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Purpose: MicroRNAs (miRNA) play pivotal oncogenic and tumor-suppressor roles in several human cancers. We sought to discover novel tumor-suppressor miRNAs in gastric cancer (GC).Experimental Design: Using Agilent miRNA microarrays, we compared miRNA expression profiles of 40 primary gastric tumors and 40 gastric normal tissues, identifying miRNAs significantly downregulated in gastric tumors.Results: Among the top 80 miRNAs differentially expressed between gastric tumors and normals (false discovery rate < 0.01), we identified hsa-miR-486 (miR-486) as a significantly downregulated miRNA in primary GCs and GC cell lines. Restoration of miR-486 expression in GC cell lines (YCC3, SCH and AGS) caused suppression of several pro-oncogenic traits, whereas conversely inhibiting miR-486 expression in YCC6 GC cells enhanced cellular proliferation. Array-CGH analysis of 106 primary GCs revealed genomic loss of the miR-486 locus in approximately 25% to 30% of GCs, including two tumors with focal genomic losses specifically deleting miR-486, consistent with miR-486 playing a tumor-suppressive role. Bioinformatic analysis identified the secreted antiapoptotic glycoprotein OLFM4 as a potential miR-486 target. Restoring miR-486 expression in GC cells decreased endogenous OLFM4 transcript and protein levels, and also inhibited expression of luciferase reporters containing an OLFM4 3′ untranslated region with predicted miR-486 binding sites. Supporting the biological relevance of OLFM4 as a miR-486 target, proliferation in GC cells was also significantly reduced by OLFM4 silencing.Conclusions:miR-486 may function as a novel tumor-suppressor miRNA in GC. Its antioncogenic activity may involve the direct targeting and inhibition of OLFM4. Clin Cancer Res; 17(9); 2657–67. ©2011 AACR.

Supplementary Figures S1-S4; Supplementary Table S1.

Q fever is a major public health problem in French Guiana. In recent years, a considerable number of cases has been reported in French Guiana’s penitentiary center. The main objective of this study was to describe the epidemiology of these cases. A retrospective study was conducted at the prison to identify cases of acute Q fever in people incarcerated between 2010 and 2021. During the study period, 16 patients were diagnosed with acute Q fever. The positivity rate varied between 13 and 57%. The annual incidence rate in 2019, 2020 and 2021 was 269 (95% CI: 0-640) 1,120 (95% CI: 290-1950) and 1,931 (95% CI: 60-3810) per 100,000 person-years, respectively. While several vertebrate species have already been shown to play an important role in the transmission of Coxiella burnetii, the full epidemiology picture in the tropics is far from clear, and the prison context, with its controlled environment, could help provide answers.

G protein-coupled receptors (GPCRs) and their ligands have been implicated in progression and metastasis of several cancers. GPCRs signal through heterotrimeric G proteins, and among the different types of G proteins, GNA12/13 have been most closely linked to tumor progression. In this study, we explored the role of GNA13 in prostate cancer cell invasion and the mechanism of up-regulation of GNA13 in these cells. An initial screen for GNA13 protein expression showed that GNA13 is highly expressed in the most aggressive cancer cell lines. Knockdown of GNA13 in highly invasive PC3 cells revealed that these cells depend on GNA13 expression for their invasion, migration, and Rho activation. As mRNA levels in these cells did not correlate with protein levels, we assessed the potential involvement of micro-RNAs (miRNAs) in post-transcriptional control of GNA13 expression. Expression analysis of miRNAs predicted to bind the 3'-UTR of GNA13 revealed that miR-182 and miR-141/200a showed an inverse correlation to the protein expression in LnCAP and PC3 cells. Ectopic expression of miR-182 and miR-141/200a in PC3 cells significantly reduced protein levels, GNA13-3'-UTR reporter activity and in vitro invasion of these cells. This effect was blocked by restoration of GNA13 expression in these cells. Importantly, inhibition of miR-182 and miR-141/200a in LnCAP cells using specific miRNA inhibitors elevated the expression of GNA13 and enhanced invasion of these cells. These data provide strong evidence that GNA13 is an important mediator of prostate cancer cell invasion, and that miR-182 and miR-200 family members regulate its expression post-transcriptionally.

MicroRNA–mRNA interactions are commonly validated and deconstructed in cell lines transfected with luciferase reporters. However, due to cell type-specific variations in microRNA or RNA-binding protein abundance, such assays may not reliably reflect microRNA activity in other cell types that are less easily transfected. In order to measure miRNA activity in primary cells, we constructed miR-Sens, a MSCV-based retroviral vector that encodes both a Renilla luciferase reporter gene controlled by microRNA binding sites in its 3′ UTR and a Firefly luciferase normalization gene. miR-Sens sensors can be efficiently transduced in primary cells such as human fibroblasts and mammary epithelial cells, and allow the detection of overexpressed and, more importantly, endogenous microRNAs. Notably, we find that the relative luciferase activity is correlated to the miRNA expression, allowing quantitative measurement of microRNA activity. We have subsequently validated the miR-Sens 3′ UTR vectors with known human miRNA-372, miRNA-373, and miRNA-31 targets (LATS2 and TXNIP). Overall, we observe that miR-Sens-based assays are highly reproducible, allowing detection of the independent contribution of multiple microRNAs to 3′ UTR–mediated translational control of LATS2. In conclusion, miR-Sens is a new tool for the efficient study of microRNA activity in primary cells or panels of cell lines. This vector will not only be useful for studies on microRNA biology, but also more broadly on other factors influencing the translation of mRNAs.

Serial quantitation of BCR-ABL mRNA levels is an important indicator of therapeutic response for patients with chronic myelogenous leukemia and Philadelphia chromosome–positive acute lymphoblastic leukemia, but there is substantial variation in the real-time quantitative polymerase chain reaction methodologies used by different testing laboratories. To help improve the comparability of results between centers we sought to develop accredited reference reagents that are directly linked to the BCR-ABL international scale. After assessment of candidate cell lines, a reference material panel comprising 4 different dilution levels of freeze-dried preparations of K562 cells diluted in HL60 cells was prepared. After performance evaluation, the materials were assigned fixed percent BCR-ABL/control gene values according to the International Scale. A recommendation that the 4 materials be established as the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL translocation by real-time quantitative polymerase chain reaction was approved by the Expert Committee on Biological Standardization of the World Health Organization in November 2009. We consider that the development of these reagents is a significant milestone in the standardization of this clinically important test, but because they are a limited resource we suggest that their availability is restricted to manufacturers of secondary reference materials.

Protein kinases are involved in a wide variety of physiological and pathological processes. Small kinase inhibitor molecules are frequently used to dissect signaling pathways or to counteract oncogenic events. However, in many cases the precise role of a given inhibitor is not well understood. Besides a known primary target, potential secondary targets might be involved in the biological response to the drug. We describe recent advances in chemical genetics and chemical proteomics that allow a new comprehensive analysis of kinase signaling pathways. One approach consists of engineering the kinase pocket to design inhibitor sensitive and resistant alleles. A second strategy is to modify the kinase pocket to specifically accept radio-labeled ATP analogs, allowing the identification of direct downstream substrates after transphosphorylation. The third method takes advantage of recently improved inhibitor affinity chromatography to identify inhibitor targets by mass spectrometry. Ultimately, these new technologies will help define the actions of kinase inhibitors useful for human disease treatment.

Real-time quantitative RT-PCR (RQ-PCR) is a sensitive tool to monitor minimal residual disease (MRD) in leukemic patients through the amplification of a fusion gene (FG) transcript. In order to correct variations in RNA quality and quantity and to calculate the sensitivity of each measurement, a control gene (CG) transcript should be amplified in parallel to the FG transcript. To identify suitable CGs, a study group within the Europe Against Cancer (EAC) program initially focused on 14 potential CGs using a standardized RQ-PCR protocol. Based on the absence of pseudogenes and the level and stability of the CG expression, three genes were finally selected: Abelson (ABL), beta-2-microglobulin (B2M), and beta-glucuronidase (GUS). A multicenter prospective study on normal (n=126) and diagnostic leukemic (n=184) samples processed the same day has established reference values for the CG expression. A multicenter retrospective study on over 250 acute and chronic leukemia samples obtained at diagnosis and with an identified FG transcript confirmed that the three CGs had a stable expression in the different types of samples. However, only ABL gene transcript expression did not differ significantly between normal and leukemic samples at diagnosis. We therefore propose to use the ABL gene as CG for RQ-PCR-based diagnosis and MRD detection in leukemic patients. Overall, these data are not only eligible for quantification of fusion gene transcripts, but also for the quantification of aberrantly expressed genes.

Purpose: MicroRNAs (miRNA) play pivotal oncogenic and tumor-suppressor roles in several human cancers. We sought to discover novel tumor-suppressor miRNAs in gastric cancer (GC). Experimental Design: Using Agilent miRNA microarrays, we compared miRNA expression profiles of 40 primary gastric tumors and 40 gastric normal tissues, identifying miRNAs significantly downregulated in gastric tumors. Results: Among the top 80 miRNAs differentially expressed between gastric tumors and normals (false discovery rate < 0.01), we identified hsa-miR-486 (miR-486) as a significantly downregulated miRNA in primary GCs and GC cell lines. Restoration of miR-486 expression in GC cell lines (YCC3, SCH and AGS) caused suppression of several pro-oncogenic traits, whereas conversely inhibiting miR-486 expression in YCC6 GC cells enhanced cellular proliferation. Array-CGH analysis of 106 primary GCs revealed genomic loss of the miR-486 locus in approximately 25% to 30% of GCs, including two tumors with focal genomic losses specifically deleting miR-486, consistent with miR-486 playing a tumor-suppressive role. Bioinformatic analysis identified the secreted antiapoptotic glycoprotein OLFM4 as a potential miR-486 target. Restoring miR-486 expression in GC cells decreased endogenous OLFM4 transcript and protein levels, and also inhibited expression of luciferase reporters containing an OLFM4 3′ untranslated region with predicted miR-486 binding sites. Supporting the biological relevance of OLFM4 as a miR-486 target, proliferation in GC cells was also significantly reduced by OLFM4 silencing. Conclusions: miR-486 may function as a novel tumor-suppressor miRNA in GC. Its antioncogenic activity may involve the direct targeting and inhibition of OLFM4. Clin Cancer Res; 17(9); 2657–67. ©2011 AACR.

Conventional cytogenetic analysis currently stratifies acute myelogenous leukaemia (AML) into prognostically relevant groups. However, approximately 50% of adult AMLs have normal cytogenetics (NC-AMLs), and represent a heterogeneous and poorly understood group. We analysed gene expression in 55 AML samples including 53 cases from adult patients with NC-AML (n = 36), trisomy 8, t(15;17), t(8;21), t(11;19), 7q deletion, and two cell lines using 9000-gene DNA microarrays. Global hierarchical clustering showed that NC-AMLs are a heterogeneous group. Supervised analysis distinguished two subgroups of NC-AML: one subgroup constituted a homogeneous NC cluster ('pure NC-AML'), and the other NC-AMLs were close to the AML cases with translocations ('translocation like'). Gene expression signatures were also derived for patients with trisomy 8, as well as FLT3 and MLL gene duplications. Importantly, samples from 24 NC-AML patients who could be evaluated for clinical outcome were analysed. In all, 43 genes that discriminated two classes of patients with significantly different prognosis were identified. The poor prognosis class contained a majority of 'pure NC-AMLs', whereas the 'translocation-like' AMLs were in the good prognosis class. Discriminator genes included genes involved in drug resistance (TOP2B), protein transport (MTX2, SLC35A2), and cell signalling (MAPK1, PRKAB2). Our results demonstrate the transcriptional heterogeneity of NC-AMLs, and suggest the existence of 'translocation-like' NC-AMLs and of a gene expression signature that may predict response to chemotherapy.

Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemias such as childhood acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and acute promyelocytc leukemia. This report focuses on the accurate quantitative measurement of fusion gene (FG) transcripts as can be applied in 35-45% of ALL and acute myeloid leukemia, and in more than 90% of CML. A total of 26 European university laboratories from 10 countries have collaborated to establish a standardized protocol for TaqMan-based real-time quantitative PCR (RQ-PCR) analysis of the main leukemia-associated FGs within the Europe Against Cancer EAC) program. Four phases were scheduled: (1) training, (2) optimization, (3) sensitivity testing and (4) patient sample testing. During our program, three quality control rounds on a large series of coded RNA samples were performed including a balanced randomized assay, which enabled final validation of the EAC primer and probe sets. The expression level of the nine major FG transcripts in a large series of stored diagnostic leukemia samples (n = 278) was evaluated. After normalization, no statistically significant difference in expression level was observed between bone marrow and peripheral blood on paired samples at diagnosis. However, RQ-PCR revealed marked differences in FG expression between transcripts in leukemic samples at diagnosis that could account for differential assay sensitivity. The development of standardized protocols for RQ-PCR analysis of FG transcripts provides a milestone for molecular determination of MRD levels. This is likely to prove invaluable to the management of patients entered into multicenter therapeutic trials.

Heterotrimeric guanine nucleotide binding proteins (G proteins) transmit a variety of extracellular signals from cell surface G protein-coupled receptors (GPCRs) to intracellular effector molecules. Heterotrimeric G proteins are classified according to the α subunit into four subfamilies: Gs, Gi, Gq, and G12. The G12 subfamily, which is comprised of two members, Gα12 (GNA12) and Gα13 (GNA13), has been implicated in cancer cell invasion and metastasis. G12 signaling promotes prostate, breast and ovarian cancer cell invasion in vitro, and these proteins are highly expressed in metastatic cancer tissues. As part of a program to elucidate the mechanisms by which GNA12 and GNA13 expression is upregulated in cancer cells, we assessed the potential involvement of micro-RNAs (miRNAs) in post-transcriptional control of GNA13 expression. The initial focus was on prostate cancer; LnCAP (with the lowest GNA13 protein level) and PC3 (with the highest GNA13 level) were employed as the model system. Expression analysis of miRNAs predicted to bind the 3′UTR of GNA13 revealed that miR-182 and miR-141/200a showed an inverse correlation to the protein expression in LnCAP and PC3 cells. Ectopic expression of miR-182 and miR-141/200a in PC3 cells significantly reduced mRNA and protein levels, as well as GNA13 3′UTR- reporter activity. Further, expression of these miRNAs significantly inhibited PC3 cell invasion in an in vitro matrigel invasion assay, and this effect was blocked by overexpression of GNA13 in these cells. Importantly, inhibition of miR-182 and miR-141/200a in LnCAP cells using specific miRNA inhibitors (anti-miRs) elevated the expression of GNA13 and enhanced the basal invasion of these cells. These data provide strong evidence that GNA13 expression is regulated by post-transcriptional mechanisms involving miR-182 and miR-200 family members. Further studies are being performed to identify the role of these miRNAs in cancer progression and to determine the importance of impact on GNA13 expression as an important mediator of this effect. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-278. doi:1538-7445.AM2012-LB-278

Mutations in the kinase domain of BCR-ABL result in impaired drug binding and are thought to be the leading cause of acquired resistance to the tyrosine kinase inhibitor imatinib (Gleevec®). While imatinib is a highly effective therapy in all stages of chronic myelogenous leukemia (CML), relapse after an initial response is common in patients with advanced disease. The T315I point mutation is one of the most common imatinib-resistant mutations and patients with this mutation are also resistant to two second generation tyrosine kinase inhibitors, dasatinib and nilotinib. Recently, the U.S. FDA approved dasatinib for treatment of imatinib-resistant, Philadelphia chromosome-positive acute and chronic leukemias. Thus, appropriate detection of this mutation is essential to optimal management of patients with imatinib resistance and may be useful for clinical trials of agents that target patients with the T315I mutation. Current methods for mutation detection, such as direct DNA sequencing, are not sensitive enough for detection of point mutations at low levels of BCR-ABL transcript. Conversely, ultrasensitive detection methods such as allele specific PCR (AS-PCR) may be too sensitive and can be plagued by false positive test results. In addition the clinical relevance/significance of mutation detection at ultra sensitive levels ( < 1% mutant) is questionable and not yet known. We developed a novel T315I mutation detection assay, using Fluorescent Resonance Energy Transfer (FRET)-based hybridization probes and melting curve analysis. BCR-ABL amplicons generated from a first round of PCR are amplified using primers flanking the ABL kinase region encoding for codon 315. The resultant amplicon is hybridized with fluorescein-labelled anchor probe and a LC Red 640-labelled T315I mutation specific probe. Wild-type and T315I mutant amplicons are distinguished by melting curve analysis (Roche Light Cycler 480™). Using a series of plasmid and cell line dilutions we determined that the sensitivity of this assay for detection of T315I mutations was 5–10%. Using patient-derived samples we were able to successfully genotype samples containing as few as 20–50 BCR-ABL transcripts. To date, the assay sensitivity and specificity are 100%. The assay is performed in a plate based format (96 or 384 wells) and commercially available software allows automated genotype assignment. This approach is suitable for high-throughput detection of T315I mutations for clinical management of CML patients and/or screening of patients to determine eligibility for clinical studies.

Introduction: Major Molecular Response (MMR) is defined as a three-log reduction from a standardized baseline of BCR-ABL/control gene transcript ratio in CML patients at diagnosis. MMR has prognostic significance for progression-free survival for patients on Imatinib® therapy. Day-to-day monitoring of the MMR value in clinical laboratories is challenging due to the absence of a commercially available standardized MMR control RNA. To improve the reliability of BCR-ABL quantitation, MolecularMD has evaluated the feasibility of a single MMR control RNA valid for blood samples drawn in EDTA or PAXgene™ tubes. Material and Methods: Patient sample RNAs were interchanged between our laboratory and an International Randomized Interferon versus STI571 study (IRIS) laboratory, which had established an MMR value and international scale reporting. This exchange enabled our laboratory to establish an MMR value and reporting on an international scale using a validated conversion factor. A serial dilution of a BCR-ABL positive cell line into a human BCR-ABL negative cell line was prepared. These dilutions were tested in IRIS laboratories with established MMR value and international scale reporting and at our laboratory by QRT-PCR to determine the BCR-ABL/control gene ratio using respectively BCR and ABL control genes. We compared the BCR-ABL/ABL ratio in 104 paired PB CML patient samples drawn either in EDTA and PAXgene tubes and the BCR-ABL/BCR ratio in 32 patient samples. Stability studies were performed to evaluate the degradation of liquid and dried forms of the MMR RNA. Results: We established a conversion factor (CF) of 0.81 with an MMR value of 0.123%. Using this CF and MMR value, we created appropriate RNA dilutions that matched the MMR value using ABL as a control gene. Repeated analyzes of this MMR control RNA confirmed the accuracy of the sample with a median value of 0.124%, very close to the MMR value defined previously (0.123%). Stability studies demonstrated that the dried RNA samples could be stored several days at 37°C and freeze-thawed up-to 10 times without significant degradation. These RNA samples once reconstituted with water could also be used several times for BCR-ABL monitoring without any significant degradation. Comparison of BCR-ABL/ABL ratio between EDTA and PAXgene tubes revealed differences unlikely to have clinical impact on disease management suggesting that the MMR RNA created would be suitable under both EDTA and PAXgene extraction methodologies. Conclusions: We produced a stable MMR control RNA in large quantity for accurate monitoring of the MMR value. This MMR control RNA is now be tested in several laboratories to confirm the stability and reliability of this reagent. The MMR control RNA will be an important tool for standardizing MMR value in laboratories, and an integral part of a BCR-ABL QRT-PCR diagnostic kit.

Background Gα13 (GNA13) is the α subunit of a heterotrimeric G protein that mediates signaling through specific G protein-coupled receptors (GPCRs). Our recent study showed that control of GNA13 expression by specific microRNAs (miRNAs or miRs) is important for prostate cancer cell invasion. However, little is known about the control of GNA13 expression in breast cancers. This project was carried out to determine (i) whether enhanced GNA13 expression is important for breast cancer cell invasion, and (ii) if so, the mechanism of deregulation of GNA13 expression in breast cancers. Methods To determine the probable miRNAs regulating GNA13, online miRNA target prediction tool Targetscan and Luciferase assays with GNA13-3′-UTR were used. Effect of miRNAs on GNA13 mRNA, protein and invasion was studied using RT-PCR, western blotting and in vitro Boyden chamber assay respectively. Cell proliferation was done using MTT assays. Results Overexpression of GNA13 in MCF-10a cells induced invasion, whereas knockdown of GNA13 expression in MDA-MB-231 cells inhibited invasion. Expression analysis of miRNAs predicted to bind the 3′-UTR of GNA13 revealed that miR-31 exhibited an inverse correlation to GNA13 protein expression in breast cancer cells. Ectopic expression of miR-31 in MDA-MB-231 cells significantly reduced GNA13 mRNA and protein levels, as well as GNA13-3′-UTR-reporter activity. Conversely, blocking miR-31 activity in MCF-10a cells induced GNA13 mRNA, protein and 3′-UTR reporter activity. Further, expression of miR-31 significantly inhibited MDA-MB-231 cell invasion, and this effect was partly rescued by ectopic expression of GNA13 in these cells. Examination of 48 human breast cancer tissues revealed that GNA13 mRNA levels were inversely correlated to miR-31 levels. Conclusions These data provide strong evidence that GNA13 expression in breast cancer cells is regulated by post-transcriptional mechanisms involving miR-31. Additionally our data shows that miR-31 regulates breast cancer cell invasion partially via targeting GNA13 expression in breast cancer cells. Loss of miR-31 expression and increased GNA13 expression could be used as biomarkers of breast cancer progression. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0337-x) contains supplementary material, which is available to authorized users.