Publications by authors named "Jessica Petiti"

21 Publications

  • Page 1 of 1

Genetic Screening for Potential New Targets in Chronic Myeloid Leukemia Based on Drosophila Transgenic for Human BCR-ABL1.

Cancers (Basel) 2021 Jan 14;13(2). Epub 2021 Jan 14.

Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy.

Chronic myeloid leukemia is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome that originates from the reciprocal translocation t(9;22)(q34;q11.2) and encodes for the constitutively active tyrosine kinase protein BCR-ABL1 from the () sequence and the () gene. Despite BCR-ABL1 being one of the most studied oncogenic proteins, some molecular mechanisms remain enigmatic, and several of the proteins, acting either as positive or negative BCR-ABL1 regulators, are still unknown. The Drosophila melanogaster represents a powerful tool for genetic investigations and a promising model to study the BCR-ABL1 signaling pathway. To identify new components involved in BCR-ABL1 transforming activity, we conducted an extensive genetic screening using different Drosophila mutant strains carrying specific small deletions within the chromosomes 2 and 3 and the transgenic as the background. From the screening, we identified several putative candidate genes that may be involved either in sustaining chronic myeloid leukemia (CML) or in its progression. We also identified, for the first time, a tight connection between the BCR-ABL1 protein and Rab family members, and this correlation was also validated in CML patients. In conclusion, our data identified many genes that, by interacting with BCR-ABL1, regulate several important biological pathways and could promote disease onset and progression.
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http://dx.doi.org/10.3390/cancers13020293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830713PMC
January 2021

Standardization of BCR-ABL1 p210 Monitoring: From Nested to Digital PCR.

Cancers (Basel) 2020 Nov 6;12(11). Epub 2020 Nov 6.

Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy.

The introduction of tyrosine kinase inhibitors in 2001 as a targeted anticancer therapy has significantly improved the quality of life and survival of patients with chronic myeloid leukemia. At the same time, with the introduction of tyrosine kinase inhibitors, the need for precise monitoring of the molecular response to therapy has emerged. Starting with a qualitative polymerase chain reaction, followed by the introduction of a quantitative polymerase chain reaction to determine the exact quantity of the transcript of interest-p210 BCR-ABL1, molecular monitoring in patients with chronic myeloid leukemia was internationally standardized. This enabled precise monitoring of the therapeutic response, unification of therapeutic protocols, and comparison of results between different laboratories. This review aims to summarize the steps in the diagnosis and molecular monitoring of p210 BCR-ABL1, as well as to consider the possible future application of a more sophisticated method such as digital polymerase chain reaction.
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http://dx.doi.org/10.3390/cancers12113287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694607PMC
November 2020

Deferasirox-Dependent Iron Chelation Enhances Mitochondrial Dysfunction and Restores p53 Signaling by Stabilization of p53 Family Members in Leukemic Cells.

Int J Mol Sci 2020 Oct 16;21(20). Epub 2020 Oct 16.

Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy.

I is crucial to satisfy several mitochondrial functions including energy metabolism and oxidative phosphorylation. Patients affected by Myelodysplastic Syndromes (MDS) and acute myeloid leukemia (AML) are frequently characterized by iron overload (IOL), due to continuous red blood cell (RBC) transfusions. This event impacts the overall survival (OS) and it is associated with increased mortality in lower-risk MDS patients. Accordingly, the oral iron chelator Deferasirox (DFX) has been reported to improve the OS and delay leukemic transformation. However, the molecular players and the biological mechanisms laying behind remain currently mostly undefined. The aim of this study has been to investigate the potential anti-leukemic effect of DFX, by functionally and molecularly analyzing its effects in three different leukemia cell lines, harboring or not p53 mutations, and in human primary cells derived from 15 MDS/AML patients. Our findings indicated that DFX can lead to apoptosis, impairment of cell growth only in a context of IOL, and can induce a significant alteration of mitochondria network, with a sharp reduction in mitochondrial activity. Moreover, through a remarkable reduction of Murine Double Minute 2 (MDM2), known to regulate the stability of p53 and p73 proteins, we observed an enhancement of p53 transcriptional activity after DFX. Interestingly, this iron depletion-triggered signaling is enabled by p73, in the absence of p53, or in the presence of a p53 mutant form. In conclusion, we propose a mechanism by which the increased p53 family transcriptional activity and protein stability could explain the potential benefits of iron chelation therapy in terms of improving OS and delaying leukemic transformation.
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http://dx.doi.org/10.3390/ijms21207674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589297PMC
October 2020

Bcl-xL represents a therapeutic target in Philadelphia negative myeloproliferative neoplasms.

J Cell Mol Med 2020 09 13;24(18):10978-10986. Epub 2020 Aug 13.

Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.

Myeloproliferative neoplasms are divided into essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). Although ruxolitinib was proven to be effective in reducing symptoms, patients rarely achieve complete molecular remission. Therefore, it is relevant to identify new therapeutic targets to improve the clinical outcome of patients. Bcl-xL protein, the long isoform encoded by alternative splicing of the Bcl-x gene, acts as an anti-apoptotic regulator. Our study investigated the role of Bcl-xL as a marker of severity of MPN and the possibility to target Bcl-xL in patients. 129 MPN patients and 21 healthy patients were enrolled in the study. We analysed Bcl-xL expression in leucocytes and in enriched CD34+ and CD235a+ cells. Furthermore, ABT-737, a Bcl-xL inhibitor, was tested in HEL cells and in leucocytes from MPN patients. Bcl-xL was found progressively over-expressed in cells from ET, PV and PMF patients, independently by JAK2 mutational status. Moreover, our data indicated that the combination of ABT-737 and ruxolitinib resulted in a significantly higher apoptotic rate than the individual drug. Our study suggests that Bcl-xL plays an important role in MPN independently from JAK2 V617F mutation. Furthermore, data demonstrate that targeting simultaneously JAK2 and Bcl-xL might represent an interesting new approach.
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http://dx.doi.org/10.1111/jcmm.15730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7521327PMC
September 2020

Iron overload alters the energy metabolism in patients with myelodysplastic syndromes: results from the multicenter FISM BIOFER study.

Sci Rep 2020 06 8;10(1):9156. Epub 2020 Jun 8.

Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.

Myelodysplastic syndromes (MDS) are hematological malignancies characterized by ineffective hematopoiesis and increased apoptosis in the bone marrow, which cause peripheral cytopenia. Mitochondria are key regulators of apoptosis and a site of iron accumulation that favors reactive oxygen species (ROS) production with detrimental effects on cell survival. Although the energy metabolism could represent an attractive therapeutic target, it was poorly investigated in MDS. The purpose of the study was to analyze how the presence of myelodysplastic hematopoiesis, iron overload and chelation impact on mitochondrial metabolism. We compared energy balance, OxPhos activity and efficiency, lactic dehydrogenase activity and lipid peroxidation in mononuclear cells (MNCs), isolated from 38 MDS patients and 79 healthy controls. Our data show that ATP/AMP ratio is reduced during aging and even more in MDS due to a decreased OxPhos activity associated with an increment of lipid peroxidation. Moreover, the lactate fermentation enhancement was observed in MDS and elderly subjects, probably as an attempt to restore the energy balance. The biochemical alterations of MNCs from MDS patients have been partially restored by the in vitro iron chelation, while only slight effects were observed in the age-matched control samples. By contrast, the addition of iron chelators on MNCs from young healthy subjects determined a decrement in the OxPhos efficiency and an increment of lactate fermentation and lipid peroxidation. In summary, MDS-MNCs display an altered energy metabolism associated with increased oxidative stress, due to iron accumulation. This condition could be partially restored by iron chelation.
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http://dx.doi.org/10.1038/s41598-020-66162-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7280296PMC
June 2020

Transplantation Induces Profound Changes in the Transcriptional Asset of Hematopoietic Stem Cells: Identification of Specific Signatures Using Machine Learning Techniques.

J Clin Med 2020 Jun 1;9(6). Epub 2020 Jun 1.

Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy.

During the phase of proliferation needed for hematopoietic reconstitution following transplantation, hematopoietic stem/progenitor cells (HSPC) must express genes involved in stem cell self-renewal. We investigated the expression of genes relevant for self-renewal and expansion of HSPC (operationally defined as CD34+ cells) in steady state and after transplantation. Specifically, we evaluated the expression of ninety-one genes that were analyzed by real-time PCR in CD34+ cells isolated from (i) 12 samples from umbilical cord blood (UCB); (ii) 15 samples from bone marrow healthy donors; (iii) 13 samples from bone marrow after umbilical cord blood transplant (UCBT); and (iv) 29 samples from patients after transplantation with adult hematopoietic cells. The results show that transplanted CD34+ cells from adult cells acquire an asset very different from transplanted CD34+ cells from cord blood. Multivariate machine learning analysis (MMLA) showed that four specific gene signatures can be obtained by comparing the four types of CD34+ cells. In several, but not all cases, transplanted HSPC from UCB overexpress reprogramming genes. However, these remarkable changes do not alter the commitment to hematopoietic lineage. Overall, these results reveal undisclosed aspects of transplantation biology.
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http://dx.doi.org/10.3390/jcm9061670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355574PMC
June 2020

Novel Multiplex Droplet Digital PCR Assays to Monitor Minimal Residual Disease in Chronic Myeloid Leukemia Patients Showing Atypical Transcripts.

J Clin Med 2020 May 13;9(5). Epub 2020 May 13.

Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy.

fusion transcript is the minimal residual disease marker in chronic myeloid leukemia; 2% of patients show unusual breakpoints generating atypical transcripts, not quantifiable by standardized real-time PCR (RT-PCR). Response monitoring is performed by non-quantitative NESTED PCR, useless for evaluating patients' molecular remission, excluding them from treatment-free-remission protocols. Droplet digital PCR (ddPCR) is highly sensitive technology, allowing an absolute quantification independent of standard curves. Based on this, we have developed assays able to evaluate the molecular response in atypical patients. We designed new ddPCR-based molecular assays able to quantify atypical transcripts, with a detection limit of 0.001%, validated in a cohort of 65 RNA from 11 patients. Fifty samples were identified congruently by ddPCR and NESTED PCR (40 positives and 10 negatives for atypical transcript), while 11 positive samples were detected only by ddPCR. Our results highlight ddPCR usefulness, primarily when the level is less than 1.5% and NESTED PCR results are often inaccurate. Furthermore, we identified 3 patients who maintained a deep molecular response for at least one year, who could be considered good candidates for treatment-free remission approaches. Here, we describe a new promising molecular approach, highly sensitive, to monitor atypical patients, paving the foundation to include them in treatment-free remission protocols.
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http://dx.doi.org/10.3390/jcm9051457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290999PMC
May 2020

Landscape of Tumor Suppressor Mutations in Acute Myeloid Leukemia.

J Clin Med 2020 Mar 16;9(3). Epub 2020 Mar 16.

Department of Clinical and Biological Sciences, University of Turin, 10124 Turin, Italy.

Acute myeloid leukemia is mainly characterized by a complex and dynamic genomic instability. Next-generation sequencing has significantly improved the ability of diagnostic research to molecularly characterize and stratify patients. This detailed outcome allowed the discovery of new therapeutic targets and predictive biomarkers, which led to develop novel compounds (e.g., IDH 1 and 2 inhibitors), nowadays commonly used for the treatment of adult relapsed or refractory AML. In this review we summarize the most relevant mutations affecting tumor suppressor genes that contribute to the onset and progression of AML pathology. Epigenetic modifications (TET2, IDH1 and IDH2, DNMT3A, ASXL1, WT1, EZH2), DNA repair dysregulation (TP53, NPM1), cell cycle inhibition and deficiency in differentiation (NPM1, CEBPA, TP53 and GATA2) as a consequence of somatic mutations come out as key elements in acute myeloid leukemia and may contribute to relapse and resistance to therapies. Moreover, spliceosomal machinery mutations identified in the last years, even if in a small cohort of acute myeloid leukemia patients, suggested a new opportunity to exploit therapeutically. Targeting these cellular markers will be the main challenge in the near future in an attempt to eradicate leukemia stem cells.
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http://dx.doi.org/10.3390/jcm9030802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141302PMC
March 2020

Clone wars: co-occurrence of IDH2 R140Q and R172K in myelodysplastic syndromes.

Ann Hematol 2020 Apr 4;99(4):891-893. Epub 2020 Feb 4.

Department of Clinical and Biological Sciences of the University of Turin, San Luigi Hospital, Regione Gonzole 10, 10043 ORBASSANO, Turin, Italy.

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http://dx.doi.org/10.1007/s00277-020-03913-xDOI Listing
April 2020

Highly Sensitive Detection of Mutations in Acute Myeloid Leukemia.

J Clin Med 2020 Jan 19;9(1). Epub 2020 Jan 19.

Department of Clinical and Biological Sciences of the University of Turin, San Luigi Gonzaga Hospital, Regione Gonzole 10, 10043 Orbassano (Turin), Italy.

Background: Acute myeloid leukemia is a heterogeneous hematological disease, characterized by karyotypic and molecular alterations. Mutations in have a role in diagnosis and as a minimal residue disease marker. Often the variant allele frequency during follow up is less than 20%, which represents the limit of detection of Sanger sequencing. Therefore, the development of sensitive methodologies to identify mutations might help to monitor patients' response to therapy. We compared three different methods to identify and monitor mutations in patients' specimens.

Methods: Performances of PNA-PCR clamping, droplet digital PCR and Sanger for status identification were evaluated and compared in 96 DNA patients' specimens.

Results: In contrast with Sanger sequencing, our results highlighted the concordance between PNA clamping and digital PCR. Furthermore, PNA-PCR clamping was able to detect more mutated DNA with respect to Sanger sequencing that showed several false negatives independently from the allelic frequency.

Conclusions: We found that PNA-PCR clamping and digital PCR identified mutations in DNA samples with comparable results in a percentage significantly higher compared to Sanger sequencing. PNA-PCR clamping can be used even in laboratories not equipped for sophisticated analyses, decreasing cost and time for characterization.
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http://dx.doi.org/10.3390/jcm9010271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019902PMC
January 2020

Reduced Expression of Sprouty1 Contributes to the Aberrant Proliferation and Impaired Apoptosis of Acute Myeloid Leukemia Cells.

J Clin Med 2019 Jul 4;8(7). Epub 2019 Jul 4.

Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy.

In most of the acute myeloid leukemia patients there is an aberrant tyrosine kinase activity. The prototype of Sprouty proteins was originally identified in as antagonists of Breathless, the mammalian ortholog of fibroblast growth factor receptor. Usually, SPRY family members are inhibitors of RAS signaling induced by tyrosine kinases receptors and they are implicated in negative feedback processes regulating several intracellular pathways. The present study aims to investigate the role of a member of the Sprouty family, Sprouty1, as a regulator of cell proliferation and growth in patients affected by acute myeloid leukemia. Sprouty1 mRNA and protein were both significantly down-regulated in acute myeloid leukemia cells compared to the normal counterpart, but they were restored when remission is achieved after chemotherapy. Ectopic expression of Sprouty1 revealed that it plays a key role in the proliferation and apoptotic defect that represent a landmark of the leukemic cells. Our study identified Sprouty1 as negative regulator involved in the aberrant signals of adult acute myeloid leukemia. Furthermore, we found a correlation between Sprouty1 and FoxO3a delocalization in acute myeloid leukemia (AML) patients at diagnosis, suggesting a multistep regulation of RAS signaling in human cancers.
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http://dx.doi.org/10.3390/jcm8070972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678378PMC
July 2019

Digital PCR in Myeloid Malignancies: Ready to Replace Quantitative PCR?

Int J Mol Sci 2019 May 7;20(9). Epub 2019 May 7.

Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy.

New techniques are on the horizon for the detection of small leukemic clones in both, acute leukemias and myeloproliferative disorders. A promising approach is based on digital polymerase chain reaction (PCR). Digital PCR (dPCR) is a breakthrough technology designed to provide absolute nucleic acid quantification. It is particularly useful to detect a low amount of target and therefore it represents an alternative method for detecting measurable residual disease (MRD). The main advantages are the high precision, the very reliable quantification, the absolute quantification without the need for a standard curve, and the excellent reproducibility. Nowadays the main disadvantages of this strategy are the costs that are still higher than standard qPCR, the lack of standardized methods, and the limited number of laboratories that are equipped with instruments for dPCR. Several studies describing the possibility and advantages of using digital PCR for the detection of specific leukemic transcripts or mutations have already been published. In this review we summarize the available data on the use of dPCR in acute myeloid leukemia and myeloproliferative disorders.
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http://dx.doi.org/10.3390/ijms20092249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6540058PMC
May 2019

Curcumin induces apoptosis in JAK2-mutated cells by the inhibition of JAK2/STAT and mTORC1 pathways.

J Cell Mol Med 2019 06 29;23(6):4349-4357. Epub 2019 Apr 29.

Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.

Myeloproliferative neoplasms are chronic myeloid cancers divided in Philadelphia positive and negative. The JAK2 V617F is the most common mutation in Philadelphia negative patients and results in a constitutive activation of the JAK/STAT pathway, conferring a proliferative advantage and apoptosis inhibition. Recent studies identified a functional crosstalk between the JAK/STAT and mTOR pathways. The identification of an effective therapy is often difficult, so the availability of new therapeutic approaches might be attractive. Previous studies showed that curcumin, the active principle of the Curcuma longa, can suppress JAK2/STAT pathways in different type of cancer and injuries. In this study, we investigated the anti-proliferative and pro-apoptotic effects of curcumin in JAK2 V617F-mutated cells. HEL cell line and cells from patients JAK2 V617F mutated have been incubated with increasing concentrations of curcumin for different time. Apoptosis and proliferation were evaluated. Subsequently, JAK2/STAT and AKT/mTOR pathways were investigated at both RNA and protein levels. We found that curcumin induces apoptosis and inhibition of proliferation in HEL cells. Furthermore, we showed that curcumin inhibits JAK2/STAT and mTORC1 pathways in JAK2 V617F-mutated cells. This inhibition suggests that curcumin could represent an alternative strategy to be explored for the treatment of patients with myeloproliferative neoplasms.
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http://dx.doi.org/10.1111/jcmm.14326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533565PMC
June 2019

Prognostic significance of The Wilms' Tumor-1 (WT1) rs16754 polymorphism in acute myeloid leukemia.

Leuk Res 2018 04 5;67:6-11. Epub 2018 Feb 5.

Department of Clinical and Biological Sciences, University of Turin, Turin, Italy. Electronic address:

Acute myeloid leukemia is a genetically heterogeneous disease characterized by the accumulation of mutations in hematopoietic progenitor cells. For its heterogeneity, prognostic markers are very useful for therapeutic choice. The most important prognostic markers are age, white blood cell count, chromosomal alterations and gene mutations. Recent works have studied the prognostic significance of WT1 polymorphisms and mutations, highlighting the role of SNP rs16754 as a positive prognostic factor in AML patients. Nevertheless, the data are still unclear. To investigate the role of WT1 rs16754 polymorphism in AML, we designed a new tool for the detection using PNA directed PCR Clamping technology. Our data were able to establish a correlation between SNP rs16754 and the clinical outcome. Our results support the hypothesis that rs16754 polymorphism is an independent positive prognostic molecular marker that could be useful for therapeutic choice. In view of this, we described a novel assay faster, more sensitive and cheaper than DNA sequencing. The assay allows evaluating WT1 rs16754 polymorphism in diagnostic routine to improve prognostic information faster and without over-costing for diagnostic laboratories.
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http://dx.doi.org/10.1016/j.leukres.2018.01.016DOI Listing
April 2018

A novel assay to detect calreticulin mutations in myeloproliferative neoplasms.

Oncotarget 2017 Jan;8(4):6399-6405

Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.

The myeloproliferative neoplasms are chronic myeloid cancers divided in Philadelphia positive (Ph+), chronic myeloid leukemia, or negative: polycythemia vera (PV) essential thrombocythemia (ET), and primary myelofibrosis (PMF). Most Ph negative cases have an activating JAK2 or MPL mutation. Recently, somatic mutations in the calreticulin gene (CALR) were detected in 56-88% of JAK2/MPL-negative patients affected by ET or PMF. The most frequent mutations in CARL gene are type-1 and 2. Currently, CALR mutations are evaluated by sanger sequencing. The evaluation of CARL mutations increases the diagnostic accuracy in patients without other molecular markers and could represent a new therapeutic target for molecular drugs.We developed a novel detection assay in order to identify type-1 and 2 CALR mutations by PNA directed PCR clamping. Seventy-five patients affected by myeloproliferative neoplasms and seven controls were examined by direct DNA sequencing and by PNA directed PCR clamping. The assay resulted to be more sensitive, specific and cheaper than sanger sequencing and it could be applied even in laboratory not equipped for more sophisticated analysis. Interestingly, we report here a case carrying both type 1 and type2 mutations in CALR gene.
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http://dx.doi.org/10.18632/oncotarget.14113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5351640PMC
January 2017

Variable but consistent pattern of Meningioma 1 gene (MN1) expression in different genetic subsets of acute myelogenous leukaemia and its potential use as a marker for minimal residual disease detection.

Oncotarget 2016 Nov;7(45):74082-74096

Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.

Meningioma 1 (MN1) gene overexpression has been reported in acute myeloid leukaemia (AML) patients and identified as a negative prognostic factor. In order to characterize patients presenting gene overexpression and to verify if MN1 transcript could be a useful marker for minimal residual disease detection, MN1 was quantified in 136 AML patients with different cytogenetic risk and in 50 normal controls. In 20 patients bearing a fusion gene transcript suitable for minimal residual disease quantitative assessment and in 8 patients with NPM1 mutation, we performed a simultaneous analysis of MN1 and the fusion-gene transcript or NPM1 mutation during follow-up. Sequential MN1 and WT1 analysis was also performed in 13 AML patients lacking other molecular markers. The data obtained show that normal cells consistently express low levels of MN1 transcript. In contrast, high levels of MN1 expression are present in 47% of patients with normal karyotype and in all cases with inv(16). MN1 levels during follow-up were found to follow the pattern of other molecular markers (fusion gene transcripts, NPM1 and WT1). Increased MN1 expression in the BM during follow up was always found to be predictive of an impending hematological relapse.
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http://dx.doi.org/10.18632/oncotarget.12269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342037PMC
November 2016

The Wilms' tumor (WT1) gene expression correlates with the International Prognostic Scoring System (IPSS) score in patients with myelofibrosis and it is a marker of response to therapy.

Cancer Med 2016 07 11;5(7):1650-3. Epub 2016 May 11.

Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.

The Wilms tumor gene WT1 is a useful marker of clonal hematopoiesis and it has been shown to be a good marker of residual disease and it reflects the response to therapy. Although myelofibrosis is characterized by mutations of JAK2 and calreticulin (CALR), these mutations are not useful to monitor response to therapy. In this study we demonstrated that in patients affected by myelofibrosis WT1 correlates with the International Prognostic Scoring System (IPSS) score at diagnosis. Furthermore WT1 is a good marker of response to JAK2 inhibitors especially for patients without blasts and for patients who develop anemia or thrombocytopenia not for progression but as therapy related toxicity. Finally, WT1 transcript reduction can mirror a benefit of therapy on the disease burden. This study demonstrated that WT1 is a good marker for monitoring the response to therapy in patients affected by myelofibrosis.
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http://dx.doi.org/10.1002/cam4.735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4867666PMC
July 2016

Detection of BCR-ABL T315I mutation by peptide nucleic acid directed PCR clamping and by peptide nucleic acid FISH.

Biomark Res 2015 3;3:15. Epub 2015 Jul 3.

Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.

Background: Mutations of the BCR-ABL1 fusion gene represent a well established cause of resistance to tyrosine kinase inhibitors. Among the different mutations identified T315I is of particular concern since it is not effectively targeted by the majority of Tyrosine Kinase Inhibitors so far available. We developed a novel assay based on peptide nucleic acid (PNA) technology coupled to immunofluorescence microscopy (PNA-FISH) for the specific detection at a single cell level of BCR-ABL (T315I) mutation thus improving both, diagnostic resolution and the study of clonal prevalence. Furthermore we developed an additional method based on PNA directed PCR-clamping for the fast and easy detection of the mutation.

Results: The PNA directed PCR clamping allows to detect an amount of mutated template as low as 0.5 %. This method is highly sensitive, specific and cheap and could be applied even in laboratory not equipped for more sophisticated analysis. Furthermore, the PNA FISH method allows to identify a small amount of progenitor cells still present after therapy with specific inhibitors.

Conclusions: We present here two different methods based on PNA for the detection of T315I useful for different purposes. PNA-FISH can be used to study clonal evolution. In addition, this method could help in the study of compound mutations being able to identify two different mutations in a single cell. PNA directed PCR clamping although not superior to sequencing can be applied worldwide even in laboratory not equipped to search for mutations.
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http://dx.doi.org/10.1186/s40364-015-0039-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4490729PMC
July 2015