Genetic Variants Detected Using Cell-Free DNA from Blood and Tumor Samples in Patients with Inflammatory Breast Cancer.

We studied genomic alterations in 19 inflammatory breast cancer (IBC) patients with advanced disease using samples of tissue and paired blood serum or plasma (cell-free DNA, cfDNA) by targeted next generation sequencing (NGS). At diagnosis, the disease was triple negative (TN) in eleven patients (57.8%), ER+ Her2- IBC in six patients (31.6%), ER+ Her2+ IBC in one patient (5.3%), and ER- Her2+ IBC in one other patient (5.3%). Pathogenic or likely pathogenic variants were frequently detected in TP53 (47.3%), PMS2 (26.3%), MRE11 (26.3%), RB1 (10.5%), BRCA1 (10.5%), PTEN (10.5%) and AR (10.5%); other affected genes included PMS1, KMT2C, BRCA2, PALB2, MUTYH, MEN1, MSH2, CHEK2, NCOR1, PIK3CA, ESR1 and MAP2K4. In 15 of the 19 patients in which tissue and paired blood were collected at the same time point, 80% of the variants detected in tissue were also detected in the paired cfDNA. Higher concordance between tissue and cfDNA was found for variants with higher allele fraction in tissue (AFtissue ≥ 5%). Furthermore, 86% of the variants detected in cfDNA were also detected in paired tissue. Our study suggests that the genetic profile measured in blood cfDNA is complementary to that of tumor tissue in IBC patients.

Ibrutinib and venetoclax target distinct subpopulations of CLL cells: implication for residual disease eradication.

Ibrutinib inhibits Bruton tyrosine kinase while venetoclax is a specific inhibitor of the anti-apoptotic protein BCL2. Both drugs are highly effective as monotherapy against chronic lymphocytic leukemia (CLL), and clinical trials using the combination therapy have produced remarkable results in terms of rate of complete remission and frequency of undetectable minimal residual disease. However, the laboratory rationale behind the success of the drug combination is still lacking. A better understanding of how these two drugs synergize would eventually help develop other rational combination strategies. Using an ex vivo model that promotes CLL proliferation, we show that modeled ibrutinib proliferative responses, but not viability responses, correlate well with patients' actual clinical responses. Importantly, we demonstrate for the first time that ibrutinib and venetoclax act on distinct CLL subpopulations that have different proliferative capacities. While the dividing subpopulation of CLL responds to ibrutinib, the resting subpopulation preferentially responds to venetoclax. The combination of these targeted therapies effectively reduced both the resting and dividing subpopulations in most cases. Our laboratory findings help explain several clinical observations and contribute to the understanding of tumor dynamics. Additionally, our proliferation model may be used to identify novel drug combinations with the potential of eradicating residual disease.

Focus on RNA isolation: obtaining RNA for microRNA (miRNA) expression profiling analyses of neural tissue.

MicroRNAs (miRNAs) are present in all known plant and animal tissues and appear to be somewhat concentrated in the mammalian nervous system. Many different miRNA expression profiling platforms have been described. However, relatively little research has been published to establish the importance of 'upstream' variables in RNA isolation for neural miRNA expression profiling. We tested whether apparent changes in miRNA expression profiles may be associated with tissue processing, RNA isolation techniques, or different cell types in the sample. RNA isolation was performed on a single brain sample using eight different RNA isolation methods, and results were correlated using a conventional miRNA microarray and then cross-referenced to Northern blots. Differing results were seen between samples obtained using different RNA isolation techniques and between microarray and Northern blot results. Another complication of miRNA microarrays is tissue-level heterogeneity of cellular composition. To investigate this phenomenon, miRNA expression profiles were determined and compared between highly-purified primary cerebral cortical cell preparations of rat primary E15-E18 neurons versus rat primary E15-E18 astrocytes. Finally, to assess the importance of dissecting human brain gray matter from subjacent white matter in cerebral cortical studies, miRNA expression profiles were compared between gray matter and immediately contiguous white matter. The results suggest that for microarray studies, cellular composition is important, and dissecting white matter from gray matter improves the specificity of the results. Based on these data, recommendations for miRNA expression profiling in neural tissues, and considerations worthy of further study, are discussed.

Macromolecule biosynthesis: a key function of sleep.

The function(s) of sleep remains a major unanswered question in biology. We assessed changes in gene expression in the mouse cerebral cortex and hypothalamus following different durations of sleep and periods of sleep deprivation. There were significant differences in gene expression between behavioral states; we identified 3,988 genes in the cerebral cortex and 823 genes in the hypothalamus with altered expression patterns between sleep and sleep deprivation. Changes in the steady-state level of transcripts for various genes are remarkably common during sleep, as 2,090 genes in the cerebral cortex and 409 genes in the hypothalamus were defined as sleep specific and changed (increased or decreased) their expression during sleep. The largest categories of overrepresented genes increasing expression with sleep were those involved in biosynthesis and transport. In both the cerebral cortex and hypothalamus, during sleep there was upregulation of multiple genes encoding various enzymes involved in cholesterol synthesis, as well as proteins for lipid transport. There was also upregulation during sleep of genes involved in synthesis of proteins, heme, and maintenance of vesicle pools, as well as antioxidant enzymes and genes encoding proteins of energy-regulating pathways. We postulate that during sleep there is a rebuilding of multiple key cellular components in preparation for subsequent wakefulness.

Form-deprivation myopia in chick induces limited changes in retinal gene expression.

PURPOSE: Evidence has implicated the retina as a principal controller of refractive development. In the present study, the retinal transcriptome was analyzed to identify alterations in gene expression and potential signaling pathways involved in form-deprivation myopia of the chick. METHODS: One-week-old white Leghorn chicks wore a unilateral image-degrading goggle for 6 hours or 3 days (n = 6 at each time). Total RNA from the retina/(retinal pigment epithelium) was used for expression profiling with chicken gene microarrays (Chicken GeneChips; Affymetrix, Santa Clara, CA). To identify gene expression level differences between goggled and contralateral nongoggled eyes, normalized microarray signal intensities were analyzed by the significance analysis of microarrays (SAM) approach. Differentially expressed genes were validated by real-time quantitative reverse transcription-polymerase chain reaction (qPCR) in independent biological replicates. RESULTS: Small changes were detected in differentially expressed genes in form-deprived eyes. In chickens that had 6 hours of goggle wear, downregulation of bone morphogenetic protein 2 and connective tissue growth factor was validated. In those with 3 days of goggle wear, downregulation of bone morphogenetic protein 2, vasoactive intestinal peptide, preopro-urotensin II-related peptide and mitogen-activated protein kinase phosphatase 2 was validated, and upregulation of endothelin receptor type B and interleukin-18 was validated. CONCLUSIONS: Form-deprivation myopia, in its early stages, is associated with only minimal changes in retinal gene expression at the level of the transcriptome. While the list of validated genes is short, each merits further study for potential involvement in the signaling cascade mediating myopia development.

Onset of human preterm and term birth is related to unique inflammatory transcriptome profiles at the maternal fetal interface.

BACKGROUND: Preterm birth is a main determinant of neonatal mortality and morbidity and a major contributor to the overall mortality and burden of disease. However, research of the preterm birth is hindered by the imprecise definition of the clinical phenotype and complexity of the molecular phenotype due to multiple pregnancy tissue types and molecular processes that may contribute to the preterm birth. Here we comprehensively evaluate the mRNA transcriptome that characterizes preterm and term labor in tissues comprising the pregnancy using precisely phenotyped samples. The four complementary phenotypes together provide comprehensive insight into preterm and term parturition. METHODS: Samples of maternal blood, chorion, amnion, placenta, decidua, fetal blood, and myometrium from the uterine fundus and lower segment (n = 183) were obtained during cesarean delivery from women with four complementary phenotypes: delivering preterm with (PL) and without labor (PNL), term with (TL) and without labor (TNL). Enrolled were 35 pregnant women with four precisely and prospectively defined phenotypes: PL (n = 8), PNL (n = 10), TL (n = 7) and TNL (n = 10). Gene expression data were analyzed using shrunken centroid analysis to identify a minimal set of genes that uniquely characterizes each of the four phenotypes. Expression profiles of 73 genes and non-coding RNA sequences uniquely identified each of the four phenotypes. The shrunken centroid analysis and 10 times 10-fold cross-validation was also used to minimize false positive finings and overfitting. Identified were the pathways and molecular processes associated with and the cis-regulatory elements in gene's 5' promoter or 3'-UTR regions of the set of genes which expression uniquely characterized the four phenotypes. RESULTS: The largest differences in gene expression among the four groups occurred at maternal fetal interface in decidua, chorion and amnion. The gene expression profiles showed suppression of chemokines expression in TNL, withdrawal of this suppression in TL, activation of multiple pathways of inflammation in PL, and an immune rejection profile in PNL. The genes constituting expression signatures showed over-representation of three putative regulatory elements in their 5'and 3' UTR regions. CONCLUSIONS: The results suggest that pregnancy is maintained by downregulation of chemokines at the maternal-fetal interface. Withdrawal of this downregulation results in the term birth and its overriding by the activation of multiple pathways of the immune system in the preterm birth. Complications of the pregnancy associated with impairment of placental function, which necessitated premature delivery of the fetus in the absence of labor, show gene expression patterns associated with immune rejection.

MiRNA-9 and MiRNA-200a distinguish hemangioblastomas from metastatic clear cell renal cell carcinomas in the CNS.

Central nervous system (CNS) tumors in von Hippel-Lindau syndrome (VHL) include hemangioblastomas and metastatic clear cell renal cell carcinomas (Met CCRCC). While these tumors often show similar histologic features, differentiating them is of significant importance as Met CCRCC are higher-grade tumors with worse prognosis. No single current immunohistochemical marker unequivocally differentiates between these two entities. MicroRNAs (miRNAs) are noncoding cellular small RNA molecules that play an important role in cancer. We hypothesized that hemangioblastomas and Met CCRCC display distinct miRNA signatures enabling their histologic differentiation. MiRNAs were profiled in 10 cases each of hemangioblastomas, Met CCRCC and primary CCRCC. Ten miRNAs had greater abundance (including miR-9 (∼10-fold) and miR-135a (∼7-fold)) and 39 miRNAs were lower [including miR-200a (∼22-fold) and miR-200b (∼12-fold)] in hemangioblastomas compared with Met CCRCC. Quantitative real-time RT-PCR in 20 hemangioblastomas and 13 Met CCRCC showed a 12-fold increase in miR-9 and a 15-fold decrease of miR-200a in hemangioblastomas compared with Met CCRCC. Finally, in situ hybridization for miR-9 in 15 hemangioblastomas and 10 Met CCRCC confirmed these results. Our data suggest that miR-9 and miR-200a can distinguish between hemangioblastomas and Met CCRCC. Further, these results may also provide insight in understanding the biology of hemangioblastomas.

Image defocus and altered retinal gene expression in chick: clues to the pathogenesis of ametropia.

PURPOSE: Because of the retina's role in refractive development, this study was conducted to analyze the retinal transcriptome in chicks wearing a spectacle lens, a well-established means of inducing refractive errors, to identify gene expression alterations and to develop novel mechanistic hypotheses about refractive development. METHODS: One-week-old white Leghorn chicks wore a unilateral spectacle lens of +15 or -15 D for 6 hours or 3 days. With total RNA from the retina/(retinal pigment epithelium, RPE), chicken gene microarrays were used to compare gene expression levels between lens-wearing and contralateral control eyes (n = 6 chicks for each condition). Normalized microarray signal intensities were evaluated by analysis of variance, using a false discovery rate of <10% as the statistical criterion. Selected differentially expressed genes were validated by qPCR. RESULTS: Very few retina/RPE transcripts were differentially expressed after plus lens wear. In contrast, approximately 1300 transcripts were differentially expressed under each of the minus lens conditions, with minimal overlap. For each condition, low fold-changes typified the altered transcriptome. Differentially regulated genes under the minus lens conditions included many potentially informative signaling molecules and genes whose protein products have roles in intrinsic retinal circadian rhythms. CONCLUSIONS: Plus or minus lens wear induce markedly different, not opposite, alterations in retina/RPE gene expression. The initial retinal responses to defocus are quite different from those when the eye growth patterns are well established, suggesting that different mechanisms govern the initiation and persistence or progression of refractive errors. The gene lists identify promising signaling candidates and regulatory pathways for future study, including a potential role for circadian rhythms in refractive development.

Diagnostic microRNAs in myelodysplastic syndrome.

OBJECTIVE: The myelodysplastic syndromes (MDS) are aging-associated disorders characterized by ineffective maturation of hematopoietic elements, which are often diagnostically challenging. This study identifies microRNAs (miRNA) and miRNA targets that might represent diagnostic markers for MDS. MATERIALS AND METHODS: This study utilized a total of 42 MDS samples and 45 controls. A discovery set of 20 frozen bone marrow mononuclear cell samples (10 MDS, 10 controls) was profiled on a custom Agilent miRNA microarray. Classifier miRNAs were validated in a separate set of 49 paraffin-embedded particle preparations by real-time polymerase chain reaction (24 MDS, 25 controls). Target prediction analysis was compared to a de novo transcriptional profile of MDS derived from the Microarray Innovations in Leukemia study. c-Myb and Sufu were further investigated by immunohistochemical stains on a set of 26 paraffin-embedded samples. RESULTS: We identified 13 miRNAs of interest from the discovery set, 8 of which proved statistically significant on real-time polymerase chain reaction verification. These eight miRNAs were then examined in an independent real-time polymerase chain reaction validation set. Notably, hsa-miR-378, hsa-miR-632, and hsa-miR-636 demonstrated particularly high discrimination between MDS and normal controls. Target prediction identified potential targets of miRNA regulation that correspond to many of the genes that characterize MDS. Immunohistochemical staining performed on a third validation set confirmed that c-Myb and Sufu are differentially expressed in MDS. CONCLUSIONS: Our data utilize both discovery and validation sets and two complementary platforms to identify miRNAs associated with MDS. We have analyzed predicted targets and identified c-Myb and Sufu as potential diagnostic markers of MDS.

Comparison between NuGEN's WT-Ovation Pico and one-direct amplification systems.

Differential gene expression between groups of homogenous cell types is a biological question whose time has come. RNA can be extracted from small numbers of cells, such as those isolated by laser-capture microdissection, but the small amounts obtained often require amplification to enable whole genome transcriptome profiling by technologies such as microarray analysis and RNA-seq. Recently, advances in amplification procedures make amplification directly from whole cell lysates possible. The aim of this study was to compare two amplification systems for variations in observed RNA abundance attributable to the amplification procedure for use with small quantities of cells isolated by laser-capture microdissection. Arabidopsis root cells undergoing giant cell formation as a result of nematode infestation and uninfested control root cells were laser-captured and used to evaluate two amplification systems. One, NuGEN's WT-Ovation Pico (Pico) amplification system, uses total RNA as starting material, and the other, NuGEN's WT-One-Direct (One-Direct) amplification system, uses lysate containing the captured cells. The reproducibility of whole genome transcript profiling and correlations of both systems were investigated after microarray analysis. The One-Direct system was less reproducible and more variable than the Pico system. The Pico amplification kit resulted in the detection of thousands of differentially expressed genes between giant cells and control cells. This is in marked contrast to the relatively few genes detected after amplification with the One-Direct amplification kit.

Tough beginnings: alterations in the transcriptome of cloned embryos during the first two cell cycles.

Cloned embryos produced by somatic cell nuclear transfer (SCNT) display a plethora of phenotypic characteristics that make them different from fertilized embryos, indicating defects in the process of nuclear reprogramming by the recipient ooplasm. To elucidate the extent and timing of nuclear reprogramming, we used microarrays to analyze the transcriptome of mouse SCNT embryos during the first two cell cycles. We identified a large number of genes mis-expressed in SCNT embryos. We found that genes involved in transcription and regulation of transcription are prominent among affected genes, and thus may be particularly difficult to reprogram, and these likely cause a ripple effect that alters the transcriptome of many other functions, including oxidative phosphorylation, transport across membrane, and mRNA transport and processing. Interestingly, we also uncovered widespread alterations in the maternal (i.e., non-transcribed) mRNA population of SCNT embryos. We conclude that gene expression in early SCNT embryos is grossly abnormal, and that this is at least in part the result of incomplete reprogramming of transcription factor genes.

Cooperativity of paired oligonucleotide probes for microarray hybridization assays.

Synthetic DNA probes attached to microarrays usually range in length from 25 to 70 nucleotides. There is a compromise between short probes with lower sensitivity, which can be accurately synthesized in higher yields, and long probes with greater sensitivity but lower synthesis yields. Described here are microarrays printed with spots containing a mixture of two short probes, each designed to hybridize at noncontiguous sites in the same targeted sequence. We have shown that, for a printed microarray, mixed probe spots containing a pair of 30mers show significantly greater hybridization than spots containing a single 30mer and can approach the amount of hybridization to spots containing a 60mer or a 70mer. These spots with mixed oligonucleotide probes display cooperative hybridization signals greater than those that can be achieved by either probe alone. Both the higher synthesis yields of short probes and the greater sensitivity of long oligonucleotides can be utilized. This strategy provides new design options for microarray hybridization assays to detect RNA abundance, RNA splice variants, or sequence polymorphisms.