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Germ cell cancers (GCC) are the most frequent malignancy in young Caucasian males. GCC can consist of seminomas (SE) and non-seminomas (malignant NS: embryonal carcinoma (EC), yolk sac tumor (YS), choriocarcinoma (CH) and teratoma (TE)). Current serum-markers used for diagnosis and follow-up (AFP, hCG) are predominantly related to YS and CH and marker positivity can vary during disease. Therefore, stable markers consistently identifying more GCC components, specifically the stem cell components SE and EC, are of interest. Expression of the embryonic stem cell miR-371-3 and miR-302/367 clusters in SE/EC/YS suggest possible application of these micro-RNAs as GCC tumor-markers. The TSmiR protocol constitutes a complete, quality-controlled pipeline for the detection of miRs in serum, based on magnetic bead-based purification and qPCR quantification. As a proof of principle, TSmiR was applied to five independent serum sample series including 80 GCCs, 47 controls, 11 matched pre/post orchidectomy samples and 12 no-GCC testicular masses. GCC serum samples showed a consistent, significant (p < 0.0064) increase of miR-371/372/373/367 levels. Analogous, serum levels returned to baseline after orchidectomy (stage-I disease). Moreover, there was a trend toward higher miR levels in patients with metastasis. These results imply suitability for diagnosis and follow-up. TSmiR showed an overall sensitivity of 98%, clearly outperforming the traditional serum markers AFP/hCG (36%/57%, sensitivityAFP = 3%/45%; sensitivityhCG = 62%/66%, SE/NS). TSmiR misclassified one tumor as a control. Serum AFP/hCG and TSmiR combined identified all T samples correctly. In conclusion, TSmiR constitutes a highly sensitive and reproducible serum test for GCC patients, suitable to be prospectively tested for diagnostic and follow-up purposes. [Display omitted] •TSmiR is a complete quality controlled pipeline for the detection of miRs in serum.•Based on magnetic bead-based purification/qPCR quantification, no pre-amplification.•High sensitivity/specificity for GCC detection in five independent sample series.•miR levels return to baseline after surgery/are increased in metastasized disease.•Therefore, TSmiR is a potential valuable tool for diagnosis & follow-up of GCC.

Recently, a variant allele in the 3'UTR of the KRAS gene (rs61764370 T>G) was shown to be associated with an increased risk for developing non-small cell lung cancer, as well as ovarian cancer, and was most enriched in ovarian cancer patients from hereditary breast and ovarian cancer families. This functional variant has been shown to disrupt a let-7 miRNA binding site leading to increased expression of KRAS in vitro. In the current study, we have genotyped this KRAS-variant in breast cancer index cases from 268 BRCA1 families, 89 BRCA2 families, 685 non-BRCA1/BRCA2 families, and 797 geographically matched controls. The allele frequency of the KRAS-variant was found to be increased among patients with breast cancer from BRCA1, but not BRCA2 or non-BRCA1/BRCA2 families as compared to controls. As BRCA1 carriers mostly develop ER-negative breast cancers, we also examined the variant allele frequency among indexes from non-BRCA1/ BRCA2 families with ER-negative breast cancer. The prevalence of the KRAS-variant was, however, not significantly increased as compared to controls, suggesting that the variant allele not just simply associates with ER-negative breast cancer. Subsequent expansion of the number of BRCA1 carriers with breast cancer by including other family members in addition to the index cases resulted in loss of significance for the association between the variant allele and mutant BRCA1 breast cancer. In this same cohort, the KRAS-variant did not appear to modify breast cancer risk for BRCA1 carriers. Importantly, results from the current study suggest that KRAS-variant frequencies might be increased among BRCA1 carriers, but solid proof requires confirmation in a larger cohort of BRCA1 carriers. Keywords KRAS-variant, Let-7, Breast cancer susceptibility, Association, BRCA1

To unravel the mechanisms underlying failure of endocrine therapy of breast cancer, we have previously executed a functional genetic screen and identified the adaptor protein BCAR1 to be causative for tamoxifen resistance. As a consequence of the manifold of interactions with other proteins, we characterized the contribution of individual protein domains of BCAR1 to anti-estrogen-resistant proliferation of human breast cancer cells. We took advantage of the observation that the closely related family member HEF1 was unable to support long-term anti-estrogen-resistant cell proliferation. Chimerical proteins containing defined domains of BCAR1 and HEF1 were evaluated for anti-estrogen-resistant growth. Exchange of the SH3 and C -terminal domains did not modify the capacity to support cell proliferation. Full support of anti-estrogen resistant proliferation was observed for chimerical molecules containing the central part of BCAR1. The bi-partite SRC-binding site or the Serine-rich domain did not explain the differential capacity of BCAR1. These findings indicate that the differences between BCAR1 and HEF1 with respect to support of anti-estrogen resistance reside in the substrate domain which contains multiple sites for tyrosine phosphorylation. The crucial interactions required for anti-estrogen resistance occur within the substrate domain of BCAR1. Further deciphering of these interactions may resolve the growth regulatory mechanism and provide an explanation for the observation that primary tumors with high levels of BCAR1 are likely to fail on tamoxifen therapy. This information may also help to devise alternative personalized treatment strategies with improved outcome for breast cancer patients.

The cell of origin of the five subtypes (I-V) of germ cell tumors (GCTs) are assumed to be germ cells from different maturation stages. This is (potentially) reflected in their methylation status as fetal maturing primordial germ cells are globally demethylated during migration from the yolk sac to the gonad. Imprinted regions are erased in the gonad and later become uniparentally imprinted according to fetal sex. Here, 91 GCTs (type I-IV) and four cell lines were profiled (Illumina's HumanMethylation450BeadChip). Data was pre-processed controlling for cross hybridization, SNPs, detection rate, probe-type bias and batch effects. The annotation was extended, covering snRNAs/microRNAs, repeat elements and imprinted regions. A Hidden Markov Model-based genome segmentation was devised to identify differentially methylated genomic regions. Methylation profiles allowed for separation of clusters of non-seminomas (type II), seminomas/dysgerminomas (type II), spermatocytic seminomas (type III) and teratomas/dermoid cysts (type I/IV). The seminomas, dysgerminomas and spermatocytic seminomas were globally hypomethylated, in line with previous reports and their demethylated precursor. Differential methylation and imprinting status between subtypes reflected their presumed cell of origin. Ovarian type I teratomas and dermoid cysts showed (partial) sex specific uniparental maternal imprinting. The spermatocytic seminomas showed uniparental paternal imprinting while testicular teratomas exhibited partial imprinting erasure. Somatic imprinting in type II GCTs might indicate a cell of origin after global demethylation but before imprinting erasure. This is earlier than previously described, but agrees with the totipotent/embryonic stem cell like potential of type II GCTs and their rare extra-gonadal localization. The results support the common origin of the type I teratomas and show strong similarity between ovarian type I teratomas and dermoid cysts. In conclusion, we identified specific and global methylation differences between GCT subtypes, providing insight into their developmental timing and underlying developmental biology. Data and extended annotation are deposited at GEO (GSE58538 and GPL18809).

Background Testicular germ cell cancer (TGCC), being the most frequent malignancy in young Caucasian males, is initiated from an embryonic germ cell. This study determines intratumour heterogeneity to unravel tumour progression from initiation until metastasis. Methods In total, 42 purified samples of four treatment-resistant nonseminomatous (NS) TGCC were investigated, including the precursor germ cell neoplasia in situ (GCNIS) and metastatic specimens, using whole-genome and targeted sequencing. Their evolution was reconstructed. Results Intratumour molecular heterogeneity did not correspond to the supposed primary tumour histological evolution. Metastases after systemic treatment could be derived from cancer stem cells not identified in the primary cancer. GCNIS mostly lacked the molecular marks of the primary NS and comprised dominant clones that failed to progress. A BRCA-like mutational signature was observed without evidence for direct involvement of BRCA1 and BRCA2 genes. Conclusions Our data strongly support the hypothesis that NS is initiated by whole-genome duplication, followed by chromosome copy number alterations in the cancer stem cell population, and accumulation of low numbers of somatic mutations, even in therapy-resistant cases. These observations of heterogeneity at all stages of tumourigenesis should be considered when treating patients with GCNIS-only disease, or with clinically overt NS.

Recently, a variant allele in the 3'UTR of the KRAS gene (rs61764370 T>G) was shown to be associated with an increased risk for developing non-small cell lung cancer, as well as ovarian cancer, and was most enriched in ovarian cancer patients from hereditary breast and ovarian cancer families. This functional variant has been shown to disrupt a let-7 miRNA binding site leading to increased expression of KRAS in vitro. In the current study, we have genotyped this KRAS-variant in breast cancer index cases from 268 BRCA1 families, 89 BRCA2 families, 685 non-BRCA1/BRCA2 families, and 797 geographically matched controls. The allele frequency of the KRAS-variant was found to be increased among patients with breast cancer from BRCA1, but not BRCA2 or non-BRCA1/BRCA2 families as compared to controls. As BRCA1 carriers mostly develop ER-negative breast cancers, we also examined the variant allele frequency among indexes from non-BRCA1/BRCA2 families with ER-negative breast cancer. The prevalence of the KRAS-variant was, however, not significantly increased as compared to controls, suggesting that the variant allele not just simply associates with ER-negative breast cancer. Subsequent expansion of the number of BRCA1 carriers with breast cancer by including other family members in addition to the index cases resulted in loss of significance for the association between the variant allele and mutant BRCA1 breast cancer. In this same cohort, the KRAS-variant did not appear to modify breast cancer risk for BRCA1 carriers. Importantly, results from the current study suggest that KRAS-variant frequencies might be increased among BRCA1 carriers, but solid proof requires confirmation in a larger cohort of BRCA1 carriers.

Adult male germline stem cells (spermatogonia) proliferate by mitosis and, after puberty, generate spermatocytes that undertake meiosis to produce haploid spermatozoa. Germ cells are under evolutionary constraint to curtail mutations and maintain genome integrity. Despite constant turnover, spermatogonia very rarely form tumors, so-called spermatocytic tumors (SpT). In line with the previous identification of FGFR3 and HRAS selfish mutations in a subset of cases, candidate gene screening of 29 SpTs identified an oncogenic NRAS mutation in two cases. To gain insights in the etiology of SpT and into properties of the male germline, we performed whole-genome sequencing of five tumors (4/5 with matched normal tissue). The acquired single nucleotide variant load was extremely low (~0.2 per Mb), with an average of 6 (2-9) non-synonymous variants per tumor, none of which is likely to be oncogenic. The observed mutational signature of SpTs is strikingly similar to that of germline de novo mutations, mostly involving C>T transitions with a significant enrichment in the ACG trinucleotide context. The tumors exhibited extensive aneuploidy (50-99 autosomes/tumor) involving whole-chromosomes, with recurrent gains of chr9 and chr20 and loss of chr7, suggesting that aneuploidy itself represents the initiating oncogenic event. We propose that SpT etiology recapitulates the unique properties of male germ cells; because of evolutionary constraints to maintain low point mutation rate, rare tumorigenic driver events are caused by a combination of gene imbalance mediated via whole-chromosome aneuploidy. Finally, we propose a general framework of male germ cell tumor pathology that accounts for their mutational landscape, timing and cellular origin.

Most breast cancers depend on estrogenic growth stimulation. Functional genetic screenings in in vitro cell models have identified genes, which override growth suppression induced by anti-estrogenic drugs like tamoxifen. Using that approach, we have previously identified Breast Cancer Anti-Estrogen Resistance 4 (BCAR4) as a mediator of cell proliferation and tamoxifen-resistance. Here, we show high level of expression and function of BCAR4 in human breast cancer. BCAR4 mRNA expression was evaluated by (q)RT-PCR in a panel of human normal tissues, primary breast cancers and cell lines. A new antibody raised against C78-I97 of the putative BCAR4 protein and used for western blot and immunoprecipitation assays. Furthermore, siRNA-mediated gene silencing was implemented to study the function of BCAR4 and its downstream targets ERBB2/3. Except for placenta, all human normal tissues tested were BCAR4-negative. In primary breast cancers, BCAR4 expression was comparatively rare (10%), but associated with enhanced proliferation. Relative high BCAR4 mRNA expression was identified in IPH-926, a cell line derived from an endocrine-resistant lobular breast cancer. Moderate BCAR4 expression was evident in MDA-MB-134 and MDA-MB-453 breast cancer cells. BCAR4 protein was detected in breast cancer cells with ectopic (ZR-75-1-BCAR4) and endogenous (IPH-926, MDA-MB-453) BCAR4 mRNA expression. Knockdown of BCAR4 inhibited cell proliferation. A similar effect was observed upon knockdown of ERBB2/3 and exposure to lapatinib, implying that BCAR4 acts in an ERBB2/3-dependent manner. BCAR4 encodes a functional protein, which drives proliferation of endocrine-resistant breast cancer cells. Lapatinib, a clinically approved EGFR/ERBB2 inhibitor, counteracts BCAR4-driven tumor cell growth, a clinical relevant observation.

Autologous transplantation of spermatogonial stem cells is a promising new avenue to restore fertility in infertile recipients. Expansion of the initial spermatogonial stem cell pool through cell culturing is a necessary step to obtain enough cells for effective repopulation of the testis after transplantation. Since in vitro propagation can lead to (epi-)genetic mutations and possibly malignant transformation of the starting cell population, we set out to investigate genome-wide DNA methylation status in uncultured and cultured primary testicular ITGA6+ sorted cells and compare them with germ cell tumor samples of the seminoma subtype. Seminomas displayed a severely global hypomethylated profile, including loss of genomic imprinting, which we did not detect in cultured primary testicular ITGA6+ cells. Differential methylation analysis revealed altered regulation of gamete formation and meiotic processes in cultured primary testicular ITGA6+ cells but not in seminomas. The pivotal POU5F1 marker was hypomethylated in seminomas but not in uncultured or cultured primary testicular ITGA6+ cells, which is reflected in the POU5F1 mRNA expression levels. Lastly, seminomas displayed a number of characteristic copy number variations that were not detectable in primary testicular ITGA6+ cells, either before or after culture. Together, the data show a distinct DNA methylation patterns in cultured primary testicular ITGA6+ cells that does not resemble the pattern found in seminomas, but also highlight the need for more sensitive methods to fully exclude the presence of malignant cells after culture and to further study the epigenetic events that take place during in vitro culture.

Liquid biopsy-based biomarkers, such as microRNAs, represent valuable tools for patient management, but often do not make it to integration in the clinic. We aim to explore issues impeding this transition, in the setting of germ cell tumors, for which novel biomarkers are needed. We describe a model for identifying and validating clinically relevant microRNAs for germ cell tumor patients, using both in vitro, in vivo (mouse model) and patient-derived data. Initial wide screening of candidate microRNAs is performed, followed by targeted profiling of potentially relevant biomarkers. We demonstrate the relevance of appropriate (negative) controls, experimental conditions (proliferation), and issues related to sample origin (serum, plasma, cerebral spinal fluid) and pre-analytical variables (hemolysis, contaminants, temperature), all of which could interfere with liquid biopsy-based studies and their conclusions. Finally, we show the value of our identification model in a specific scenario, contradicting the presumed role of miR-375 as marker of teratoma histology in liquid biopsy setting. Our findings indicate other putative microRNAs (miR-885-5p, miR-448 and miR-197-3p) fulfilling this clinical need. The identification model is informative to identify the best candidate microRNAs to pursue in a clinical setting.