Identification of Genes Associated with Resistance and Response in Vivo to Therapy with Rituximab, Fludarabine and Cyclophosphamide in Patients with Chronic Lymphocytic Leukemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1622-1622 ◽  
Author(s):  
Medhat Shehata ◽  
Dita Demirtas ◽  
Stefanie Tauber ◽  
Susanne Schnabl ◽  
Martin Bilban ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Signature ◽  
Poor Response ◽  
Response To Therapy ◽  
Lymphocytic Leukemia ◽  
Pcr Analysis ◽  
Poor Responders ◽  
Rt Pcr ◽  
Number Of Patients

Abstract Fludarabine and cyclophosphamide are the backbone of therapy for patients with CLL. The addition of rituximab leads to further improvement of response rates and progression free survival (results from the randomized CLL8 study of the GCLLSG). However, a considerable number of patients have insufficient or short responses to FC or RFC and there is a need to identify factors influencing response or resistance to therapy. The aims of this study are to identify gene expression associated with response or resistance before the start of therapy, to investigate changes in the expression of specific genes or pathways associated with response or resistance during the first cycle of FC and RFC, to provide a rationale for the additional use of novel drugs to improve remission and overcome resistance. We investigated peripheral blood samples from 20 patients receiving FC (n=10) or RFC (n=10) by gene expression profiling, flow cytometry, RT-PCR and western blotting before and during therapy. Sixteen patients received FC or RFC as first line (8 within the CLL8 study) and 4 as second line treatment. All patients were in stage Binet B or C. Gene expression was analyzed and correlated to good (CR or PR) or poor clinical response (SD or PD) at the end of therapy based on NCI-WG/IWCLL criteria. CD19+ cells were harvested by cell sorting before therapy, 24 hours after FC (FC arm), 24 hours after rituximab, and 24 hours after FC (RFC arm). Microarray analysis was performed using Affymetrix U133A gene chips. Genes with a consistent pattern of expression (high or low) in the majority of samples in the good or poor response group were further analyzed. Overall, 9 patients responded adequately to therapy (3 CR, 7 PR), while 11 did not (7 SD, 3PD). Unmutated IgVH status and poor risk cytogenetics were more frequent in poor responders. Gene expression signature before treatment showed that overexpression of 39 genes strongly correlated with response, while overexpression of 20 genes (including HSPA1B, IFI6, APP, CEACAM1, CD9, GAB1, INPP5F) was associated with resistance. Changes in expression after initiation of treatment was also analyzed. Seven genes (including CENTD1, HBA2, COL9A2 and APRIN) were significantly upregulated after rituximab in non-responders. Upregulation of 13 genes (including PMAIP1, SFRS11, CLK1, EFHC1, MRPL39, TUG1, TBRG1, CD49d, PTPRC) after R-FC and 7 genes (including ITPKB, LOC641298, CD44, TAF5) after FC was associated with poor response (resistance) to RFC and FC respectively. Many of these genes are involved in regulation of apoptosis, cell cycle, integrin and PI3-K signaling Therapeutic antibodies or inhibitors against some of these targets are already available. RT-PCR analysis demonstrated a significant downregulation of Akt1 mRNA 24 hours after rituximab infusion in RFC group but no significant changes were observed in patients receiving FC alone. In vitro exposure to rituximab confirmed its in vivo effect and resulted in a significant downregulation of Akt1 and PI3-K-p85 mRNA expression. FACS analysis demonstrated a decrease in the percentage and mean fluorescence intensity (MFI) of surface CD20 after rituximab infusion. This effect was associated with a significant change in total amount and phosphorylation state of CD20 in the RFC group. There was also a decrease in the MFI of CD44 and CD23 after rituximab in the majority of patients in the RFC group but this effect was not consistent in the FC group. In conclusion, we have identified a set of markers associated with good or poor response to FC or RFC before therapy and during the first cycle of treatment. The data provide a rationale for targeted drug combinations to overcome resistance and improve response to therapy in CLL.

scholarly journals The Gonococcal Fur-Regulated tbpA and tbpB Genes Are Expressed during Natural Mucosal Gonococcal Infection

2005 ◽  
Vol 73 (7) ◽  
pp. 4281-4287 ◽  
Author(s):  
Sarika Agarwal ◽  
Carol A. King ◽  
Ellen K. Klein ◽  
David E. Soper ◽  
Peter A. Rice ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Iron Concentration ◽  
Pcr Analysis ◽  
Positive Trend ◽  
Gonococcal Infection ◽  
Rt Pcr ◽  
Male Subjects

ABSTRACT Iron is limiting in the human host, and bacterial pathogens respond to this environment by regulating gene expression through the ferric uptake regulator protein (Fur). In vitro studies have demonstrated that Neisseria gonorrhoeae controls the expression of several critical genes through an iron- and Fur-mediated mechanism. While most in vitro experiments are designed to determine the response of N. gonorrhoeae to an exogenous iron concentration of zero, these organisms are unlikely to be exposed to such severe limitations of iron in vivo. To determine if N. gonorrhoeae expresses iron- and Fur-regulated genes in vivo during uncomplicated gonococcal infection, we examined gene expression profiles of specimens obtained from male subjects with urethral infections. RNA was isolated from urethral swab specimens and used as a template to amplify, by reverse transcriptase PCR (RT-PCR), gonococcal genes known to be regulated by iron and Fur (tbpA, tbpB, and fur). The constitutively expressed gonococcal rmp gene was used as a positive control. RT-PCR analysis indicated that gonorrhea-positive specimens where rmp expression was seen were also 93% (51/55) fbpA positive, 87% (48/55) tbpA positive, and 86% (14 of 16 tested) tbpB positive. In addition, we detected a fur transcript in 79% (37 of 47 tested) of positive specimens. We also measured increases in levels of immunoglobulin G antibody against TbpA (91%) and TbpB (73%) antigens in sera from infected male subjects compared to those in uninfected controls. A positive trend between tbpA gene expression and TbpA antibody levels in sera indicated a relationship between levels of gene expression and immune response in male subjects infected with gonorrhea for the first time. These results indicate that gonococcal iron- and Fur-regulated tbpA and tbpB genes are expressed in gonococcal infection and that male subjects with mucosal gonococcal infections exhibit antibodies to these proteins.

Overexpression of the Receptor for Hyaluronan-Mediated Motility (RHAMM) Characterizes the Malignant Clone in Multiple Myeloma: Identification of Three Distinct RHAMM Variants

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1684-1696 ◽  
Author(s):  
Mary Crainie ◽  
Andrew R. Belch ◽  
Michael J. Mant ◽  
Linda M. Pilarski
Keyword(s):  
Multiple Myeloma ◽  
Crohn’S Disease ◽  
B Cells ◽  
Crohn's Disease ◽  
Plasma Cells ◽  
Lymphocytic Leukemia ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Activated B Cells

The receptor for hyaluronan (HA)-mediated motility (RHAMM) controls motility by malignant cells in myeloma and is abnormally expressed on the surface of most malignant B and plasma cells in blood or bone marrow (BM) of patients with multiple myeloma (MM). RHAMM cDNA was cloned and sequenced from the malignant B and plasma cells comprising the myeloma B lineage hierarchy. Three distinct RHAMM gene products, RHAMMFL, RHAMM−48, and RHAMM−147, were cloned from MM B and plasma cells. RHAMMFL was 99% homologous to the published sequence of RHAMM. RHAMM−48 and RHAMM−147 variants align with RHAMMFL, but are characterized by sequence deletions of 48 bp (16 amino acids [aa]) and 147 bp (49 aa), respectively. The relative frequency of these RHAMM transcripts in MM plasma cells was determined by cloning of reverse-transcriptase polymerase chain reaction (RT-PCR) products amplified from MM plasma cells. Of 115 randomly picked clones, 49% were RHAMMFL, 47% were RHAMM−48, and 4% were RHAMM−147. All of the detected RHAMM variants contain exon 4, which is alternatively spliced in murine RHAMM, and had only a single copy of the exon 8 repeat sequence detected in murine RHAMM. RT-PCR analysis of sorted blood or BM cells from 22 MM patients showed that overexpression of RHAMM variants is characteristic of MM B cells and BM plasma cells in all patients tested. RHAMM also appeared to be overexpressed in B lymphoma and B-chronic lymphocytic leukemia (CLL) cells. In B cells from normal donors, RHAMMFL was only weakly detectable in resting B cells from five of eight normal donors or in chronically activated B cells from three patients with Crohn’s disease. RHAMM−48 was detectable in B cells from one of eight normal donors, but was undetectable in B cells of three donors with Crohn’s disease. RHAMM−147 was undetectable in normal and Crohn’s disease B cells. In situ RT-PCR was used to determine the number of individual cells with aggregate RHAMM transcripts. For six patients, 29% of BM plasma cells and 12% of MM B cells had detectable RHAMM transcripts, while for five normal donors, only 1.2% of B cells expressed RHAMM transcripts. This work suggests that RHAMMFL, RHAMM−48, and RHAMM−147 splice variants are overexpressed in MM and other B lymphocyte malignancies relative to resting or in vivo–activated B cells, raising the possibility that RHAMM and its variants may contribute to the malignant process in B-cell malignancies such as lymphoma, CLL, and MM.

Overexpression of the Receptor for Hyaluronan-Mediated Motility (RHAMM) Characterizes the Malignant Clone in Multiple Myeloma: Identification of Three Distinct RHAMM Variants

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1684-1696 ◽  
Author(s):  
Mary Crainie ◽  
Andrew R. Belch ◽  
Michael J. Mant ◽  
Linda M. Pilarski
Keyword(s):  
Multiple Myeloma ◽  
Crohn’S Disease ◽  
B Cells ◽  
Crohn's Disease ◽  
Plasma Cells ◽  
Lymphocytic Leukemia ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Activated B Cells

Abstract The receptor for hyaluronan (HA)-mediated motility (RHAMM) controls motility by malignant cells in myeloma and is abnormally expressed on the surface of most malignant B and plasma cells in blood or bone marrow (BM) of patients with multiple myeloma (MM). RHAMM cDNA was cloned and sequenced from the malignant B and plasma cells comprising the myeloma B lineage hierarchy. Three distinct RHAMM gene products, RHAMMFL, RHAMM−48, and RHAMM−147, were cloned from MM B and plasma cells. RHAMMFL was 99% homologous to the published sequence of RHAMM. RHAMM−48 and RHAMM−147 variants align with RHAMMFL, but are characterized by sequence deletions of 48 bp (16 amino acids [aa]) and 147 bp (49 aa), respectively. The relative frequency of these RHAMM transcripts in MM plasma cells was determined by cloning of reverse-transcriptase polymerase chain reaction (RT-PCR) products amplified from MM plasma cells. Of 115 randomly picked clones, 49% were RHAMMFL, 47% were RHAMM−48, and 4% were RHAMM−147. All of the detected RHAMM variants contain exon 4, which is alternatively spliced in murine RHAMM, and had only a single copy of the exon 8 repeat sequence detected in murine RHAMM. RT-PCR analysis of sorted blood or BM cells from 22 MM patients showed that overexpression of RHAMM variants is characteristic of MM B cells and BM plasma cells in all patients tested. RHAMM also appeared to be overexpressed in B lymphoma and B-chronic lymphocytic leukemia (CLL) cells. In B cells from normal donors, RHAMMFL was only weakly detectable in resting B cells from five of eight normal donors or in chronically activated B cells from three patients with Crohn’s disease. RHAMM−48 was detectable in B cells from one of eight normal donors, but was undetectable in B cells of three donors with Crohn’s disease. RHAMM−147 was undetectable in normal and Crohn’s disease B cells. In situ RT-PCR was used to determine the number of individual cells with aggregate RHAMM transcripts. For six patients, 29% of BM plasma cells and 12% of MM B cells had detectable RHAMM transcripts, while for five normal donors, only 1.2% of B cells expressed RHAMM transcripts. This work suggests that RHAMMFL, RHAMM−48, and RHAMM−147 splice variants are overexpressed in MM and other B lymphocyte malignancies relative to resting or in vivo–activated B cells, raising the possibility that RHAMM and its variants may contribute to the malignant process in B-cell malignancies such as lymphoma, CLL, and MM.

scholarly journals Relation of Gene Expression Phenotype to Immunoglobulin Mutation Genotype in B Cell Chronic Lymphocytic Leukemia

2001 ◽  
Vol 194 (11) ◽  
pp. 1639-1648 ◽  
Author(s):  
Andreas Rosenwald ◽  
Ash A. Alizadeh ◽  
George Widhopf ◽  
Richard Simon ◽  
R. Eric Davis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Germ Line ◽  
Gene Expression Signature ◽  
Lymphocytic Leukemia ◽  
Common Mechanism ◽  
Human Leukemia ◽  
Mutational Status ◽  
Cell Chronic Lymphocytic Leukemia

The most common human leukemia is B cell chronic lymphocytic leukemia (CLL), a malignancy of mature B cells with a characteristic clinical presentation but a variable clinical course. The rearranged immunoglobulin (Ig) genes of CLL cells may be either germ-line in sequence or somatically mutated. Lack of Ig mutations defined a distinctly worse prognostic group of CLL patients raising the possibility that CLL comprises two distinct diseases. Using genomic-scale gene expression profiling, we show that CLL is characterized by a common gene expression “signature,” irrespective of Ig mutational status, suggesting that CLL cases share a common mechanism of transformation and/or cell of origin. Nonetheless, the expression of hundreds of other genes correlated with the Ig mutational status, including many genes that are modulated in expression during mitogenic B cell receptor signaling. These genes were used to build a CLL subtype predictor that may help in the clinical classification of patients with this disease.

Cloning of rabbit prolactin cDNA and prolactin gene expression in the rabbit mammary gland

1996 ◽  
Vol 16 (1) ◽  
pp. 27-37 ◽  
Author(s):  
L Gabou ◽  
M Boisnard ◽  
I Gourdou ◽  
H Jammes ◽  
J-P Dulor ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Amino Acid ◽  
Untranslated Regions ◽  
Pcr Analysis ◽  
Cdna Clones ◽  
Rt Pcr ◽  
Prolactin Gene ◽  
New Zealand White Rabbits ◽  
Rna Probe

ABSTRACT cDNA clones coding for rabbit prolactin were isolated from a pituitary library using a rat prolactin RNA probe. One cDNA contained 873 bases including the entire coding sequence of rabbit prolactin, its signal peptide and the 5′ and 3′ untranslated regions of 44 and 145 nucleotides respectively. The deduced amino acid sequence of the cloned prolactin cDNA presented a 93–78% identity with mink, porcine and human prolactins. The prolactin gene transcription was investigated by RT-PCR analysis in several organs of midlactating New Zealand White rabbits. The ectopic transcription of the prolactin gene was examined in more detail in the mammary gland. A strong PCR signal was detected in the mammary gland of virgin does and was also observed during pregnancy and at the beginning of lactation. This PCR signal was very weak in mid-lactating and absent in post-weaning mammary gland.

Fas-Mediated Modulation of Bcr/Abl in Chronic Myelogenous Leukemia Results in Differential Effects on Apoptosis

Blood ◽  
1998 ◽  
Vol 92 (3) ◽  
pp. 981-989 ◽  
Author(s):  
Carmine Selleri ◽  
Jaroslaw P. Maciejewski ◽  
Fabrizio Pane ◽  
Luigia Luciano ◽  
Anna Maria Raiola ◽  
...  
Keyword(s):  
Chronic Myelogenous Leukemia ◽  
Chronic Phase ◽  
Poor Response ◽  
Inhibitory Response ◽  
Myelogenous Leukemia ◽  
Poor Responders ◽  
Cd34 Cells ◽  
In Cells

Abstract Fas-R is expressed constitutively in CD34+ cells of patients with chronic myelogenous leukemia (CML); Fas-R triggering results in decreased proliferation rate due to apoptosis of clonogenic cells. We have already shown that α-interferon (IFN-α) enhances Fas-R expression on CML progenitor cells, thus increasing their sensitivity to Fas-R agonists. Although it appears that IFN-α can prime CML cells for the effects of Fas, the response to IFN-α in vivo is not a constant feature in CML patients. We studied the mechanisms of Fas-mediated apoptosis in 11 patients suffering from CML in chronic phase and tried to see whether there was a correlation between in vitro inducibility of apoptosis in CD34+ CML cells after Fas-R triggering and the clinical response to IFN-α. After priming with IFN-α, Fas triggering resulted in in vitro suppression of hematopoietic cell growth in seven of eight patients who had optimal hematologic response to IFN-α; in the same conditions, no inhibitory response to Fas-R agonist was observed in cells from three of three patients who proved to be poor responders to IFN-α. In responders to IFN-α, Fas-R agonist induced dose-dependent apoptosis of CD34+ cells; this effect was associated with a decrease in the bcr/abl protein level. In cells derived from patients with a poor response to IFN-α, the rate of apoptosis in culture remained unchanged in the presence of Fas-R agonist and nobcr/abl downmodulation was observed. Finally, we measuredbcr/abl mRNA by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and found that decreased bcr/ablprotein after Fas triggering was not associated with decreased amounts of specific mRNA, a finding which is consistent with a posttranscriptional regulation of the bcr/abl protein expression. It appears that Fas-mediated downmodulation of p210bcr/abl restores susceptibility to apoptosis of CML cells; in addition, in vitro studies on CML cells may predict response to IFN-α treatment. © 1998 by The American Society of Hematology.

scholarly journals Gene Expression Signature-Based Approach Identifies Antifungal Drug Ciclopirox As a Novel Inhibitor of HMGA2 in Colorectal Cancer

Biomolecules ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 688 ◽  
Author(s):  
Yu-Min Huang ◽  
Chia-Hsiung Cheng ◽  
Shiow-Lin Pan ◽  
Pei-Ming Yang ◽  
Ding-Yen Lin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Colorectal Cancer ◽  
Cell Cycle ◽  
Therapeutic Potential ◽  
Specific Inhibitor ◽  
Gene Expression Signature ◽  
Hsp90 Inhibitor ◽  
Response To Therapy ◽  
Mesenchymal Transition ◽  
Potential Inhibitor

Human high-mobility group A2 (HMGA2) encodes for a non-histone chromatin protein which influences a variety of biological processes, including the cell cycle process, apoptosis, the DNA damage repair process, and epithelial–mesenchymal transition. The accumulated evidence suggests that high expression of HMGA2 is related to tumor progression, poor prognosis, and a poor response to therapy. Thus, HMGA2 is an important molecular target for many types of malignancies. Our recent studies revealed the positive connections between heat shock protein 90 (Hsp90) and HMGA2 and that the Hsp90 inhibitor has therapeutic potential to inhibit HMGA2-triggered tumorigenesis. However, 43% of patients suffered visual disturbances in a phase I trial of the second-generation Hsp90 inhibitor, NVP-AUY922. To identify a specific inhibitor to target HMGA2, the Gene Expression Omnibus (GEO) database and the Library of Integrated Network-based Cellular Signatures (LINCS) L1000platform were both analyzed. We identified the approved small-molecule antifungal agent ciclopirox (CPX) as a novel potential inhibitor of HMGA2. In addition, CPX induces cytotoxicity of colorectal cancer (CRC) cells by induction of cell cycle arrest and apoptosis in vitro and in vivo through direct interaction with the AT-hook motif (a small DNA-binding protein motif) of HMGA2. In conclusion, this study is the first to report that CPX is a novel potential inhibitor of HMGA2 using a drug-repurposing approach, which can provide a potential therapeutic intervention in CRC patients.

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