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Introduction: In situ hybridization (ISH) plays an important role in the field of molecular diagnostics, especially in an anatomical pathology laboratory. ISH is a technique that can detect the targeted DNA or RNA sequences in tissue sections from frozen or fixed materials with labeled DNA or RNA probes. Radioactive and non-radioactive probes are the two major probes that can be used to label the targeted nucleic acids., Areas Covered: Two decades after the Human Genome Project, ISH has not only simply been applied to identify the chromosomal location of a human gene but has also been extensively applied to gene expressions studies and utilized for clinical diagnosis, especially for the determination of biomarkers for breast and ovarian cancers - human epidermal growth factor receptor 2. Duchenne muscular dystrophy, Cri-du-chat syndrome, Angelman syndrome, PraderWilli syndrome, cystic fibrosis, and trisomy are diseases that can also be detected by ISH. In this review, the basic principles, historical development, advantages and disadvantages, enhancement in reporting molecules and probes, advancement in detection methods, in situ PCR, clinical applications and novel applications of ISH will be discussed., Expert Opinion: With the advancement in ISH technologies and appropriate training, diagnosis can be improved in Anatomical Pathology.

Objectives: Acute lymphoblastic leukemia (ALL) is one of the most common malignancies in children. Our aim was to identify a novel miRNA that can predict prognosis of childhood ALL patients and explore its potential mechanism., Methods: The miRNA expression profiles of childhood ALL were analyzed using GEO database and HiSeq instruments. The expression of miR-155 was examined by RT-PCR in 42 ALL patients. To investigate the role of miR-155 in ALL, four ALL cell lines (CEM-C1, Jurkat, MOLT-3 and MOLT-4) were transfected with miR-155 mimics, miR-155 inhibitors or corresponding controls. Dual-luciferase reporter system was applied to confirm the miR-155 target ZNF238. Moreover, proliferation and apoptosis were evaluated by MTT and flow cytometry., Results: Dataset GSE56489 and GSE23024 demonstrated that miR-155 was up-regulated and ZNF238 was down-regulated at diagnosis status of ALL. High miR-155 expression was associated with poor outcome. Overexpressed miR-155 promoted ALL cell proliferation and inhibited apoptosis. Dual-luciferase reporter result showed that miR-155 directly regulated ZNF238. Silencing ZNF238 promoted cell proliferation in ALL cells., Discussion: Our research indicating that miR-155 might possess potential value as a biomarker for predicting the prognosis of individuals. However, the role of ZNF238 in childhood ALL remain unknown. In the present study, we found the possible role of ZNF238 as a new tumor suppressor in ALL, which might be necessary for the antiproliferative functions of normal cells to counteract ALL formation., Conclusion: Our results propose that miR-155 is in association with poor prognosis of childhood ALL. Furthermore, miR-155 could promote cell proliferation targeting ZNF238.

The prognosis of patients with acute myeloid leukemia (AML) caused by the FLT3-ITD mutation is poor. Arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) can down-regulate FLT3-ITD level and selectively kill leukemia cells carrying the FLT3-ITD mutation. However, the mechanisms of action of these two compounds are unknown. Here, we found that ATO could bind FLT3-ITD at Lys91 and Asp225, whereas ATRA could bind FLT3-ITD at Lys5 and Gln6. Both compounds could not bind wild-type FLT3. Further studies revealed that ATO/ATRA may suppress the Expression of FLT3-ITD by promoting the UBE2L6-mediated ubiquitination pathway and decreasing the expression of C-MYC. However, further studies are needed to define the mechanisms of these compounds on AML. Our research provides an experimental basis for the use of ATO/ATRA in FLT3-ITD AML in clinical practice.

Objective: To investigate to what extent paternal involvement and support during pregnancy were associated with preterm (PTB) and small-for-gestational age (SGA) births., Methods: Using data from the Boston Birth Cohort (n = 7047), multiple logistic regression models were performed to estimate the log odds of either PTB or SGA birth, with paternal involvement, paternal social support, and family and friend social support variables as the primary independent variables., Results: About 10% of participating mothers reported their husbands not being involved or supportive during their pregnancies. Lack of paternal involvement was associated with 21% higher risk of PTB (OR = 1.21, 95% CI: 1.01-1.45). Similarly, lack of paternal support was borderline associated with PTB (OR = 1.13, 95% CI: 0.94-1.35). Also marginally significant, lack of paternal involvement (OR = 1.18, 95% CI: 0.95-1.47) and father's support (OR = 1.19, 95% CI: 0.96-1.48) were associated with higher odds of SGA birth. No associations were found between familial and friend support during pregnancy and PTB or SGA., Conclusions: Among predominantly low-income African Americans, lack of paternal involvement and lack of paternal support during pregnancy were associated with an increased risk of PTB, and suggestive of SGA birth. These findings, if confirmed in future research, underscore the important role a father can play in reducing PTB and/or SGA.

Maternal smoking during pregnancy may affect newborn DNA methylation (DNAm). However, little is known about how these associations vary by a newborn's sex and/or maternal nutrition. To fill in this research gap, we investigated epigenome-wide DNAm associations with maternal smoking during pregnancy in African American mother-newborn pairs. DNAm profiling in cord (n = 379) and maternal blood (n = 300) were performed using the Illumina HumanMethylation450 BeadChip array. We identified 12 CpG sites whose DNAm levels in cord blood were associated with maternal smoking, at a false discovery rate <5%. The identified associations in the GFI1 gene were more pronounced in male newborns than in females (P = 0.002 for maternal smoking × sex interaction at cg18146737). We further observed that maternal smoking and folate level may interactively affect cord blood DNAm level at cg05575921 in the AHRR gene (P = 5.0 × 10 -4 for interaction): compared to newborns unexposed to maternal smoking and with a high maternal folate level (>19.2 nmol/L), the DNAm level was about 0.03 lower (P = 3.6 × 10 -4 ) in exposed newborns with a high maternal folate level, but was 0.08 lower (P = 1.2 × 10 -8 ) in exposed newborns with a low maternal folate level. Our data suggest that adequate maternal folate levels may partly counteract the impact of maternal smoking on DNAm. These findings may open new avenues of inquiry regarding sex differences in response to environmental insults and novel strategies to mitigate their intergenerational health effects through optimization of maternal nutrition.

Developmental exposure to endocrine-disrupting chemicals (EDCs), 17β-estradiol-3-benzoate (EB) and bisphenol A (BPA), increases susceptibility to prostate cancer (PCa) in rodent models. Here, we used the methylated-CpG island recovery assay (MIRA)-assisted genomic tiling and CpG island arrays to identify treatment-associated methylome changes in the postnatal day (PND)90 dorsal prostate tissues of Sprague-Dawley rats neonatally (PND1, 3, and 5) treated with 25 µg/pup or 2,500 µg EB/kg body weight (BW) or 0.1 µg BPA/pup or 10 µg BPA/kg BW. We identified 111 EB-associated and 86 BPA-associated genes, with 20 in common, that have significant differentially methylated regions. Pathway analysis revealed cancer as the top common disease pathway. Bisulfite sequencing validated the differential methylation patterns observed by array analysis in 15 identified candidate genes. The methylation status of 7 (Pitx3, Wnt10b, Paqr4, Sox2, Chst14, Tpd52, Creb3l4) of these 15 genes exhibited an inverse correlation with gene expression in tissue samples. Cell-based assays, using 5-aza-cytidine-treated normal (NbE-1) and cancerous (AIT) rat prostate cells, added evidence of DNA methylation-mediated gene expression of 6 genes (exception: Paqr4). Functional connectivity of these genes was linked to embryonic stem cell pluripotency. Furthermore, clustering analyses using the dataset from The Cancer Genome Atlas revealed that expression of this set of 7 genes was associated with recurrence-free survival of PCa patients. In conclusion, our study reveals that gene-specific promoter methylation changes, resulting from early-life EDC exposure in the rat, may serve as predictive epigenetic biomarkers of PCa recurrence, and raises the possibility that such exposure may impact human disease.

The JWA gene was initially cloned as a novel cell differentiation-associated gene and was subsequently found to be an environmental responsive gene. The JWA gene also produced a marked effect during chemical-induced multidirectional differentiations of primary and human myeloid leukemia cells. Recently, a novel single nucleotide polymorphism (SNP) in exon2 of the JWA gene (454CA) was identified that may play a role in risk of bladder cancer. The aim of this study was to investigate the association between the 454CA (NM&#95;006407.2) in JWA exon2 variants and risk of leukemia in a hospital-based case-control study of 202 leukemia patients and 289 cancer-free controls. Results indicated that 454A allele was found to associate with significantly increased risk of leukemia, although the 454CA is a synonymous polymorphism in coding region of the JWA gene. In conclusion, the potentially functional genetic polymorphism 454CA of the JWA gene appears to contribute to the risk of multiple kinds of leukemia in a south Chinese population.

Recently, a novel single nucleotide polymorphism (SNP) in the promoter of the JWA gene (-76G --> C) was identified that may alter the transcription activity and thus play a role in increased risk of bladder cancer. In this study, a screen for more novel variants in the JWA exons was undertaken by using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) followed by a PCR-restriction fragment length polymorphism (PCR-RFLP) method and evaluating the functions of newl identified JWA -76G --> C using the reporter gene assay. In addition to the -76G --> C polymorphism, another novel SNP (723T --> G) in exon 3 of JWA was identified. In a case-control study of these two SNPs in 413 gastric cancer and 250 esophageal squamous-cell carcinoma (ESCC) patients and 814 cancer-free controls in a Chinese population, data showed that both SNPs were associated with enhanced risk of these cancers. The reporter gene assay showed that the -76C variant allele lost its response to benzo[a]pyrene (BaP) exposure, compared to the -76G allele. In addition, the JWA -76C allele was found to be associated with increased gastric and esophageal cancer risks in this study population. Further studies are needed to substantiate the biological significance and related mechanisms underlying the associations.