Testicular germ cell tumors (TGCT) are a rare neoplasia but are still the most common malignancy in males between the ages of 15 and 44. TGCTs can be divided into two main types: Seminomas (SE) and non-seminomas (NS), the latter with an earlier age of onset and a worst prognosis. One of the most consistent features of TGCTs is the gain of material in the short arm of chromosome 12, that occurs in almost 100% of TGCT cases; 80% of them involve the formation of an isochromosome of the short arm i(12p). This might be the key step that allows the lesion to progress from a germ cell neoplasia in situ (GCNIS), which is a microscopic finding preceding the TGCT and without gain in 12p, to a TGCT. Some tumors, specially SE, present a more restricted amplification of certain 12p regions such as the 12p11.2-12.1 amplicon instead of the i(12p). The mechanism that associates the gain of 12p and the development of invasiveness is not yet well understood but it is believed a number of genes are involved, including DPPA3/STELLA, SOX5, PHC2, ATF7IP and proto-oncogenes Cyclin D2 and KRAS. Genome wide association studies have allowed us to acquire a better knowledge of the pathogenesis of this type of tumor, in which multiple genes show an increase in copy numbers, higher expression or activating mutations in genes related to the KIT/ KITLG pathway like KRAS, BRAF or KIT and KITLG itself. A less frequent subtype of TGCT found in older patients are spermatocytic tumors (ST). It does not develop from a GCNIS and presents a gain of genetic material in chromosome 9 instead of 12. It is believed the overexpression of the gene DMRT1, at 9p24.2, might have a role in the development of ST. In this review we are trying to delineate the most important loci involved in testicular germ tumors, the genes involved in this pathogenesis, and attempting to describe the possible mechanisms behind this tumorigenesis.

Background: Hepatocellular carcinoma (HCC) is a malignancy exhibiting the highest lethality. The present study aimed to identify different immune-related clusters in HCC and a robust tumor gene signature to facilitate the prognosis prediction for HCC patients.

Methods: For the 375 HCC cases collected from the dataset of Cancer Genome Atlas (TCGA), their overall survival (OS) and immune-related genes (IRGs) expression patterns were collected. Thereafter, consensus clustering was employed for grouping and functional enrichment, whereas the ESTIMATE algorithm and the CIBERSORT algorithm were used in subsequent assessment. Immunohistochemistry (IHC) was conducted to verify the protein expression of model genes in HCC and adjacent tissues.

Results: According to consensus clustering with 93-survival related IRGs, a total of five subgroups were found. These five clusters had different prognoses, immune statuses, and expression of immune checkpoints. Afterwards, 11 genes were enrolled for constructing the OS-related prediction model for TCGA HCC cases, which was then validated using the database of International Cancer Genome Consortium (ICGC). The protein expression of LCN2, S100A10, FABP6, PLXNA1, KITLG and OXTR were enhanced in HCC tissues relative to that in normal hepatic tissues, while the protein expression of S100A1, CCL26, CMTM4, IL1RN and RARG were reduced in HCC compared with normal tissues. In addition, different immunocyte infiltration levels between low- and high- groups were further examined.

Conclusions: According to our results, the IRGs-based classifications assist in explaining the HCC heterogeneity, which may help to develop the more efficient individualized treatments.

Keywords: Hepatocellular carcinoma; Immune system; Precision medicine; Prognosis.

While the typical Arabian camel is characterized by a single colored coat, there are rare populations with white spotting patterns. White spotting coat patterns are found in virtually all domesticated species, but are rare in wild species. Theories suggest that white spotting is linked to the domestication process, and is occasionally associated with health disorders. Though mutations have been found in a diverse array of species, fewer than 30 genes have been associated with spotting patterns, thus providing a key set of candidate genes for the Arabian camel. We obtained 26 spotted camels and 24 solid controls for candidate gene analysis. One spotted and eight solid camels were whole genome sequenced as part of a separate project. The spotted camel was heterozygous for a frameshift deletion in KIT (c.1842delG, named KITW1 for White spotting 1), whereas all other camels were wild-type (KIT+/KIT+). No additional mutations unique to the spotted camel were detected in the EDNRB, EDN3, SOX10, KITLG, PDGFRA, MITF, and PAX3 candidate white spotting genes. Sanger sequencing of the study population identified an additional five kITW1/KIT+ spotted camels. The frameshift results in a premature stop codon five amino acids downstream, thus terminating KIT at the tyrosine kinase domain. An additional 13 spotted camels tested KIT+/KIT+, but due to phenotypic differences when compared to the KITW1/KIT+ camels, they likely represent an independent mutation. Our study suggests that there are at least two causes of white spotting in the Arabian camel, the newly described KITW1 allele and an uncharacterized mutation.

The alarming decline in sperm count has become a global concern in the recent decades. The division and differentiation of male germ cells (Gc) into sperm are governed by Sertoli cells (Sc) upon their functional maturation during puberty. However, the roles of genes regulating pubertal maturation of Sc have not been fully determined. We have observed that Tetraspanin 8 (Tspan8) is down regulated in Sc during puberty in rats. However there has been no in-vivo evidence for a causal link between the down regulation of Tspan8 expression and the onset of spermatogenesis as yet. To investigate this, we generated a novel transgenic (Tg) rat, in which the natural down-regulation of Tspan8 was prevented specifically in Sc from puberty up to adulthood. Adult Tg male rats showed around 98% reduction in sperm count despite having a similar level of serum testosterone (T) as the controls. Functional maturation of Sc was impaired as indicated by elevated levels of Amh and low levels of Kitlg and Claudin11 transcripts. The integrity of the blood testis barrier was compromised due to poor expression of Gja1 and Gc apoptosis was discernible. This effect was due to a significant rise in both Mmp7 and phospho P38 MAPK in Tg rat testis. Taken together, we demonstrated that the natural down-regulation of Tspan8 in Sc during puberty is a prerequisite for establishing male fertility. This study divulges one of the etiologies of certain forms of idiopathic male infertility where somatic cell defect, but not hormonal deficiency, is responsible for impaired spermatogenesis.

Keywords: Sertoli cells; Tetraspanin8; male infertility; transgenic rat.

The mechanisms of idiopathic severe aplastic anemia (SAA) in children are not completely understood. Insufficiency of the bone marrow microenvironment, in which mesenchymal stem cells (MSCs) are an important element, can be a potential factor associated with hematopoietic impairment. In the current study, we studied whether aberrant gene expression could be found in MSCs from children with SAA. Using microarray analysis, two different patterns of global gene expression were detected in the SAA MSCs. Fourteen genes (POLE2, HGF, KIF20A, TK1, IL18R1, KITLG, FGF18, RRM2, TTK, CXCL12, DLG7, TOP2A, NUF2, and TYMS), which are related to DNA synthesis, cytokines, or growth factors, were significantly downregulated. Further, knockdown of gene expression was performed using the small hairpin RNA (shRNA)-containing lentivirus method. We found that knockdown of CXCL12, HGF, IL-18R1, FGF18, or RRM2 expression compelled MSCs from the controls to behave like those from the SAA children, with decreased survival and differentiation potential. Among them, inhibition of CXCL12 gene expression had the most profound effects on the behavior of MSCs. Further experiments regarding re-introduction of the CXCL12 gene could largely recover the survival and differentiation potential in MSCs with inhibition of CXCL12 expression. Our findings suggest that MSCs from children with SAA exhibit aberrant gene expression profiles and downregulation of CXCL12 gene may be associated with alterations in the bone marrow microenvironment.

BACKGROUND

Ovulation and luteinization of follicles are complex biological processes initiated by the preovulatory luteinizing hormone surge. The objective of this study was to identify genes that are differentially expressed in bovine granulosa cells (GC) of ovulatory follicles.

METHODS

Granulosa cells were collected during the first follicular wave of the bovine estrous cycle from dominant follicles (DF) and from ovulatory follicles (OF) obtained 24 h following injection of human chorionic gonadotropin (hCG). A granulosa cell subtracted cDNA library (OF-DF) was generated using suppression subtractive hybridization and screened.

RESULTS

Detection of genes known to be upregulated in bovine GC during ovulation, such as ADAMTS1, CAV1, EGR1, MMP1, PLAT, PLA2G4A, PTGES, PTGS2, RGS2, TIMP1, TNFAIP6 and VNN2 validated the physiological model and analytical techniques used. For a subset of genes that were identified for the first time, gene expression profiles were further compared by semiquantitative RT-PCR in follicles obtained at different developmental stages. Results confirmed an induction or upregulation of the respective mRNAs in GC of OF 24 h after hCG-injection compared with those of DF for the following genes: ADAMTS9, ARAF, CAPN2, CRISPLD2, FKBP5, GFPT2, KIT, KITLG, L3MBLT3, MRO, NUDT10, NUDT11, P4HA3, POSTN, PSAP, RBP1, SAT1, SDC4, TIMP2, TNC and USP53. In bovine GC, CRISPLD2 and POSTN mRNA were found as full-length transcript whereas L3MBLT3 mRNA was alternatively spliced resulting in a truncated protein missing the carboxy-terminal end amino acids, 774KNSHNEL780. Conversely, L3MBLT3 is expressed as a full-length mRNA in a bovine endometrial cell line. The 774KNSHNEL780 sequence is well conserved in all mammalian species and follows a SAM domain known to confer protein/protein interactions, which suggest a key function for these amino acids in the epigenetic control of gene expression.

CONCLUSIONS

We conclude that we have identified novel genes that are upregulated by hCG in bovine GC of OF, thereby providing novel insight into peri-ovulatory regulation of genes that contribute to ovulation and/or luteinization processes.

Targeted cancer immunotherapy with irradiated, granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting, allogeneic cancer cell lines has been an effective approach to reduce tumor burden in several patients. It is generally assumed that to be effective, these cell lines need to express immunogenic antigens coexpressed in patient tumor cells, and antigen-presenting cells need to take up such antigens then present them to patient T cells. We have previously reported that, in a phase I pilot study (ClinicalTrials.gov NCT00095862), a subject with stage IV breast cancer experienced substantial regression of breast, lung, and brain lesions following inoculation with clinical formulations of SV-BR-1-GM, a GM-CSF-secreting breast tumor cell line. To identify diagnostic features permitting the prospective identification of patients likely to benefit from SV-BR-1-GM, we conducted a molecular analysis of the SV-BR-1-GM cell line and of patient-derived blood, as well as a tumor specimen. Compared to normal human breast cells, SV-BR-1-GM cells overexpress genes encoding tumor-associated antigens (TAAs) such as PRAME, a cancer/testis antigen. Curiously, despite its presumptive breast epithelial origin, the cell line expresses major histocompatibility complex (MHC) class II genes (HLA-DRA, HLA-DRB3, HLA-DMA, HLA-DMB), in addition to several other factors known to play immunostimulatory roles. These factors include MHC class I components (B2M, HLA-A, HLA-B), ADA (encoding adenosine deaminase), ADGRE5 (CD97), CD58 (LFA3), CD74 (encoding invariant chain and CLIP), CD83, CXCL8 (IL8), CXCL16, HLA-F, IL6, IL18, and KITLG. Moreover, both SV-BR-1-GM cells and the responding study subject carried an HLA-DRB3*02:02 allele, raising the question of whether SV-BR-1-GM cells can directly present endogenous antigens to T cells, thereby inducing a tumor-directed immune response. In support of this, SV-BR-1-GM cells (which also carry the HLA-DRB3*01:01 allele) treated with yellow fever virus (YFV) envelope (Env) 43-59 peptides reactivated YFV-DRB3*01:01-specific CD4+ T cells. Thus, the partial HLA allele match between SV-BR-1-GM and the clinical responder might have enabled patient T lymphocytes to directly recognize SV-BR-1-GM TAAs as presented on SV-BR-1-GM MHCs. Taken together, our findings are consistent with a potentially unique mechanism of action by which SV-BR-1-GM cells can act as APCs for previously primed CD4+ T cells.

Most current methods for detecting natural selection from DNA sequence data are limited in that they are either based on summary statistics or a composite likelihood, and as a consequence, do not make full use of the information available in DNA sequence data. We here present a new importance sampling approach for approximating the full likelihood function for the selection coefficient. Our method CLUES treats the ancestral recombination graph (ARG) as a latent variable that is integrated out using previously published Markov Chain Monte Carlo (MCMC) methods. The method can be used for detecting selection, estimating selection coefficients, testing models of changes in the strength of selection, estimating the time of the start of a selective sweep, and for inferring the allele frequency trajectory of a selected or neutral allele. We perform extensive simulations to evaluate the method and show that it uniformly improves power to detect selection compared to current popular methods such as nSL and SDS, and can provide reliable inferences of allele frequency trajectories under many conditions. We also explore the potential of our method to detect extremely recent changes in the strength of selection. We use the method to infer the past allele frequency trajectory for a lactase persistence SNP (MCM6) in Europeans. We also infer the trajectory of a SNP (EDAR) in Han Chinese, finding evidence that this allele's age is much older than previously claimed. We also study a set of 11 pigmentation-associated variants. Several genes show evidence of strong selection particularly within the last 5,000 years, including ASIP, KITLG, and TYR. However, selection on OCA2/HERC2 seems to be much older and, in contrast to previous claims, we find no evidence of selection on TYRP1.

Bronchopulmonary dysplasia (BPD) is a chronic inflammatory lung disease that affects infants born preterm. Family studies indicate that BPD has a significant genetic component.

BACKGROUND

We assessed the gene encoding Kit ligand (KITLG) as a candidate for genetic predisposition to moderate-to-severe BPD (controls were infants with no or mild BPD).

METHODS

Eight KITLG-tagging single nucleotide polymorphisms (SNPs) were analyzed in cohorts of very preterm infants originating from northern Finland (56 cases and 197 controls), southern Finland (n = 59 + 52), and Canada (n = 58 + 68). Additional replication populations included infants born in Finland (n = 41 + 241) and Hungary (n = 29 + 40). All infants were of European origin. Results were controlled for risk factors of BPD. Kit ligand concentration in umbilical cord blood, collected from very preterm infants (n = 120), was studied.

RESULTS

Six SNPs of KITLG and a haplotype including all eight genotyped SNPs were associated with moderate-to-severe BPD in the northern Finnish population. When all the populations were combined, SNP rs11104948 was significantly associated with BPD. Kit ligand concentration in umbilical cord blood of infants born very preterm was an independent risk factor of BPD.

CONCLUSIONS

We show that KITLG polymorphisms are associated with susceptibility to moderate-to-severe BPD. In addition, higher Kit ligand concentrations were observed in infants that subsequently developed BPD. These results support the possibility that KITLG gene is involved in predisposition to BPD. Pediatr Pulmonol. 2015; 50:260-270. © 2014 Wiley Periodicals, Inc.

Waardenburg syndrome (WS) is a group of genetic disorders that often determined by abnormal pigmentation and hearing impairment. Four subgroups of disease are recognized according to physical characteristics and involved genes. Mutation in the genes, MITF, SOX10, SNAI2, PAX3, KIT and KITLG are related to Waardenburg syndrome type II. In this study, we performed exome sequencing in a WS2 proband and detected a heterozygous non-sense variation in MITF. Clinical features, pedigrees investigations and molecular segregation revealed autosomal dominant inheritance with incomplete penetrance. To our knowledge it's the first evidence about incomplete penetrance of WS2 related to MITF gene.

Keywords: Abnormal pigmentation; Hearing impairment; Incomplete penetrance; MITF; Waardenburg syndrome.

Phosphodiesterases (PDEs) are important regulators of the intracellular cAMP concentration, which is a central second messenger that affects a multitude of intracellular functions. In the ovaries, cAMP exerts diverse functions, including regulation of ovulation and it has been suggested that augmented cAMP levels stimulate primordial follicle growth. The present study examined the gene expression, enzyme activity and immunolocalization of the different cAMP hydrolysing PDEs families in the rat ovary. Further, the effect of PDE4 inhibition on primordial follicle activation in cultured neonatal rat ovaries was also evaluated. We found varied expression of all eight families in the ovary with Pde7b and Pde8a having the highest expression each accounting for more than 20% of the total PDE mRNA. PDE4 accounted for 15-26% of the total PDE activity. Immunoreactive PDE11A was found in the oocytes and PDE2A in the corpora lutea. Incubating neonatal rat ovaries with PDE4 inhibitors did not increase primordial follicle activation or change the expression of the developing follicle markers Gdf9, Amh, Inha, the proliferation marker Mki67 or the primordial follicle marker Tmeff2. In addition, the cAMP analogue 8-bromo-cAMP did not increase AKT1 or FOXO3A phosphorylation associated with follicle activation or increase the expression of Kitlg known to be associated with follicle differentiation but did increase the Tmeff2, Mki67 and Inha expression in a dose-dependent manner. In conclusion, this study shows that both Pde7b and Pde8a are highly expressed in the rodent ovary and that PDE4 inhibition does not cause an increase in primordial follicle activation.

BACKGROUND

Chronic idiopathic urticaria (CIU) is a complicated skin disease with unknown pathophysiology. MicroRNAs (miRNA) have been shown to be active in cellular regulation. The goal of this pilot study was to examine whether miRNAs may be involved in the regulation of CIU or as biomarkers for CIU.

METHODS

Four groups of three patients each were selected: patients with either active hives or no hives and with positive or negative chronic urticaria (CU) index results. MiRNAs were isolated from patient plasma and analyzed by using miRNA microarray technology to determine the amount of each of the 2567 known human miRNAs.

RESULTS

A total of 16 miRNAs were found to be differentially expressed in patients with active hives. Among them, five (2355-3p, 4264, 2355-5p, 29c-5p, and 361-3p) were significantly increased in samples with positive CU index results, which could be useful biomarkers for patients with chronic autoimmune urticaria. The miRNA data bases were used to find the targets of these selected miRNA sequences. These potential targets were then compared against a list of 154 urticaria-related genes. Twenty-five genes were found to match. These included eight that were significantly downregulated and eight that were significantly upregulated; however, seven of the eight downregulated genes (FBXL20, OPHN1, YPEL2, STARD9, EZH1, KLHL24, ING4) and five of the eight upregulated genes (BYSL, PNO1, ADAMTS9, STEAP4, SRGN) have no reported roles in signaling. For the 13 genes with reported roles in signaling, the following pathways were found: transforming growth factor beta signaling pathway (NRC31, KITLG, THBS1, CCL2), glucocorticoid receptor signaling pathway (NR3C1, SELE, CCL2), p53 signaling pathway (CCNG2, THBS1, CCL2), p21-activated kinase pathway (PAK1IP1, KITLG, CCL2), phosphoinositide-3 kinase protein kinase B signaling pathway (KITLG, CHRM, THBS1), and neuroactive ligand-receptor interaction (NRC31, HRH1, CHRM), which could play important roles in CIU.

CONCLUSIONS

A better understanding of those genes with undefined function and simultaneous quantitation of both miRNAs and messenger RNAs are needed to fully understand CIU disease.

BACKGROUND

Vitiligo vulgaris is a depigmentary disorder resulting from the disappearance of functional melanocytes. Currently, the pathogenesis of this disorder remains obscure.

OBJECTIVE

Genetic analysis of patients with vitilgo may provide important clues for elucidating the complex pathomechanisms involved in the disease process. Because dysfunctional keratinocytes have recently been implicated in the pathogenesis of vitiligo vulgaris, we conducted a case-control association study to investigate this phenomenon.

METHODS

Fifty-one patients with vitiligo vulgaris and 118 healthy controls from Taiwan were recruited to investigate the association between relevant keratinocyte-related genes and the occurrence of vitiligo vulgaris. This study genotyped 11 single-nucleotide polymorphisms (SNPs) in five genes including stem cell factor (SCF, also known as KITLG), basic fibroblast growth factor (bFGF, also known as NuDT6), endothelin-1 (EDN1), hepatocyte growth factor (HGF) and stem cell growth factor (SCGF, also known as CLEC11A).

RESULTS

Our results revealed that the A allele for SNP rs11104947 in the SCF gene and the T allele for SNP rs13866 in the SCGF gene were, respectively, associated with a 1.95- and a 2.14-fold risk of developing vitiligo vulgaris. A higher risk was also detected among subjects who carried the SCF rs995029/rs11104947 C/A haplotype (odds ratio = 2.45). Furthermore, the at-risk alleles for SCF rs11104947 (A allele) and for SCGF SNP rs13866 (T allele) were found to display a 7.92-fold increased gene-gene combined risk. No significant relationship between polymorphic frequency for genes bFGF, EDN1 as well as HGF and occurrence of vitiligo vulgaris was observed.

CONCLUSIONS

These novel genetic findings provide new insights in relation to the mechanisms that might be involved in the development of vitiligo vulgaris.

Testicular microlithiasis (TM) is one of the symptoms of testicular dysgenesis syndrome (TDS). TM is particularly interesting as an informative marker of testicular germ cell tumors (TGCTs). KIT ligand gene (KITLG), BCL2 antagonist/killer 1 (BAK1), and sprouty RTK signaling antagonist 4 (SPRY4) genes are associated with a high risk of TGCTs, whereas bone morphogenetic protein 7 gene (BMP7), transforming growth factor beta receptor 3 gene (TGFBR3), and homeobox D cluster genes (HOXD) are related to TDS. Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, we investigated allele and genotype frequencies for KITLG (rs995030, rs1508595), SPRY4 (rs4624820, rs6897876), BAK1 (rs210138), BMP7 (rs388286), TGFBR3 (rs12082710), and HOXD (rs17198432) in 142 TGCT patients, 137 TM patients, and 153 fertile men (control group). We found significant differences in the KITLG GG_rs995030 genotype in TM (P = 0.01) and TGCT patients (P = 0.0005) compared with the control. We also revealed strong associations between KITLG_rs1508595 and TM (G allele, P = 0.003; GG genotype, P = 0.01) and between KITLG_rs1508595 and TGCTs (G allele, P = 0.0001; GG genotype, P = 0.0007). Moreover, there was a significant difference in BMP7_rs388286 between the TGCT group and the control (T allele, P = 0.00004; TT genotype, P = 0.00006) and between the TM group and the control (T allele, P = 0.04). HOXD also demonstrated a strong association with TGCTs (rs17198432 A allele, P = 0.0001; AA genotype, P = 0.001). Furthermore, significant differences were found between the TGCT group and the control in the BAK1_rs210138 G allele (P = 0.03) and the GG genotype (P = 0.01). KITLG and BMP7 genes, associated with the development of TGCTs, may also be related to TM. In summary, the KITLG GG_rs995030, GG_rs1508595, BMP7 TT_rs388286, HOXD AA_rs17198432, and BAK1 GG_rs210138 genotypes were associated with a high risk of TGCT development.

Recently, a functional polymorphism in KITLG, rs4590952, was identified to be associated with testicular cancer susceptibility through increasing the p53-dependent KITLG expression and disrupting the function of p53. We performed a hospital-based case-control study, including 1241 breast cancer (BC) patients and 1259 cancer-free controls, to investigate the role of this polymorphism in the risk of BC in Chinese Han population. However, no significant association between rs4590952 and BC risk was identified in allelic model with the odds ratio (OR) of 1.04 (95% confidence interval (CI) = 0.73-1.46, P = 0.839) or in any other genetic models. When performed stratified analysis according to the Estrogen Receptor (ER) and Progesterone Receptor (PR) status, rs4590952 was neither associated with ER+/PR+ nor ER-PR- subgroups. Our results suggested that rs4590952 was not associated with the risk of BC in Chinese population, implying that heterogeneous distinct mechanisms might exist in the etiology of different cancers.

In the course of a reverse genetic screen in the Belgian Blue cattle breed, we uncovered a 10-bp deletion (c.87_96del) in the first coding exon of the melanophilin gene (MLPH), which introduces a premature stop codon (p.Glu32Aspfs*1) in the same exon, truncating 94% of the protein. Recessive damaging mutations in the MLPH gene are well known to cause skin, hair, coat or plumage color dilution phenotypes in numerous species, including human, mice, dog, cat, mink, rabbit, chicken and quail. Large-scale array genotyping undertaken to identify p.Glu32Aspfs*1 homozygous mutant animals revealed a mutation frequency of 5% in the breed and allowed for the identification of 10 homozygous mutants. As expression of a colored coat requires at least one wild-type allele at the co-dominant Roan locus encoded by the KIT ligand gene (KITLG), homozygous mutants for p.Ala227Asp corresponding with the missense mutation were excluded. The six remaining colored calves displayed a distinctive dilution phenotype as anticipated. This new coat color was named 'cool gray'. It is the first damaging mutation in the MLPH gene described in cattle and extends the already long list of species with diluted color due to recessive mutations in MLPH and broadens the color palette of gray in this breed.

Ionizing radiation has been shown to arrest spermatogenesis despite the presence of surviving stem spermatogonia, by blocking their differentiation. This block is a result of damage to the somatic environment and is reversed when gonadotropins and testosterone are suppressed, but the mechanisms are still unknown. We examined spermatogonial differentiation and Sertoli cell factors that regulate spermatogonia after irradiation, during hormone suppression, and after hormone suppression combined with Leydig cell elimination with ethane dimethane sulfonate. These results showed that the numbers and cytoplasmic structure of Sertoli cells are unaffected by irradiation, only a few type A undifferentiated (Aund) spermatogonia and even fewer type A1 spermatogonia remained, and immunohistochemical analysis showed that Sertoli cells still produced KIT ligand (KITLG) and glial cell line-derived neurotrophic factor (GDNF). Some of these cells expressed KIT receptor, demonstrating that the failure of differentiation was not a result of the absence of the KIT system. Hormone suppression resulted in an increase in Aund spermatogonia within 3 days, a gradual increase in KIT-positive spermatogonia, and differentiation mainly to A3 spermatogonia after 2 weeks. KITL (KITLG) protein expression did not change after hormone suppression, indicating that it is not a factor in the stimulation. However, GDNF increased steadily after hormone suppression, which was unexpected since GDNF is supposed to promote stem spermatogonial self-renewal and not differentiation. We conclude that the primary cause of the block in spermatogonial development is not due to Sertoli cell factors such (KITL\GDNF) or the KIT receptor. As elimination of Leydig cells in addition to hormone suppression resulted in differentiation to the A3 stage within 1 week, Leydig cell factors were not necessary for spermatogonial differentiation.

Three recent genome-wide association studies of testicular germ cell tumors have uncovered predisposition alleles in or near several genes, including KITLG, BAK1, SPRY4, TERT, ATF7IP, and DMRT1. The calculated per-allele odds ratio for variants in the region of KITLG is the highest reported for any malignancy so far. These findings are in agreement with epidemiological data indicating that testicular cancer has a higher heritability than most other cancers. Here, we discuss the question of whether the newly identified risk polymorphisms can be used to guide patient care.

Characterization of molecules within important oncogenetic pathways may have future implications for development of therapies and biomarkers in lung cancer. One such target is the tyrosine kinase receptor KIT (c-KIT). We evaluated alterations and expression of KIT and its ligand, KITLG (also known as SCF), in 72 clinical lung tumor specimens of different histologies. Gene copy number, mRNA expression levels, and protein expression were assayed using array-based comparative genomic hybridization, real-time quantitative reverse transcription PCR and immunohistochemistry, respectively. For validation, we investigated copy number alterations and mRNA expression in external microarray data sets of 1,600 and 555 primary lung tumors, respectively. Positivity for KIT staining was most common in large cell neuroendocrine carcinoma (LCNEC) which also showed the highest KIT mRNA expression levels whereas expression was lowest in squamous cell carcinoma (SqCC). KIT mRNA expression levels were higher in KIT immunopositive samples, but expression was not affected by KIT copy numbers. Copy number gains of KIT were significantly more frequent in SqCC compared with adenocarcinoma in our own series and in the 1,600-sample data set. Immunopositivity for both KIT and KITLG in the same tumor was rare except in LCNEC. Our results highlight an increased KIT mRNA expression and frequent KIT immunopositivity in LCNEC but point out a poor correlation between KIT copy numbers and expression in SqCC, perhaps reflecting the existence of a protective mechanism against KIT alterations in this subgroup.

Rationale: The existence of primary and acquired drug resistance is the main obstacle for the effect of multi-kinase inhibitor sorafenib and regorafenib in advanced hepatocellular carcinoma (HCC). However, plenty of patients did not significantly benefit from sorafenib treatment and little is known about the mechanism of drug resistance. Methods: Laser capture microdissection was used to acquire matched normal liver and tumor tissues on formalin-fixed paraffin-embedded specimens collected before sorafenib therapy from the first surgery of 119 HCC patients. Ultra-deep sequencing (~1000×) targeting whole exons of 440 genes in microdissected specimens and siRNA screen in 7 cell lines were performed to find mutations associated with differential responses to sorafenib. Patient-derived xenograft models were employed to determine the role of TP53 in response to sorafenib. Lentiviruses harboring wild-type and c.G52C-mutant OCT4 were applied to explore the function of OCT4 in resistance to sorafenib. ChIP-PCR assay for analysis of OCT4 transcriptional activity was performed to explore the affinity with the KITLG promoter. Statistical analyses were used to associate levels of p53 and OCT4 with tumor features and patient outcomes. Results: Total 1,050 somatic mutations and 26 significant driver genes were identified. SiRNA screening in 7 HCC cell lines was further performed to identify mutations associated with differential responses to sorafenib. A recurrent nonsynonymous mutation c.G52C in OCT4 (OCT4^mut) was strongly associated with good response to sorafenib, whereas the stop-gain mutation in TP53 showed the opposite outcome both in vitro and in vivo. OCT4^wt-induced stem cell factor (encoded by KITLG gene, SCF) expression and cross-activation of c-KIT/FLT3-BRAF signals were identified indispensably for sorafenib resistance, which could be reversed by the combination of c-KIT tyrosine kinase inhibitors or neutralizing antibody against SCF. Mechanistically, an OCT4 binding site in upstream of KITLG promoter was identified with a higher affinity to wildtype of OCT4 rather than G52C-mutant form, which is indispensable for OCT4-induced expression of KITLG and sorafenib resistance. Conclusion: Our study reported a novel somatic mutation in OCT4 (c.G52C) responsible for the sorafenib effect, and also shed new light on the treatment of HCC through the combination of specific tyrosine kinase inhibitors according to individual genetic patterns.

Testicular germ cell cancer (TGCT) is the most common malignancy among young adult males, which has become important due to its increased incidence and mortality in the population worldwide. The etiology is multifactorial. Recent studies have shown some associations between the development of isolated TGCT and certain risk factors, such as exposure to endocrine disruptors, cryptorchidism, and family history of cancer, in order to identify the key pieces in carcinogenesis. Some of the most important findings in recent years is the association of different genes, such as c-KIT/KITLG, expression of the miR-371-373 cluster and protein expression as c-KIT and POU5F1 in the development of this neoplasia, and the identification of new molecular markers as TGFBR3 gene, identifying aberrant methylation patterns in promoter regions of several genes, expression of miR-1297 which regulates PTEN and protein expression as DMTR1. In the future, a multidisciplinary research strategy could provide valuable new insights into the etiology of TGCTs, which support clinical diagnosis of TGCT in the next years to increase survival in this kind of patients.

Our previous study has showed that a novel gene is differentially expressed in the liver of cyadox-fed piglets, but its sequence and function are unknown. Here, rapid amplification of cDNA ends (RACE) and bioinformatics analysis showed that the novel gene is 953 bp without protein-coding ability and locates in chromosome 11. Hence, we identified the novel gene as long non-coding RNA (lncRNA) and named it cyadox-related novel gene (CRNG). Fluorescence in situ hybridization (FISH) showed that CRNG mainly distributes in cytoplasm. Moreover, microarray assay in combination with CRNG interference and overexpression showed that the differential genes such as ANPEP, KITLG, STAT5A, FOXP3, miR-451, IL-2, IL-10, IL-6, and TNF-α are mainly involved in viral and pathogens infection and the immune-inflammatory responses in PK-15 cells. This work reveals that CRNG might play a role in preventing the host from being infected by pathogens and viruses and exerting immune regulatory effects in the cytoplasm, which may be involved in prophylaxis of cyadox in piglets.

Malignant gonadal germ cell tumors, referred to as germ cell cancers (GCC), occur with increased frequency in individuals who have specific types of differences (disorders) of sex development (DSD). Recent population-based studies have identified new environmental and genetic risk factors that have led to a 'genvironment' hypothesis, which may potentially be helpful in risk assessment in DSD-related GCC. In DSD, the malignancy risk is highly heterogeneous, but recent studies allow now to discriminate between high- and low-risk conditions. Gonadal biopsy is in some cases the best procedure of choice to assess the risk, and with the availability of immunohistochemical biomarkers [OCT3/4 (POU5F1), TSPY, SOX9, FOXL2 and KITLG (SCF)], a reliable classification of GCC and its precursors can be made. The opportunities in the field of virtual diagnostic pathology will be presented, having possibilities for rare diseases in general and DSD specifically. It is expected that the International DSD Registry will stimulate international collaborations, facilitating better diagnostic procedures as well as research.