Agnieszka Janus

Università Vita-Salute San Raffaele, Milano, Lombardy, Italy

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Publications (23)89.08 Total impact

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    ABSTRACT: European Scientific Journal Background Many prognostic factors have been identified in chronic lymphocytic leukemia (CLL) but new ones are still desired. The biological characterization of CLL is now being translated into novel treatment strategies. One new prognostic factor, and therapeutic target, may be BFL1. It is both a serum and a molecular marker that contributes to the progression of CLL and its resistance to chemotherapy. The aim of this study was to evaluate the prognostic value of BFL1 and to assess its correlation with other known prognostic markers in CLL for the cladribine and cyclophosphamide regimen (CC). Methods qPCR TaqMan® Low Density Array was used for gene expression measurements. Assessment of CD38, ZAP70 and BFL-1 proteins expression was done by means of flow cytometry. Serum TK activity was measured by immunoassay. Results Protein BFL1 expression was found to be significantly higher in CLL patients than healthy volunteers (p=0.001). Moreover its level was significantly higher in patients with no response (NR) to CC therapy (p=0.009). The expression of BFL1 was considerably down regulated during CC treatment and BFL1 mRNA levels were inversely correlated with apoptotic response. In addition, protein BFL1 expression was found to be similar to thymidine kinase (TK) concentration regarding treatment response. As far as other markers are concerned, a positive correlation was identified between BFL1 and TK (r=0.52, p=0.01). Conclusions Our findings suggest that BFL1 contributes to chemoresistance and may be a co-existing prognostic factor in CLL in the future.
    Full-text · Article · Sep 2015
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    ABSTRACT: The differences in clinical course of chronic lymphocytic leukemia could have an impact on variations in a patient's response to therapy. Our published results revealed that thermal transition (95 ± 5°C) in differential scanning calorimetry profiles appear to be characteristic for the advanced stage of CLL. Moreover, a decrease/loss of this transition in nuclei from leukemic cells exposed to drugs ex vivo could indicate their diverse efficacy. It seems that the lack of changes in thermal profile could predict patient's drug resistance. In this study, we demonstrate the results obtained after drug treatment of leukemic cells by calorimetry, apoptosis-related parameters involved in expression of genes using cDNA microarray and western blot. These data were compared with the patients' clinical parameters before and after RCC therapy (rituximab + cladribine + cyclophosphamide). The complementary analysis of studied cases with opposite clinical response (CR or NR) revealed a strong relationship between clinical data, differences in thermal scans and apoptosis-related gene expression. We quantified expression of eight of apoptosis-related 89 genes, i.e., NOXA, PUMA, APAF1, ESRRBL1, CASP3, BCL2, BCL2A1 and MCL1. Particular differences in NOXA and BCL2 expression were revealed. NOXA expression in cells of patients who achieved a complete response to RCC therapy was 0.44 times higher in comparison to control ones. Interestingly, in the case of patients who did not respond to immunotherapy, NOXA expression was highly downregulated (RQ = 4.39) as compared with untreated cells. These results were confirmed by distinct cell viability, protein expression as well as by differences in calorimetry profiles.
    Full-text · Article · Oct 2012 · Cancer biology & therapy
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    ABSTRACT: The study was aimed to investigate modifications of apoptotic gene expression profile by microarray technique in 10 patients with chronic lymphocytic leukemia by treatment with rituximab, cladribine and cyclophosphamide (RCC) according to IGHV mutational status. The TaqMan Low Density Array for 96 gene transcripts was used. Those modifications followed two distinctive patterns largely overlapping the IGHV mutational status. In the IGHV-mutated group, the expression of many proapoptotic genes increased after treatment as compared to initial value. Our results suggest that RCC drugs may act through influence on the expression of some apoptosis-involved genes dependently on the IGVH mutational status.
    No preview · Article · May 2012 · Leukemia research

  • No preview · Article · Mar 2012 · Blood
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    ABSTRACT: Mounting evidence indicates that grouping of chronic lymphocytic leukemia (CLL) into distinct subsets with stereotyped BCRs is functionally and prognostically relevant. However, several issues need revisiting, including the criteria for identification of BCR stereotypy and its actual frequency as well as the identification of "CLL-biased" features in BCR Ig stereotypes. To this end, we examined 7596 Ig VH (IGHV-IGHD-IGHJ) sequences from 7424 CLL patients, 3 times the size of the largest published series, with an updated version of our purpose-built clustering algorithm. We document that CLL may be subdivided into 2 distinct categories: one with stereotyped and the other with nonstereotyped BCRs, at an approximate ratio of 1:2, and provide evidence suggesting a different ontogeny for these 2 categories. We also show that subset-defining sequence patterns in CLL differ from those underlying BCR stereotypy in other B-cell malignancies. Notably, 19 major subsets contained from 20 to 213 sequences each, collectively accounting for 943 sequences or one-eighth of the cohort. Hence, this compartmentalized examination of VH sequences may pave the way toward a molecular classification of CLL with implications for targeted therapeutic interventions, applicable to a significant number of patients assigned to the same subset.
    Full-text · Article · Mar 2012 · Blood
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    ABSTRACT: Leukemia is one of the leading journals in hematology and oncology. It is published monthly and covers all aspects of the research and treatment of leukemia and allied diseases. Studies of normal hemopoiesis are covered because of their comparative relevance.
    No preview · Article · Nov 2011 · Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K
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    ABSTRACT: Malignant B lymphocytes from chronic lymphocytic leukemia (CLL) patients maintain the capacity to respond to CD40 ligation, among other microenvironmental stimuli. In this study, we show that (i) leukemic CLL cells stimulated with the soluble form of CD40L in vitro show differential responses in terms of upregulation of surface markers (CD95 and CD80) and induction of chemokines (CCL22 and CCL17) expression/secretion, and that (ii) these changes are mirrored by a distinct activation of intracellular signalling pathways including increase in IKKalpha/beta phosphorylation and upregulation of antiapoptotic proteins (BCL-2 and MCL-1). CLL patients can then be segregated into two distinct functional subsets. We defined the responsive subset of cases CD40L dependent, considering the capacity to respond as a sign of persistent need of this stimulation for the leukemic expansion. Conversely, we named the unresponsive cases CD40L independent, considering them less dependent on this microenvironmental signal, presumably because of a higher autonomous proliferative and survival potential. Importantly, we report that (iii) the two functional subsets show an opposite clinical outcome, with CD40L-independent cases having a shorter time to progression. This indicates that the functional differences observed in vitro may reflect a different leukemic potential in vivo likely responsible for a distinct clinical course.
    No preview · Article · Nov 2011 · Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K
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    ABSTRACT: Monoclonal B-cell lymphocytosis (MBL) is classified as chronic lymphocytic leukemia (CLL)-like, atypical CLL, and CD5(-) MBL. The number of B cells per microliter divides CLL-like MBL into MBL associated with lymphocytosis (usually detected in a clinical setting) and low-count MBL detected in the general population (usually identified during population screening). After a median follow-up of 34 months we reevaluated 76 low-count MBLs with 5-color flow cytometry: 90% of CLL-like MBL but only 44.4% atypical CLL and 66.7% CD5(-) MBL persisted over time. Population-screening CLL-like MBL had no relevant cell count change, and none developed an overt leukemia. In 50% of the cases FISH showed CLL-related chromosomal abnormalities, including monoallelic or biallelic 13q deletions (43.8%), trisomy 12 (1 case), and 17p deletions (2 cases). The analysis of the T-cell receptor β (TRBV) chains repertoire showed the presence of monoclonal T-cell clones, especially among CD4(high)CD8(low), CD8(high)CD4(low) T cells. TRBV2 and TRBV8 were the most frequently expressed genes. This study indicates that (1) the risk of progression into CLL for low-count population-screening CLL-like MBL is exceedingly rare and definitely lower than that of clinical MBL and (2) chromosomal abnormalities occur early in the natural history and are possibly associated with the appearance of the typical phenotype.
    Full-text · Article · Aug 2011 · Blood
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    ABSTRACT: Malignant B lymphocytes from chronic lymphocytic leukemia (CLL) patients maintain the capacity to respond to CD40 ligation, among other microenvironmental stimuli. In this study, we show that (i) leukemic CLL cells stimulated with the soluble form of CD40L in vitro show differential responses in terms of upregulation of surface markers (CD95 and CD80) and induction of chemokines (CCL22 and CCL17) expression/secretion, and that (ii) these changes are mirrored by a distinct activation of intracellular signalling pathways including increase in IKKalpha/beta phosphorylation and upregulation of antiapoptotic proteins (BCL-2 and MCL-1). CLL patients can then be segregated into two distinct functional subsets. We defined the responsive subset of cases CD40L dependent, considering the capacity to respond as a sign of persistent need of this stimulation for the leukemic expansion. Conversely, we named the unresponsive cases CD40L independent, considering them less dependent on this microenvironmental signal, presumably because of a higher autonomous proliferative and survival potential. Importantly, we report that (iii) the two functional subsets show an opposite clinical outcome, with CD40L-independent cases having a shorter time to progression. This indicates that the functional differences observed in vitro may reflect a different leukemic potential in vivo likely responsible for a distinct clinical course.
    Full-text · Article · Jun 2011 · Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K
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    Full-text · Article · Jan 2011 · Leukemia
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    ABSTRACT: The mammalian target of rapamycin (mTOR) kinase is a key regulator of cell growth and proliferation. Overexpression of the mTOR signaling pathway has been described in several tumor cells, including the majority of acute myeloid leukemia (AML) cases. The anti-tumor efficacy of mTOR inhibitors was shown in several preclinical and clinical studies. In AML, however, the potential antineoplastic effect of mTOR inhibitors has received little attention thus far. In this in-vitro study of the human AML cell line, HL-60, we aimed to assess the antileukemic activity of rapamycin (RAPA), an mTOR inhibitor, alone and in combination with cytarabine (Ara-C). The study showed that RAPA in concentrations of 1-10 nmol/l arrested the cell cycle progression of Hl-60 cells in the G1 phase, without evident cytotoxic effect. This effect was associated with significant inhibition of cyclin E expression. At concentrations higher than 10 nmol/l, RAPA exerted a significant proapoptotic effect, with the collapse of mitochondrial potential and caspase-3 activation. The most prominent proapoptotic effect was observed for a combination of 1 nmol/l of RAPA and 50 nmol/l of Ara-C, especially when Ara-C was added at a 24-h interval after RAPA. In conclusion, these data indicate that RAPA might be effective in the treatment of acute leukemia patients, especially in combination with Ara-C, the drug routinely used in AML treatment. On the basis of these results, attempts to combine classical induction chemotherapy with an inhibitor of the mTOR kinase in AML treatment could be warranted.
    Preview · Article · Oct 2009 · Anti-cancer drugs
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    ABSTRACT: The aim of this study was to determine the feasibility, efficacy and toxicity of the combined therapy consisting of cladribine (2-CdA), mitoxantrone and cyclophosphamide (CMC regimen) in patients with refractory or relapsed non-Hodgkin's lymphoma (NHL). Thirty six patients, 14 with mantle cell lymphoma (MCL), 10 with diffuse large B-cell lymphoma (DLBCL), 5 with follicular lymphoma (FL), 3 with small lymphocytic lymphoma (SLL), and 4 with T-cell lymphoma were enrolled to the study. The CMC protocol consisted of 2-CdA at a dose of 0.12 mg/kg in a 2-hour infusion on days 1 through 3, mitoxantrone 10 mg/m(2) i.v. on day 1 and cyclophosphamide 650 mg/m(2) i.v. on day 1. The CMC courses were repeated at intervals of 4 weeks. Thirty three patients were available for evaluation of response. Overall response rate (OR) was 58% (95% CI, 41--75%). Seven patients (21%; 95% CI, 7--35%) achieved a complete response (CR) and 12 patients (36%; 95% CI, 20--52%) achieved a partial response (PR). Seven of 19 patients with CR/PR are still in remission with a median follow-up of 3 months (range, 2-17 months). The median failure-free survival (FFS) was 5 months (range, 2-17 months). The median overall survival (OS) for the entire group was 9 months (range, 0.1-7 months). There was a significant difference in OS between responders and nonresponders after CMC therapy (log rank test, P = 0.015). When different disease status before CMC treatment was considered, a trend toward longer survival of recurrent patients was observed (log rank test, P = 0.08). Grade 3-4 neutropenia developed in 14 (39%) patients, and 16 episodes (15%) of grade 3-4 infections were observed. Grade 3-4 thrombocytopenia or anemia was seen in 9 patients (25%) and 10 patients (28%), respectively. The results of our study show that the CMC regimen is effective salvage therapy with acceptable toxicity in heavily pretreated patients with NHL including MCL and DLBCL.
    No preview · Article · Jul 2007 · Leukemia and Lymphoma
  • A. Janus · P. Smolewski
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    ABSTRACT: An array of genetic alterations is requiered for leukemic transformation. Mutations affecting genes for tyrosine kinases and their signaling pathways result in abnormal proliferation and lead to acute myeloid leukemia (AML). mTOR kinase is a key regulator of growth and proliferation in the cell. Overexpression of mTOR signaling pathway was described in more than 70% of AML cases. Antineoplastic activity of mTOR kinase inhibitors (rapamycin, RAD001, CCI-779, AP23573) has been shown in numerous preclinical studies. The results of these studies, as well as the results of the initial clinial trials suggest that mTOR kinase inhibitors, used alone and in combination with other drugs, might be effective in the treatment of AML.
    No preview · Article · Jan 2007
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    ABSTRACT: Inherited differences in xenobiotic transport and metabolism may play an important role in the development of adult acute myeloid leukemia (AML) and response to the chemotherapy. An ATP-binding cassette (ABC) family transporter P-glycoprotein (P-gp or ABCB1), encoded by ABCB1 (MDR1) gene, is involved in the protection against xenobiotics and multi-drug resistance. The aim of this study was to investigate the potential involvement of the ABCB1 gene exon 26 3435C>T single nucleotide polymorphism (SNP) in the genetic susceptibility to AML and regulation of P-gp expression and activity in AML cells. A total of 180 adult AML patients and 180 sex-matched controls were genotyped using PCR-RFLP method. Moreover, in 40 AML patients ABCB1 gene expression was studied by real-time RT-PCR and P-gp expression and activity were assessed by flow cytometry assays. The prevalence of 3435C>T ABCB1 polymorphism was similar in patient and control cohorts (P = 0.16). Furthermore, the carriers of different ABCB1 genotypes did not differ significantly according to ABCB1 gene expression (P = 0.99), P-gp expression (P = 0.42) and P-gp activity (P = 0.83) in leukemic cells. The authors conclude that isolated 3435C>T ABCB1 SNP is not a major factor of the genetic susceptibility to adult AML, and that genotyping of this polymorphism does not allow predicting P-gp expression or activity in AML cells.
    No preview · Article · Nov 2006 · Therapeutic Drug Monitoring
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    ABSTRACT: In the recent years the concept of biological, targeted treatment of malignancies has been introduced. Among the biological drugs a specific proteasome inhibitor - bortezomib (BOR), has been found to be very active in multiple myeloma (MM). It is now being often tested in MM for combinations with other drugs. Targeting the mTOR kinase pathway, important for the cell growth and proliferation, is another very promising anti-tumor approach. One of the mTOR inhibitors is rapamycin (RAPA), which has not been used in MM so far. We investigated cytoxic effect of RAPA alone or in combination with BOR on MM-derived RPMI 8226 cell line in vitro. Cells were treated for 24 hours with different concentrations of RAPA and BOR. Cytotoxicity (percentage of cells positive in propydium iodide staining; PI+ cells), apoptosis (hypoploid, subG1 fraction in a DNA content histogram) and cell cycle were assessed. Additionally, we measured expression of several proteins associated with the Akt-mTOR kinase pathway [Akt, ph-Akt, p70(S6K1), ph-p70(S6K1), 4E-BP1] as well as apoptosis-regulating proteins from Bcl-2 and p53 families (Bcl-2, Bax, p53 and p73). Cytotoxic effect of RAPA and BOR in vitro was dose-dependent. Both drugs significantly induced apoptosis of RPMI 8226 cells. There was a high correlation between the rate of PI+ and apoptotic cells (R=0,87for RAPA and R=0.92 for BOR; both p<0.001). RAPA arrested RPMI 8226 cells in G1 phase of the cell cycle and inhibited progression to S and G2M phases. Combination of 1nM of BOR and 100nM of RAPA induced significant increase in cell cytotoxicity compared to those drugs used alone (p<0.05). The combination inhibited expression of transcription factor 4E-BP1 (p<0.031 vs control). Moreover, both RAPA and BOR increased expression of p53 and p73, however their combined use did not further enhance this effect. We did not observe any significant changes in Bcl-2 family protein expression in response to RAPA+BOR combination. In conclusion, the results indicate a significant toxicity of combined treatment of RAPA and BOR on MM-derived cells. This finding needs confirmation in further studies.
    No preview · Article · Jan 2006
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    ABSTRACT: Rapamycin (RAPA), an inhibitor of mTOR (mammalian target of rapamycin) kinase was reported to exert anti- tumour efficacy in several preclinical studies. In this study we assessed cytotoxic effects of RAPA, the purine nucleoside analogue cladribine (2-chlorodeoxyadenozine, 2-CdA), each of them alone and in combination, on acute lymphoblastic leukemia (ALL) derived NALM-6 cell line and on mitogenically stimulated normal human lymphocytes. At 1 ng/ml concentration RAPA arrested mitogenically stimulated normal lymphocytes in G0/G1 but had no effect on cell cycle progression of leukemic cells, and did not induce apoptosis of either normal or leukemic cells. Pre-treatment with 1 ng/ml RAPA enhanced susceptibility of NALM-6 cells to undergo apoptosis upon treatment with 2-CdA. In contrast, pre-treatment of normal lymphocytes wim RAPA offered their partial protection from induction of apoptosis by 2-CdA. The data demonstrate increased antitumor selectivity of 2-CdA when combined with RAPA, and suggest that such combination may be considered for clinical applications in treatment of lympho-proliferative disorders. The data are consistent with the concept of "cyclotherapy", which is based on sequential treatment with agents arresting progression of normal cells through the cycle followed by chemotherapeutics that target DNA replicating and proliferating tumor cells.
    No preview · Article · Jan 2006 · Acta haematologica Polonica
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    ABSTRACT: Combination of vincristine, doxorubicin and dexamethasone (VAD regimen) is a widely-used, effective and economic initial treatment of multiple myeloma (MM). However, between 30 and 50% of MM patients do not respond to VAD, and exposure to VAD may induce multi-drug resistance. Therefore, in the era of emerging novel therapies for MM, there is a need for pre-treatment identification of potential responders to VAD. Importantly, all three VAD components have common steps in their pathways of transport and metabolism, thus even modest inherited alteration caused by single nucleotide polymorphisms (SNP) may influence VAD efficacy an toxicity. The aim of this study was to investigate whether analysis of common functional SNP in drug transporter P-glycoprotein gene (MDR1 or ABCB1), phase I cytochrome P450A metabolizing enzymes genes (CYP3A4 and CYP3A5), phase II glutathione S-transferase P metabolizing enzyme gene (GSTP1) as well as in glucocorticoid receptor gene NR3C1 can be useful to predict response to VAD in MM. Fifty-two uniformly treated MM patients were investigated for 8 common SNPs including MDR1 exon 12 C1236T, MDR1 exon 21 G2677T/A, MDR1 exon 26 C3435T, CYP3A4*1B, CYP3A5*3, GSTP1 A313G as well as NR3C1 bcl I and NR3C1 N363S polymorphisms. Genotypes were identified using direct sequencing or RFLP methods. The response to VAD was estimated after administration of 3 courses of VAD, and patients who achieved complete or partial remission of MM were assigned as responders to VAD. In the cohort of included MM patients there were 39/52 (75%) responders to VAD. The analysis of the prognostic impact of the genotyped SNPs showed that carriers of P-glycoprotein gene MDR1 2677G allele had a significantly greater probability of achievement of the response to VAD as compared to the non-carriers, odds ratio(OR)=8.0, 95% confidence interval (95%CI)= 1.2–52.9, Fisher exact test p=0.031). Moreover, the predictive effect of MDR1 2677G allele was further increased by the presence of glucocorticoid receptor NR3C1 gene 363N allele. The carriers of both MDR1 2677G and NR3C1 363N alleles had 8.4-fold greater probability to achieve complete or partial remission with 3 courses of VAD (OR=8.40, 95% CI=1.8–40.0, p=0.008). Taken together, these data support the hypothesis that analysis of common functional polymorphisms in drug transporters, metabolizing enzymes and receptors may be useful for individualization of the initial therapy of MM. Particularly MDR1 G2677T/A and NR3C1 N363S SNPs can be considered as determinants of response to VAD therapy.
    Full-text · Conference Paper · Dec 2005
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    ABSTRACT: P-glycoprotein (P-gp), a membrane transporter encoded by MDR1 gene, influences pharmacokinetics of anti-cancer drugs and contributes to multi-drug resistance phenotype in adult acute lymphoblastic leukemia (ALL). In this study, we explored prognostic and functional role of single nucleotide polymorphism C3435T in MDR1 gene in 44 adult Caucasian patients with ALL. We found that the outcome of chemotherapy as well as MDR1 gene expression, P-gp expression and P-gp activity in isolated ALL blast cells were comparable among the patients carrying different MDR1 genotypes. Our results suggest that C3435T polymorphism in MDR1 gene is not a major prognosticator in adult ALL.
    Full-text · Article · Nov 2005 · Pharmacological reports: PR
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    Full-text · Conference Paper · Sep 2005
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    Full-text · Conference Paper · Jun 2005

Publication Stats

387 Citations
89.08 Total Impact Points

Institutions

  • 2011-2012
    • Università Vita-Salute San Raffaele
      Milano, Lombardy, Italy
    • San Raffaele Scientific Institute
      Milano, Lombardy, Italy
  • 2005-2012
    • University of Lodz
      • Department of Cytobiochemistry
      Łódź, Łódź Voivodeship, Poland
    • Medical University of Łódź
      Łódź, Łódź Voivodeship, Poland