminomas: three showed predominant amplification at the centromeric border; one mainly at the telomeric border; six showed a balanced amplification of both the centromeric and telomeric regions. The only nonseminoma investigated showed a structure in which the centromeric border was only amplified. These data support a mechanistic model in which at least two 12p genes, situated at the border regions of the amplicon, are positional candidates capable of actively supporting tumor progression in TGCTs.

P53 and SOX2 Protein Expression Predicts Esophageal Adenocarcinoma in Response to Neoadjuvant Chemoradiotherapy

Embryonic development is strictly controlled by functionality of genes in which the existing networks can act both on transcription and translation regulation. Germ cell cancers (GCC) are unique because of a number of characteristics. In spite of their clinical presentation, i.e., predominantly after puberty, they arise from primordial germ cells/gonocytes that have failed appropriate maturation to either pre-spermatogonia or oogonia. GCC mimic embryonal development to a certain extent, including capacity for totipotency. This knowledge has allowed the identification of informative diagnostic markers, including OCT3/4 (POU5F1), SOX2 and SOX17. An additional marker is the overall demethylated status of the genome. Genetic mutations in GCC are rare, which is exceptional for solid cancers. Our hypothesis is that a disturbed epigenetic regulation (through combined interaction of genetic or environmental parameters; referred to as genvironment) affect embryonic germ cell development, resulting in delayed or blocked maturation, and potentially progression to GCC. In this respect, studies of patients with Disorders of Sex Development (DSD) have increased our knowledge significantly. Genvironmental influences can lead to retention of existence of embryonic germ cells, the first step in the pathogenesis of GCC, resulting into the precursor lesions gonadoblastoma or carcinoma in situ. Identification of epigenetic alterations could lead to better understanding these processes and development of specific markers for early detection, eventually leading to development of targeted treatment. This review describes an interactive model related to the role of epigenetics in GCC pathogenesis, focusing on DNA methylation, histone modifications, epigenetic memory and inheritance, as well as environmental factors.

Value of cyclin A immunohistochemistry for cancer risk stratification in Barrett esophagus surveillance A multicenter case-control study

OBJECTIVES:The value of Barrett’s esophagus (BE) surveillance based on the histological diagnosis of low-grade dysplasia (LGD) remains debated given the lack of adequate risk stratification. The aim of this study was to evaluate the predictive value (PV) of SOX2 expression for neoplastic progression in BE patients.METHODS:We conducted a case–control study within a prospective cohort of 720 BE patients. Patients with neoplastic progression, defined as the development of high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC), were classified as cases and patients without neoplastic progression were classified as controls. SOX2 expression was determined by immunohistochemistry in more than 12,000 biopsies from 635 patients; these results were combined with our previous p53 immunohistochemical data.RESULTS:Nondysplastic BE showed homogeneous nuclear staining for SOX2, whereas SOX2 was progressively lost in dysplastic BE. Loss of SOX2 was seen in only 2% of biopsy series without dysplasia, in contrast to 28% in LGD and 67% in HGD/EAC. Loss of SOX2 expression was associated with an increased risk of neoplastic progression in BE patients after adjusting for gender, age, BE length, and esophagitis (adjusted relative risk 4.8; 95% CI 3.2–7.0). The positive PV for neoplastic progression increased from 16% with LGD alone to 56% with concurrent loss of SOX2 and aberrant p53 expression.CONCLUSIONS:SOX2 expression is lost during transition from nondysplastic BE to HGD/EAC, and it is associated with an increased risk of neoplastic progression. The highest PV is achieved by concurrent loss of SOX2 and aberrant p53 expression in BE patients with LGD. The use of these markers has the potential to significantly improve risk stratification of Barrett surveillance.

Why human extragonadal germ cell tumours occur in the midline of the body: old concepts, new perspectives.

OBJECTIVEnThe aim of the study was to investigate the association between p53, SOX2, and CD44 protein expression and tumor response, and to validate potential predictive biomarker(s) in an independent cohort.nnnBACKGROUNDnNeoadjuvant chemoradiotherapy (nCRT) followed by surgery has become a standard of care for esophageal adenocarcinoma (EAC). However, the response to nCRT is highly variable among patients.nnnMETHODSnEAC patients who underwent nCRT and surgery, between January 2003 and December 2014 at the Erasmus University Medical Center, were included and divided into a primary (n = 77) and a validation cohort (n = 70). P53, SOX2, and CD44 expression was detected by immunohistochemistry in pretreatment tumor biopsies, and scored independently by 2 investigators. Response to nCRT was assessed based on tumor regression grade (TRG) in the resection specimen.nnnRESULTSnForty-one (53%) patients in the primary cohort and 33 (47%) patients in the validation cohort showed major response (TRG1 or TRG2) in the resection specimen. Aberrant p53 and absence of SOX2 were associated with major response in the primary cohort: adjusted odds ratio (OR) 6.3 [95% confidence interval (CI), 1.3-30.1) and adjusted OR 4.1 (95% CI, 1.4-12.4), respectively. The same was true for the validation cohort (p53: adjusted OR 8.6; 95% CI, 0.93-80.9 and SOX2: adjusted OR 6.1; 95% CI, 1.6-23.4). The highest probability of a major response was seen in patients with concurrent aberrant p53 and absence of SOX2 expression, with an OR of 6.7 (95% CI, 2.1-21.4) and 6.2 (95% CI, 1.8-21.2) in the primary and validation cohort.nnnCONCLUSIONSnPattern of p53 and particularly SOX2 protein expression in EAC predicts response to nCRT. These biomarkers may help to individualize treatment in EAC patients.Objective: The aim of the study was to investigate the association between p53, SOX2, and CD44 protein expression and tumor response, and to validate potential predictive biomarker(s) in an independent cohort. Background: Neoadjuvant chemoradiotherapy (nCRT) followed by surgery has become a standard of care for esophageal adenocarcinoma (EAC). However, the response to nCRT is highly variable among patients. Methods: EAC patients who underwent nCRT and surgery, between January 2003 and December 2014 at the Erasmus University Medical Center, were included and divided into a primary (n = 77) and a validation cohort (n = 70). P53, SOX2, and CD44 expression was detected by immunohistochemistry in pretreatment tumor biopsies, and scored independently by 2 investigators. Response to nCRT was assessed based on tumor regression grade (TRG) in the resection specimen. Results: Forty-one (53%) patients in the primary cohort and 33 (47%) patients in the validation cohort showed major response (TRG1 or TRG2) in the resection specimen. Aberrant p53 and absence of SOX2 were associated with major response in the primary cohort: adjusted odds ratio (OR) 6.3 [95% confidence interval (CI), 1.3–30.1) and adjusted OR 4.1 (95% CI, 1.4–12.4), respectively. The same was true for the validation cohort (p53: adjusted OR 8.6; 95% CI, 0.93–80.9 and SOX2: adjusted OR 6.1; 95% CI, 1.6–23.4). The highest probability of a major response was seen in patients with concurrent aberrant p53 and absence of SOX2 expression, with an OR of 6.7 (95% CI, 2.1–21.4) and 6.2 (95% CI, 1.8–21.2) in the primary and validation cohort. Conclusions: Pattern of p53 and particularly SOX2 protein expression in EAC predicts response to nCRT. These biomarkers may help to individualize treatment in EAC patients.