Pancreas-specific deletion of Prox1 affects development and disrupts homeostasis of the exocrine pancreas.

BACKGROUND & AIMS: The exocrine portion of the pancreas functions in digestion and preserves pancreatic homeostasis. Learning how this tissue forms during embryogenesis could improve our understanding of human pancreatic diseases. Expression of the homeobox gene Prox1 in the exocrine pancreas changes throughout development in mice. We investigated the role of Prox1 in development of the exocrine pancreas in mice. METHODS: Mice with pancreas-specific deletion of Prox1 (Prox1(ΔPanc)) were generated and their pancreatic tissues were analyzed using immunohistochemistry, transmission electron microscopy, histologic techniques, quantitative real-time polymerase chain reaction, immunoblotting, and morphometric analysis. RESULTS: Loss of Prox1 from the pancreas led to multiple exocrine alterations, most notably premature acinar cell differentiation, increased ductal cell proliferation, altered duct morphogenesis, and imbalanced expression of claudin proteins. Prox1(ΔPanc) mice also had some minor alterations in islet cells, but beta-cell development was not affected. The exocrine congenital defects of Prox1(ΔPanc) pancreata appeared to initiate a gradual process of deterioration that resulted in extensive loss of acinar cells, lipomatosis, and damage to ductal tissue in adult mice. CONCLUSIONS: Pancreas-specific deletion of Prox1 causes premature differentiation of acinar cells and poor elongation of epithelial branches; these defects indicate that Prox1 controls the expansion of tip progenitors in the early developing pancreas. During later stages of embryogenesis, Prox1 appears to regulate duct cell proliferation and morphogenesis. These findings identify Prox1 as an important regulator of pancreatic exocrine development.

The novel ETS factor TEL2 cooperates with Myc in B lymphomagenesis.

The human ETS family gene TEL2/ETV7 is highly homologous to TEL1/ETV6, a frequent target of chromosome translocations in human leukemia and specific solid tumors. Here we report that TEL2 augments the proliferation and survival of normal mouse B cells and dramatically accelerates lymphoma development in Emu-Myc transgenic mice. Nonetheless, inactivation of the p53 pathway was a hallmark of all TEL2/Emu-Myc lymphomas, indicating that TEL2 expression alone is insufficient to bypass this apoptotic checkpoint. Although TEL2 is infrequently up-regulated in human sporadic Burkitt's lymphoma, analysis of pediatric B-cell acute lymphocytic leukemia (B-ALL) samples showed increased coexpression of TEL2 and MYC and/or MYCN in over one-third of B-ALL patients. Therefore, TEL2 and MYC also appear to cooperate in provoking a cadre of human B-cell malignancies.

Cloning of chimerical translocations as positive control for molecular genetic diagnosis of leukemia.

The diagnosis of leukemia-specific mRNAs by polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) require well-known positive standard controls. In general, the positive controls are obtained from cell lines and leukemia patients who have been diagnosed at the molecular level by RT-PCR. These are expensive and restricted sources for standard positive controls. Thus, there is a need for less expensive and reproducible standard positive controls in this area. We have cloned the t (9: 22) p190, t (9: 22) p210, t (4: 11), t (1: 19), t (15: 17), t (12; 21) breakpoint junctions of fusion genes into the plasmids. Cloned fusion genes are suitable for testing PCR experiments of the molecular genetic diagnosis of leukemia samples. We cloned and optimized fusion gene junctions as a standard positive control to check PCR efficiency and as a standard positive marker for diagnosis.

Minimal Residual Disease (MRD) Detection with Translocations and T-Cell Receptor and Immunoglobulin Gene Rearrangements in Adult Acute Lymphoblastic Leukaemia Patients: A Pilot Study.

Monitoring minimal residual disease has become increasingly important in clinical practice of ALL management. Break-point fusion regions of leukaemia related chromosomal aberrations and rearranged immunoglobulin (Ig) and T cell-receptor (TCR) genes, which can be detected by polymerase chain reaction (PCR), are used as leukaemia specific markers in genetic studies of MRD. A total of 31 consecutive patients with newly diagnosed ALL were screened for eligibility criteria. Of those 26 were included in the study. One patient with partial response following induction therapy and four patients who were lost to follow-up after induction were excluded from the study; thus 21 patients were evaluated for MRD. Chromosomal aberrations were detected in 5 (24%) of the patients and were used for MRD monitoring. Three patients had t(9;22) translocation, the other 2 had t(4;11) and t(1;19). MRD-based risk stratification of the 16 patients analysed for Ig/TCR rearrangements revealed 3 low-risk, 11 intermediate-risk and 2 high-risk patients. MRD monitoring is progressively getting to be a more important predictive factor in adult ALL patients. As reported by others confirmed by our limited data there is a good correlation between MRD status and clinical outcome in patients receiving chemotherapy. The pilot-study presented here is the first that systematically and consecutively performs a molecular MRD monitoring of ALL patients in Turkey.

Preclinical validation of a monochrome real-time multiplex assay for translocations in childhood acute lymphoblastic leukemia.

PURPOSE: The purpose is to develop a real-time multiplex reverse transcription-PCR assay for detection and quantification of leukemia-specific chimeric transcripts that identify the genetic subgroups of acute lymphoblastic leukemias (ALLs) proposed by the WHO classification. EXPERIMENTAL DESIGN: Real-time multiplex assay for t(12;21), t(4;11), and t(1;19) with hypoxanthine phosphoribosyltransferase as internal standard used in tandem with a new real time quantitative-RT-PCR assay for the t(9;22). This new strategy was designed to yield an amplicon from each translocation with a distinct melting peak allowing dependable identification using only Sybr green I, without any need for expensive hybridization probes. RESULTS: We validated this method with 92 primary ALLs and identified 4 E2A-PBX1, 4 mBCR-ABL and 10 TEL-AML1. When compared with conventional RT-PCRs and Southern blot analyses, 100% concordance was obtained. During the course of these studies, we found marked variations in the levels of the TEL-AML1 transcripts in individual patients. We, therefore, extended the study to accurately and reproducibly determine TEL-AML1 mRNA levels in 47 additional patients with t(12;21). The results indicated that the level of expression of TEL-AML1 varied among individual patients, and it was independent of the WBC count. CONCLUSIONS: Our new real-time multiplex assay can be used for rapid, simple, and reliable classification of pediatric ALL. Its reproducible quantification results should also facilitate studies on minimal residual disease. The observed variation in TEL-AML1 transcript levels is of interest because it could reflect biological and/or clinical heterogeneity in the behavior of these leukemias.

Real-Time PCR analysis of af4 and dek genes expression in acute promyelocytic leukemia t (15;17) patients.

Among several newly identified oncogenes, dek and af4 are attractive targets for researchers interested with leukemia. In this study quantitative Real-Time RT-PCR technique was used to define alterations in expression of dek and af4 genes associated with acute promyelocytic leukaemia (APL) t (15; 17). RNA samples obtained from bone marrow aspirates of fourteen APL patients, cDNA portions were labelled with Syber Green 1 dye and LightCycler analysis have been performed. Expression changes in patients were found not significant in comparison to healthy donors for af4 (P = 0.192) and dek (P = 0.0895). We suggest that af4 gene may have a role in leukomogenesis restricted to lymphoblastic lineage; also further studies must carry on with a larger series of patients in order to understand the relationship between the dek gene and APL. Our study was the first attempt for analysing dek and af4 genes in APL t (15; 17) patients by quantitative Real-Time RT-PCR. This rapid and sensitive method could be used to screen these genes in different types of leukaemia.

Real-time PCR analysis of the apoptosis related genes in ATRA treated APL t(15;17) patients.

All-trans retinoic acid (ATRA) treatment of the acute promyelocytic leukemia (APL) have subsequently resulted in cell apoptosis, but the molecular mechanism of this effect remains elusive. In order to understand a possible involvement of genes regulating apoptotic signal pathways, expression levels of bcl2, bax, dapk1, myc, bad, wt1, and mcl genes were analyzed during ATRA treatment in five APL patients with t (15;17) using Real- time PCR (LightCycler). Two samples from each patient were compared to each other: primary diagnostic sample and a sample taken at remission. Effect of the ATRA treatment was demonstrated by the concomitant induction of cd14 and il1beta genes in four patients. Also other apoptosis related genes were found down-regulated in general but especially the down regulated levels of wt1 and bax attract attention. Result suggested that ATRA dependent apoptosis of APL was under the control of both internal and external pathways without relationships to the amount of the blast populations. Ratio of bcl2 to bax may be more important for this regulation than the ratio of bcl2 to bad. Either bcl2 family or less known apoptosis related genes as wt1 will still be required to further studies in this setting.

Quantification of the FLI1 and CXCR4 gene expressions in acute lymphoblastic leukemia (ALL) patients with t(12,21).

The presence of the t(12,21) is associated with good response to therapy in acute lymphoblastic leukemia (ALL) but molecular background of this pathology is not clear. FLI1 gene plays important roles in normal regulation of myeloid hematopoiesis and leukemogenesis. The chemokine receptor CXCR4 gene may play a role in the homing of hematopoietic stem cells. Our aim was to investigate possible relationships between t(12,21) existence and expression changes of these two genes (FLI1, CXCR4) in ALL. Thirty-one ALL patients were investigated. Twenty-one of these patients were t(12,21) carriers. We used the Quantitative Real-Time RT-PCR. Obtained results were compared to normal bone marrow samples of five healthy subjects. Expression differences were not found significant in both groups. Our study was the first attempt to quantify these genes in t(12,21) patients. We conclude that Quantitative RT-PCR is a reliable method for the monitoring of these gene expressions and similar studies should expand to other translocations in haematology.

Quantification of All-Trans-Retinoic Acid (ATRA) Dependent Expression of CXCR4 Gene in Acute Promyelocytic Leukaemia.

CXCR4 is the receptor of CXC chemokine SDF-1 and may play a role in the homing of hematopoietic stem cells. We have investigated the CXCR4 gene expression during ATRA treatment in acute promyelocytic leukaemia (APL) patients. APL is a characteristic disorder with a specific translocation between PML and RAR alpha genes on chromosome 15 and 17. ATRA-induced differentiation of APL cells is strictly dependent on the presence of PML-RAR alpha. In our study, five APL patiens were involved. Two samples from each patient were compared to each other: Primary diagnostic sample and a sample taken at remission. Quantitative real-time PCR (LightCycler) has been used for quantification, which is a recently developed method for rapid and sensitive detection of gene expression. CXCR4 gene ratios were found under-expressed in cases 1 and 6 with blast counts at diagnosis 18%, and 20% but only moderately under-expressed in cases two and four where the blast count was at diagnosis 50%, and 80%. It was over-expressed only in case three where the blast count was 95%. These findings indicate that ATRA treatment might be effective in CXCR4 gene expression related to amount of the blast population in APL. Further gene expression studies would be helpful to understand how ATRA works on CXCR4 related molecular pathways in APL pathogenesis.

Pendrin expression in nodular and non-nodular thyroid tissues.

INTRODUCTION: Different mechanisms for the expression of pendrin which is an apical iodide transporter have been reported in nodular thyroid tissues compared to normal thyroid. The aim of the present study was to determine the alterations of pendrin expression in nodular and surrounding non-nodular thyroid tissues and clarify the role of pendrin in the functional behaviour of nodular lesions. MATERIAL AND METHODS: Twenty-six nodular and paired non-nodular normal thyroid tissues were collected at the same centre. Patients were divided into two groups based on the function of the dominant thyroid nodule; hot nodules (n = 18) and cold nodules (n = 8). mRNA levels of pendrin were evaluated by quantitative RT-PCR. Pendrin protein expression was determined by immunohistochemical analysis. Results of dominant nodules were compared to non-nodular thyroid tissue of the same patient. RESULTS: No statistically significant difference was found with respect to qualitative and quantitative measurements of pendrin expression between hot and cold nodules. However, percent immunohistochemical staining of pendrin was significantly higher in both hot and cold nodules compared to non-nodular thyroid tissue of the same patients. RT-PCR revealed comparable mRNA levels of pendrin gene between hot nodules and corresponding normal thyroid tissues. However, in cold nodules, significantly decreased mRNA levels of pendrin were observed compared to normal thyroid tissue. mRNA levels of pendrin showed significant positive correlation with TSH in corresponding non-nodular thyroid tissues. CONCLUSIONS: The present study demonstrates that expression of pendrin could not be influenced by TSH in thyroid nodules and expression level of pendrin seems not to have an effect on nodule function.

Comparison of apoptotic gene expression profiles between Peyronie's disease plaque and tunica albuginea.

BACKGROUND: The fibrotic plaques of Peyronie/s disease and other localized fibrotic conditions have been considered to be the result of an abnormal wound healing process. The potential role of regulatory disorders of apoptosis in abnormal wound healing may also play a role in the development of Peyronie's disease. OBJECTIVES: To examine the phenomenon of apoptosis in Peyronie's disease, authors quantified differential levels of gene expression of apoptotic proteins, Fas, Fas Ligand, Bcl-2, p53, Caspase 3 and 8 in Peyronie's plaque and tunica albuginea. MATERIAL AND METHODS: Eight patients with Peyronie's disease undergoing surgical correction of the curvature had biopsy specimens taken from both the Peyronie's plaque and normal tunica albuginea. Messenger RNA expression of the apoptotic proteins in the plaque and normal tunica was measured by reverse transcriptase PCR. RESULTS: Apoptotic gene expression was lower than the housekeeping gene's in half of the tunica albuginea samples and two thirds of the plaque samples. Overall mRNA expressions in the plaque were not significantly different from the normal tunica albuginea. CONCLUSIONS: The fibrotic plaques of Peyronie's disease and other localized fibrotic conditions have been considered to be the result of an abnormal wound healing process. The potential role of regulatory disorders of apoptosis in abnormal wound healing may also play a role in the development of Peyronie's disease. In this study, the lower expression of apoptotic genes may cause the persistence of collagen producing cells which were up-regulated for unknown reasons and consequently result in plaque formation. Similar expression levels of apoptotic genes in both tunica albuginea and Peyronie's plaques may be due to the generalized physiopathologic alterations in tunica albuginea that lead to plaque formation at a vulnerable region subjected to recurrent traumas.

A genome-wide analysis of lentivector integration sites using targeted sequence capture and next generation sequencing technology.

One application of next-generation sequencing (NGS) is the targeted resequencing of interested genes which has not been used in viral integration site analysis of gene therapy applications. Here, we combined targeted sequence capture array and next generation sequencing to address the whole genome profiling of viral integration sites. Human 293T and K562 cells were transduced with a HIV-1 derived vector. A custom made DNA probe sets targeted pLVTHM vector used to capture lentiviral vector/human genome junctions. The captured DNA was sequenced using GS FLX platform. Seven thousand four hundred and eighty four human genome sequences flanking the long terminal repeats (LTR) of pLVTHM fragment sequences matched with an identity of at least 98% and minimum 50 bp criteria in both cells. In total, 203 unique integration sites were identified. The integrations in both cell lines were totally distant from the CpG islands and from the transcription start sites and preferentially located in introns. A comparison between the two cell lines showed that the lentiviral-transduced DNA does not have the same preferred regions in the two different cell lines.

Comparison of the cytogenetic and molecular analyses in the assessment of imatinib response in chronic myelocytic leukemia.

We aimed to compare the cytogenetic and molecular analyses in the assessment of imatinib mesylate response in patients suffering the chronic phase of chronic myelocytic leukemia who were refractory to alpha-interferon treatment. A total of 117 patients in the chronic phase of chronic myelocytic leukemia were included. The patients were treated with 400 mg/day imatinib mesylate. Bone marrow samples were obtained for the cytogenetic and molecular analyses. Patients without the Ph chromosome were defined as complete cytogenetic responders. Partial cytogenetic response was determined when the Ph chromosome was detected in 1-35% of the cells. Molecular response was determined by quantitative real-time reverse transcriptase polymerase chain reaction (QR-PCR) and defined as no detection of BCR-ABL mRNA. The frequencies of complete and partial cytogenetic response were 29% (n = 34) and 15% (n = 18), respectively. No cytogenetic response was achieved in 56% (n = 65) of the patients. Molecular response was achieved in 62% (n = 21) and 33% (n = 6) of the complete and partial cytogenetic responders, respectively. All of the 65 patients with no cytogenetic response were also molecular nonresponders. We conclude that there is reasonable agreement between the cytogenetic and molecular analyses. Both methods are complementary in the assessment of response to therapy.

Prognostic significance of the TEL-AML1 fusion gene in pediatric acute lymphoblastic leukemia in Turkey.

PURPOSE: The t(12;21) translocation is the most common reciprocal chromosomal rearrangement in pediatric acute lymphoblastic leukemia (ALL). This translocation fuses two genes, TEL and AML1, and results in the production of the TEL-AML1 fusion protein. The authors investigated the incidence and prognostic significance of the TEL-AML1 fusion gene in patients with ALL in Turkey. METHODS: The authors analyzed 219 children with ALL using the reverse transcription-polymerase chain reaction. RESULTS: The TEL-AML1 fusion transcript was detected in 20.1% (44/219) of newly diagnosed children with ALL. -positive patients had precursor B-cell ALL and were 3 to 10 years old at diagnosis. -positive patients had a significantly lower rate of relapse compared with -negative patients. -positive patients have a higher overall survival rate than -negative patients. CONCLUSIONS: These data support that the presence of at diagnosis is an independent favorable prognostic indicator in patients with ALL in Turkey.

NAD(P)H:quinone oxidoreductase 1 null genotype is not associated with pediatric de novo acute leukemia.

BACKGROUND: NAD(P)H:quinone oxidoreductase1 (NQO1) is a two-electron reductase that detoxifies quinones derived from the oxidation of phenolic metabolites of benzene. Exposure to benzene metabolites increases the risk of hematotoxicity and leukemia. NQO1 enzyme activity protects the cells against metabolites of benzene. C to T base substitution at nucleotide 609 of NQO1 cDNA (C609T) results in loss of enzyme activity. Low NQO1 activity may play a role in etiology of acute leukemia. PROCEDURE: We analyzed NQO1 C609T gene polymorphism using the PCR-RFLP method in 273 patients with de novo acute leukemia (189 acute lymphoblastic leukemia (ALL), and 84 acute myeloid leukemia (AML) and 286 healthy volunteers to investigate the role of NQO1 polymorphism in the etiology of acute leukemia. RESULTS AND CONCLUSIONS: The frequency of homozygosity for NOQ1 C609T polymorphism was 3.5% in the healthy control population and 2.5% in pediatric acute leukemia. The NQO1 C609T allele frequency was not statistically different in the children with acute leukemia in comparison to the controls (odds ratio (OR), 0.76; 95% confidence interval (CI), 0.58-1.01; P = 0.06). The distribution of NQO1 genotypes among children with acute leukemia was not statistically different from the control group (P = 0.13). These findings do not support the role of NQO1 C609T polymorphism in the etiology of de novo pediatric acute leukemia.