Diagnosis of Non-Small Cell Lung Cancer via Liquid Biopsy Highlighting a <em>Fluorescence-in-situ-Hybridization</em> Circulating Tumor Cell Approach

Lung cancer (LC), is the most common and lethal cancer worldwide. It affects both sexes and in its early stages is clinically silent, until it reaches a more advanced stage, when it becomes highly incurable. In order to improve the high mortality associated with LC there has been an urgent need for screening high risk patients by low dose CT scan (LDCT) for the early detection of small resectable malignant tumors. However, while highly sensitive to detect small lung nodules, LDCT is non-specific, resulting in a compelling need for a complementary diagnostic tool. For example, a non-invasive blood test or liquid biopsy, (LB), could prove quite useful to confirm a diagnosis of malignancy prior to definitive therapy. With the advent of LB becoming increasingly clinically accepted in the diagnosis and management of LC, there has been an explosion of publications highlighting new technologies for the isolation of and detection of circulating tumor cells (CTCs) and cell free tumor DNA (cfDNA). The enormous potential for LB to play an important role in the diagnosis and management of LC to obtain valuable diagnostic information via an approach that may yield equivalent information to a surgical biopsy, regarding the presence of cancer and its molecular landscape is described

Identification of genomic aberrations in hemangioblastoma by droplet digital PCR and SNP microarray highlights novel candidate genes and pathways for pathogenesis

Background: The genetic mechanisms underlying hemangioblastoma development are still largely unknown. We used high-resolution single nucleotide polymorphism microarrays and droplet digital PCR analysis to detect copy number variations (CNVs) in total of 45 hemangioblastoma tumors. Results: We identified 94 CNVs with a median of 18 CNVs per sample. The most frequently gained regions were on chromosomes 1 (p36.32) and 7 (p11.2). These regions contain the EGFR and PRDM16 genes. Recurrent losses were located at chromosome 12 (q24.13), which includes the gene PTPN11. Conclusions: Our findings provide the first high-resolution genome-wide view of chromosomal changes in hemangioblastoma and identify 23 candidate genes: EGFR, PRDM16, PTPN11, HOXD11, HOXD13, FLT3, PTCH, FGFR1, FOXP1, GPC3, HOXC13, HOXC11, MKL1, CHEK2, IRF4, GPHN, IKZF1, RB1, HOXA9, and micro RNA, such as hsa-mir-196a-2 for hemangioblastoma pathogenesis. Furthermore, our data implicate that cell proliferation and angiogenesis promoting pathways may be involved in the molecular pathogenesis of hemangioblastoma

CEACAM1 Promotes Melanoma Cell Growth through Sox-2

AbstractThe prognostic value of the carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) in melanoma was demonstrated more than a decade ago as superior to Breslow score. We have previously shown that intercellular homophilic CEACAM1 interactions protect melanoma cells from lymphocyte-mediated elimination. Here, we study the direct effects of CEACAM1 on melanoma cell biology. By employing tissue microarrays and low-passage primary cultures of metastatic melanoma, we show that CEACAM1 expression gradually increases from nevi to metastatic specimens, with a strong dominance of the CEACAM1-Long tail splice variant. Using experimental systems of CEACAM1 knockdown and overexpression of selective variants or truncation mutants, we prove that only the full-length long tail variant enhances melanoma cell proliferation in vitro and in vivo. This effect is not reversed with a CEACAM1-blocking antibody, suggesting that it is not mediated by intercellular homophilic interactions. Downstream, CEACAM1-Long increases the expression of Sox-2, which we show to be responsible for the CEACAM1-mediated enhanced proliferation. Furthermore, analysis of the CEACAM1 promoter reveals two single-nucleotide polymorphisms (SNPs) that significantly enhance the promoter's activity compared with the consensus nucleotides. Importantly, case-control genetic SNP analysis of 134 patients with melanoma and matched healthy donors show that patients with melanoma do not exhibit the Hardy-Weinberg balance and that homozygous SNP genotype enhances the hazard ratio to develop melanoma by 35%. These observations shed new mechanistic light on the role of CEACAM1 in melanoma, forming the basis for development of novel therapeutic and diagnostic technologies

A Novel Translocation Breakpoint within the BPTF Gene Is Associated with a Pre-Malignant Phenotype

Partial gain of chromosome arm 17q is an abundant aberrancy in various cancer types such as lung and prostate cancer with a prominent occurrence and prognostic significance in neuroblastoma – one of the most common embryonic tumors. The specific genetic element/s in 17q responsible for the cancer-promoting effect of these aberrancies is yet to be defined although many genes located in 17q have been proposed to play a role in malignancy. We report here the characterization of a naturally-occurring, non-reciprocal translocation der(X)t(X;17) in human lung embryonal-derived cells following continuous culturing. This aberrancy was strongly correlated with an increased proliferative capacity and with an acquired ability to form colonies in vitro. The breakpoint region was mapped by fluorescence in situ hybridization (FISH) to the 17q24.3 locus. Further characterization by a custom-made comparative genome hybridization array (CGH) localized the breakpoint within the Bromodomain PHD finger Transcription Factor gene (BPTF), a gene involved in transcriptional regulation and chromatin remodeling. Interestingly, this translocation led to elevation in the mRNA levels of the endogenous BPTF. Knock-down of BPTF restricted proliferation suggesting a role for BPTF in promoting cellular growth. Furthermore, the BPTF chromosomal region was found to be amplified in various human tumors, especially in neuroblastomas and lung cancers in which 55% and 27% of the samples showed gain of 17q24.3, respectively. Additionally, 42% percent of the cancer cell lines comprising the NCI-60 had an abnormal BPTF locus copy number. We suggest that deregulation of BPTF resulting from the translocation may confer the cells with the observed cancer-promoting phenotype and that our cellular model can serve to establish causality between 17q aberrations and carcinogenesis

Noncoding deletions reveal a gene that is critical for intestinal function.

Large-scale genome sequencing is poised to provide a substantial increase in the rate of discovery of disease-associated mutations, but the functional interpretation of such mutations remains challenging. Here we show that deletions of a sequence on human chromosome 16 that we term the intestine-critical region (ICR) cause intractable congenital diarrhoea in infants1,2. Reporter assays in transgenic mice show that the ICR contains a regulatory sequence that activates transcription during the development of the gastrointestinal system. Targeted deletion of the ICR in mice caused symptoms that recapitulated the human condition. Transcriptome analysis revealed that an unannotated open reading frame (Percc1) flanks the regulatory sequence, and the expression of this gene was lost in the developing gut of mice that lacked the ICR. Percc1-knockout mice displayed phenotypes similar to those observed upon ICR deletion in mice and patients, whereas an ICR-driven Percc1 transgene was sufficient to rescue the phenotypes found in mice that lacked the ICR. Together, our results identify a gene that is critical for intestinal function and underscore the need for targeted in vivo studies to interpret the growing number of clinical genetic findings that do not affect known protein-coding genes

Deleterious variants in TRAK1 disrupt mitochondrial movement and cause fatal encephalopathy

Cellular distribution and dynamics of mitochondria are regulated by several motor proteins and a microtubule network. In neurons, mitochondrial trafficking is crucial because of high energy needs and calcium ion buffering along axons to synapses during neurotransmission. The trafficking kinesin proteins (TRAKs) are well characterized for their role in lysosomal and mitochondrial trafficking in cells, especially neurons. Using whole exome sequencing, we identified homozygous truncating variants in TRAK1 (NM_001042646:c.287-2A > C), in six lethal encephalopathic patients from three unrelated families. The pathogenic variant results in aberrant splicing and significantly reduced gene expression at the RNA and protein levels. In comparison with normal cells, TRAK1-deficient fibroblasts showed irregular mitochondrial distribution, altered mitochondrial motility, reduced mitochondrial membrane potential, and diminished mitochondrial respiration. This study confirms the role of TRAK1 in mitochondrial dynamics and constitutes the first report of this gene in association with a severe neurodevelopmental disorder

Immunity to heat shock protein 65: an additional determinant in intimal thickening. Atherosclerosis

Abstract Inflammation occurring consequent to vessel injury is thought to play an important role in atherosclerosis and restenosis. Autoimmunity to HSP65 has been shown to accelerate early atherogenesis in rabbits and mice, whereas in humans epidemiological data support this contention. In the current study, we explored the possibility of HSP65 influencing the extent of neointimal growth in the rat carotid injury model. Rats were either immunized with recombinant mycobacterial HSP65, heat killed preparation of Mycobacterium tuberculosis (MT), or with PBS, all emulsified in incomplete Freund&apos;s adjuvant. Animals were boosted with a similar protocol 3 weeks following the primary immunization and 2 weeks later carotid injury was applied in all animals by balloon inflation. Upon sacrifice 2 weeks later, sera were obtained for measurement of anti-HSP65 antibodies by ELISA, splenocytes were assessed for proliferative response to in vitro priming with HSP65, and carotid arteries were removed for evaluation of neointimal growth. Rats immunized with HSP65 exhibited a brisk and sustained humoral immune response to HSP65, and cellular immunity was also evident by thymidine uptake to splenocytes primed with the respective protein. Neointimal/medial ratio was significantly increased in HSP65 immunized rats, in comparison with MT injected and control animals. In conclusion, immunity to HSP65 can play a role in accelerating restenosis following arterial injury. These results should be further investigated in humans as they may provide a possible link between infections and restenosis/accelerated arteriosclerosis.

Flare-up of ulcerative colitis after systemic corticosteroids: A strong case for Strongyloides

Super-imposed infection with intestinal organisms can mimic a flare-up of underlying disease in patients with inflammatory bowel disease (IBD). We report a case of patient with long standing ulcerative colitis (UC), who presented with abdominal pain, diarrhea and low-grade fever after receiving systemic corticosteroids for an unrelated disorder. Despite a negative stool examination, a peripheral eosinophilia reappeared upon tapering down of a corticosteroid dose. Subsequently, duodenal biopsies showed evidence for Strongyloides, presumably acquired 20 years ago when the patient was residing in Brazil. The patient fully recovered following anti-helmintic therapy. This case underscores the importance of considering Strongyloides in the work-up of flaring-up IBD patients, even if a history of residing or traveling to endemic areas is in the distant past

Saccharin Increases Fasting Blood Glucose but Not Liver Insulin Resistance in Comparison to a High Fructose-Fed Rat Model

Recent data indicate that artificial sweeteners (AS) may have deleterious effects on glucose metabolism. The purpose of this study was to compare the effects of AS and the effects of a high fructose diet (HFrD) on glucose metabolism and insulin resistance (IR) in Sprague-Dawley (SD) rats. SD rats were fed either regular chow, chow with saccharin (Sac) (0.1 mg/mL) placed in their water, or HFrD for seven weeks. Glucose, insulin, and triglycerides (Tg) levels were measured upon completion. A homeostatic model assessment (HOMA)-IR index was used to determine insulin resistance. The liver was stained to detect signs of a fatty liver. Hepatic mRNA expression of glucose metabolism regulation genes, Srepb-1c (sterol regulatory element binding protein) and ChREB (α &amp; β) (carbohydrate response element binding protein), as well as other glycolytic and lipogenic genes including glucose-6-phosphatase (G6pc), were considered IR markers. Both HFrD and Sac significantly increased fasting blood glucose levels compare to the control (140 ± 5 and 137 ± 6 vs. 118 ± 3 mg/dL, respectively, p &lt; 0.05). However, only HFrD increased insulin secretion (0.99 ± 0.12 vs. 0.7 ± 0.1 and 0.6 ± 0.1 ug/L), Tg levels (420 ± 43 vs. 152 ± 20 and 127 ± 13 mg/dL), and the HOMA-IR index (3.4 ± 0.4 vs. 2.3 ± 0.36 and 2.13 ± 0.3) (HFrD vs. control and sac, p &lt; 0.05). Fatty liver changes were only observed in HFrD fed rats. The expression of ChREB β, Srepb-1c, and G6pc mRNA were only significantly elevated (between 2–10 times folds, p &lt; 0.05) in HFrD fed rats. Sac may increase fasting blood glucose but has no effect on liver insulin resistance