Joanna Szczepanek

Relapse of Acute Lymphoblastic Leukemia in Children in the Context of Microarray Analyses

Over the last four decades the treatment of patients with newly diagnosed childhood acute lymphoblastic leukemia (ALL) has improved remarkably. However, still about 20% of children with ALL relapse despite risk-adapted polychemotherapy. The prognosis of relapsed ALL is relatively poor, even with modern aggressive chemotherapy. Identification of the biological and genetic mechanisms contributing to recurrence in patients with ALL is critical for the development of effective therapeutic strategies to treat refractory leukemic patients. Allogeneic hematopoietic stem-cell transplantation is the treatment of choice for many children with relapsed ALL. The gene expression profile obtained by microarray technology could provide important determinants of the drug response and clinical outcome in childhood ALL. Incorporation of the data on expression levels of newly identified genes into existing strategies of risk stratification might improve clinical management. Current microarray data show correlation of in vitro drug resistance with significant patterns of gene expression and explain clinical differences between early and late relapse. Genes involved in cell proliferation, self-renewal and differentiation, protein biosynthesis, carbohydrate metabolism, and DNA replication and repair are usually among those highly expressed in relapsed lymphoblasts. Current status and future perspectives of microarray data on gene expression and drug resistance profile in relapsed pediatric ALL are discussed in this review.

Analysis of Relative Expression Level of VEGF (Vascular Endothelial Growth Factor), HIF-1α (Hypoxia Inducible Factor 1α) and CTGF (Connective Tissue Growth Factor) Genes in Chronic Glomerulonephritis (CGN) Patients

Background/Aims: Analysis of gene expression in renal tissue is considered to be a diagnostic tool predicting the clinical course of glomerulonephritis. The present study quantified the relative transcript levels of VEGF, CTGF and HIF-1α in renal tissue to establish their relationship with some clinical variables in patients suffering from chronic glomerulonephritis (CGN). Methods: 28 patients (6F and 22M, mean age 51.2±15.0) with CGN were enrolled. Type of CNG recognized by kidney biopsy (histopatological evaluation) was as follows: minimal change disease (MCD)-3pts, IgA nephropathy-5pts, FSGS-3pts, membranous nephropathy-4pts, mesangio-proliferative glomerulonephritis-3pts; MPGN-1pts, lupus nephritis-6pts, granulomatosis with polyangitis-2 pts; hypertensive nephropathy- 3pts. Renal tissue from 3 individuals with normal eGFR and histology was taken as control. Mean clinical follow-up of patients was 12 months after biopsy eGFR and daily urinary protein excretion (DPE) was assessed at the time of biopsy and then in 6 months intervals. Real-time PCR was used to determine relative gene expression. The housekeeping gene GAPDH was used as normalization control. Results: At the time of the biopsy relative expression of 3 analyzed genes was diminished in comparison to control. There were statistically significant differences in VEGF gene relative expression level in patients which varied according to eGFR and tendency in patients which varied according to DPE. HIF-alfa and CTGF gene showed only a tendency. Conclusions: Overexpression of the VEGF gene in subjects with DPE>3,5g may point to insufficient oxygen supply in renal tissue which may result in tubulointerstitial fibrosis with further functional renal impairment and decline of eGFR.

Gene expression signatures and ex vivo drug sensitivity profiles in children with acute lymphoblastic leukemia

IntroductionCauses of treatment failure in acute lymphoblastic leukemia (ALL) are still poorly understood. Microarray technology gives new possibilities for the analysis of the biology of leukemias. We hypothesize that drug sensitivity in pediatric ALL is driven by specific molecular mechanisms that correlate with gene expression profiles assessed by microarray analysis.ObjectiveThe aim of the study was to determine the ex vivo resistance profiles of 20 antileukemic drugs and gene expression profiles, with relation to response to initial therapy.Patients and methodsLymphoblasts were analyzed after bone marrow biopsy was obtained from 56 patients. The profile of in vitro resistance to drugs was determined in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) cytotoxicity assay. High-quality total RNA was prepared and hybridized to oligonucleotide arrays HG-U133A 2.0 Chip (Affymetrix). The expression of selected genes was tested by qualitative reverse transcription polymerase chain reaction (qRT-PCR).Results and conclusionsThe exposure of leukemic blasts to drugs initiates a complex cellular response, which reflects global changes in gene expression. Changes in the expression of several genes are highly correlated with drug resistance.

The expression patterns of plasma membrane aquaporins in leaves of sugar beet and its halophyte relative, Beta vulgaris ssp. maritima, in response to salt stress

Abstract Salt tolerance is largely dependent on a plant’s ability to maintain optimal water status in leaves. The adjustment of water relations under salinity involves changes in the transcriptional activity of genes encoding plasma membrane aquaporins (PIPs). Here, we report the effects of long-term or short-term treatments with moderate or strong salt stress on the expression of BvPIP1;1, BvPIP2;1 and BvPIP2;2 in the leaves of sugar beet, Beta vulgaris cv. Huzar, and its halophyte relative, Beta vulgaris ssp. maritima. Plants subjected to long-term treatment were watered with salt-supplemented media during a 32 day long culture period. Short-term salt treatments were executed either by immersing the petioles of excised leaves into salt solutions for 48h, or incubating excised leaf blades in salt-supplemented media for 20h. B. vulgaris ssp. maritima reacted to long-term salt treatment with a decrease in BvPIP1;1, BvPIP2;1 and BvPIP2;2 expression. Contrastingly, only BvPIP2;2 transcript was down-regulated by salinity in leaves of B. vulgaris cv. Huzar, whereas BvPIP1;1 and BvPIP2;1 did not vary in response to salt-treatments. On the other hand, the expression of BvPIP1;1, BvPIP2;1 and BvPIP2;2 was enhanced by salinity if salt solutions was supplied through leaf petioles, irrespective of genotype. PIP expression in excised leaf blades revealed a complex pattern of changes. BvPIP1;1 and BvPIP2;1 expression underwent a period of transient increase in both the control and salt-treated leaves. Furthermore, BvPIP1;1 expression was enhanced by strong salinity. BvPIP2;2 expression was up-regulated by strong salinity or up- or down-regulated by moderate salinity during the treatment period.

An analysis of the expression of collagen I and III genes in the fascia of obese patients

BACKGROUND The etiology of incisional hernias in the population of morbidly obese patients remains unclear. Most likely, factors other than purely mechanical are at play; it has been ascertained that nonobese patients suffering from inguinal and incisional hernias display alterations in the architecture of the connective tissue. The goal of this study has been to evaluate and compare the relative expression of collagen type I and III genes in the rectus abdominis muscle sheath (RMS) of obese and nonobese individuals to investigate their possible influence on the quality of the connective tissue. MATERIALS AND METHODS RMS specimens were harvested in the early stages of either bariatric or non-bariatric laparotomies; total RNA was isolated and enzymatically purified from the tissue samples. The resulting material was subjected to a quantitative and qualitative analysis; reverse transcription reactions were then performed and the resulting complementary DNA was used in real-time reverse transcription polymerase chain reactions. The biopsy specimens were also examined by scanning electron microscopy. RESULTS The real-time reverse transcription polymerase chain reactions, performed on complementary DNA, provided specific amplicons for individual genes. The efficacy of the reactions was rather low. An almost twofold decrease of the relative expression level for type I and III collagen was observed between the two patient groups; the results did not reach statistical significance. Scanning electron microscope photographs have documented a marked difference in the ultrastructure of the RMS in both groups. CONCLUSIONS The authors have shown that changes in messenger RNA levels for collagen type I and III genes may be related to the pathogenesis of incisional hernia through alterations in the ultrastructure of the RMS fascia. Our report should be considered preliminary; the results should be verified on a larger group of patients.

Array Comparative Genomic Hybridization in Pediatric Acute Leukemias

Array comparative genomic hybridization has proven to be a very powerful tool in searching for new biomarkers which can find an application in clinical practise. CGH-array technology is satisfying in almost every possible way. It is highly specific, sensitive, simple, and relatively cheap. Thus, this modern method meets the demands of clinical application. An increasing knowledge about molecular pathways and pathologic genome alterations in acute leukemias enable to define unequivocal diagnosis, prognosis and to predict a response to individual compatible therapy. This review shows a various application of CGH-array in pediatric acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL).

New Candidate Genes for Lack of Sensitivity to Therapy in Pediatric Leukemias.

In the recent years, significant development of molecular genetics has contributed to the better understanding of leukemogenesis and classification of the different leukemia subtypes. Patients diagnosed with acute leukemia usually undergo chemotherapy, which involves the induction of remission, consolidation of remission and maintenance therapy. Patients are still vulnerable to relapse due to differences in the sensitivity of leukemia cells to the chemotherapeutic agent, because of the active drugs removal from the cells, improving the repair of DNA damage or abnormalities in the apoptotic pathway. Cell adhesion and miRNA expression level also affect the drug resistance. New candidate genes and genes correlated with the disease have been sought to increase the survival of patients with leukemia. Candidate gene is usually a loci located in a certain chromosomal region suspected of involvement to the disease course or its expression product regulates disease progression. There are classic and digital methods of finding candidate genes. These techniques are inexpensive, quick and easy to carry out. Unfortunately, due to insufficient knowledge of the disease etiology and low accuracy, researchers cannot detect all genes, involved in leukemogenesis and cell resistance. Currently, scientists have many tools used for selecting genes, such as expression microarrays, comparative genomic hybridization and next generation sequencing. Among the validation techniques for candidate genes, the mostly used ones are fluorescent in situ hybridization and real-time PCR. In our review we present 18 candidate genes of resistance to therapy in childhood leukemia.

Genomic and transcriptomic profiles and in vitro resistance to mitoxantrone and idarubicin in pediatric acute leukemias

A major problem in the treatment of leukemia is the development of drug resistance to chemotherapeutic agents.

Identification of the genes expression profile associated with the ex vivo resistance to etoposide in childhood acute leukemias.

INTRODUCTION Drug resistance and the gene expression profiles might discriminate the therapy outcome, and indicate the subgroup of patients with poor prognosis. In this study we analyzed the gene expression profile in correlation with the profile of ex vivo resistance to etoposide in children with acute leukemias. METHODS The ex vivo drug resistance profile was determined by the MTT cytotoxicity assay performed on leukemic blasts of 56 patients. Gene expression profiles were obtained from the results of hybridization of cRNA to Human Genome U133A 2.0 ologonucleotide arrays. The following analyses were performed: correlation analysis, hierarchical clustering, the assignment of location and function. Verification of data for four selected genes (MNDA, GH1, NUDT21, RHOG) was performed by quantitative real time polymerase chain reaction in the studied population and in an independent group of 54 leukemic patients. RESULTS Using the permutation Spearman correlation test, a set of 233 probes/209 genes was selected. The global test confirmed the significance of the correlation of gene expression profile and resistance to etoposide (p<0.001). The NUDT21 (nudix, nucleoside diphosphate linked moiety X-type, motif 21) gene showed the strongest correlation with resistance to etoposide (FDR<0.0001%). CONCLUSIONS Profiling of transcriptome may help in assessing the sensitivity to drugs used in chemotherapy. Resistance to etoposide is possibly associated with a change of expression of a large number of biologically important genes that influence several cellular mechanisms.

Zastosowanie technologii mikromacierzy w onkologii dziecięcej

Streszczenie Nowotwory wystepujące u dzieci ksztaltują wazny i trudny dzial medycyny. Wykazują ogromne zroznicowanie, a ich diagnoza jest czesto utrudniona ze wzgledu na brak specyficznych objawow. Profilowanie ekspresji dzieki technice mikromacierzy jest czesto stosowane w badaniach pediatrycznych nowotworow hematologicznych i guzow litych. W wielu badaniach uzyskano profile ekspresji umozliwiające ich precyzyjną klasyfikacje, monitorowanie przebiegu klinicznego, ocene reakcji na leczenie i w konsekwencji rokowania. Za pomocą profilu genetycznego mozliwa jest identyfikacja nowych podklas w obrebie danego nowotworu dzieciecego, poszukiwanie nowych markerow genetycznych o znaczeniu prognostycznym oraz opracowywanie terapii dostosowanej do pacjenta. W pracy zaprezentowano przegląd najnowszych analiz z zakresu wykorzystania technik monitorowania genomu i transkryptomu komorek nowotworowych w onkologii wieku dzieciecego.