Publications by authors named "Paola Francia Di Celle"

27 Publications

  • Page 1 of 1

Higher-order connections between stereotyped subsets: implications for improved patient classification in CLL.

Blood 2020 Sep 29. Epub 2020 Sep 29.

Institute of Applied Biosciences, Centre for Research and Technology Hellas, Thessaloniki, Greece.

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B cell receptor immunoglobulins (BcR IG). Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR IG stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR IG stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. In order to address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29,856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed 'satellites', were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.
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http://dx.doi.org/10.1182/blood.2020007039DOI Listing
September 2020

JAK2V617F, CALR, and MPL Mutations and Bone Marrow Histology in Patients with Essential Thrombocythaemia.

Acta Haematol 2018 7;140(4):234-239. Epub 2018 Nov 7.

Section of Pathology, Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

Introduction: Mutations in the JAK2, CALR, and MPL genes have been shown to have prognostic value in essential thrombocythaemia (ET), but no clear association with morphological changes has been reported so far. We investigated the possible correlation between gene mutations and histopathological features in bone marrow (BM) biopsies of patients with ET.

Methods: Marrow cellularity, fibrosis, and the number of total and dysmorphic megakaryocytes and clusters of megakaryocytes were compared to gene mutations in 90 cases of ET at diagnosis.

Results: The JAK2V617F mutation was found in 58.9%, CALR in 28.9%, and MPL in 4.4% of the cases, and 7.8% were triple-negative. JAK2V617F-mutated ET showed a high BM cellularity, the lowest number of clusters of megakaryocytes and the highest number of dysmorphic megakaryocytes; CALR-mutated ET showed a reduced BM cellularity, many clusters of large megakaryocytes, and very few dysmorphic megakaryocytes; MPL-mutated ET showed the lowest BM cellularity, the highest number of clustered and large megakaryocytes, and the lowest number of dysmorphic megakaryocytes. Triple-negative ET cases had the highest BM cellularity.

Conclusions: Distinct morphological patterns were associated with gene mutations in ET, supporting the classification of ET into different subtypes.
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http://dx.doi.org/10.1159/000493970DOI Listing
June 2019

Awareness of mutational artefacts in suboptimal DNA samples: possible risk for therapeutic choices.

Expert Rev Mol Diagn 2018 05 26;18(5):467-475. Epub 2018 Apr 26.

a Department of Medical Sciences , University of Turin and Pathology Unit, Città della Salute e della Scienza Hospital , Torino , Italy.

Background: Technical biases due to PCR artefacts could represent an insidious obstacle for mutational analysis and precision medicine.

Methods: The authors report a retrospective analysis by fast COLD-PCR and sequencing of 31 suboptimal tumor DNA samples obtained from FFPE tissues and liquid biopsies.

Results: In FFPE tumor tissues and plasma liquid biopsies of patients with lung and colorectal adenocarcinoma, we observed a significant rate of artefactual KRAS mutations, unveiled by repeated analysis following UDG pretreatment as well as by simple repetition without UDG pretreatment step, thus suggesting a DNA damage different from cytosine deamination. UDG pretreatment was not only unnecessary to contrast artefacts occurrence, but also hampered the efficiency of mutational screening, reducing the analytical sensitivity. Taken individually or considered together, the reduced DNA input per reaction and UDG pretreatment limited the detection of 'real' mutated alleles, decreasing PCR sensitivity enough to hamper distinction between artefactual and true subclonal mutations of KRAS.

Conclusions: Careful validation of analytical sensitivities should always be carried out through standard controls, and strategies other than UDG pretreatment need to be identified to avoid both amplification of artefactual mutations and failure to identify real subclonal mutations.
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http://dx.doi.org/10.1080/14737159.2018.1468254DOI Listing
May 2018

Post-remissional and pre-transplant role of minimal residual disease detected by WT1 in acute myeloid leukemia: A retrospective cohort study.

Leuk Res 2017 10 30;61:10-17. Epub 2017 Aug 30.

Department of Hematology, University-Hospital Città della Salute e della Scienza, Torino, Italy.

In acute myeloid leukemia (AML), the detection of minimal residual disease (MRD) is still under investigation. The aim of the present retrospective study was to assess the role of Wilms tumor gene 1 (WT1) overexpression in a large monocentric cohort of AML patients. Among 255 enrolled patients, MRD was investigated in those in complete remission (CR) with an available WT1 at baseline (>250 copies) and at two further time-points: after induction (n=117) and prior allogeneic hematopoietic cell transplantation (allo-HCT), n=65. Baseline BM WT1 overexpression was not associated with response to induction (p=0.244). Median overall survival (OS) and disease-free survival (DFS) were significantly shorter in patients with >350 WT1 copies after induction compared to those with ≤350 (HR for mortality 2.13; 95% CI 1.14-3.97, p=0.018 and HR for relapse 2.81; 95% CI 1.14-6.93, p=0.025). Patients with WT1>150 copies pre allo-HCT had a significantly higher 2-year cumulative incidence of relapse (CIR) compared to those with WT1≤150 (HR 4.61; 95% CI 1.72-12.31, p=0.002). The prognostic role of WT1 overexpression resulted independent from other well-established risk factors. According to these results, WT1 overexpression might represent an additional MRD tool for risk stratification in patients classified nowadays in CR.
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http://dx.doi.org/10.1016/j.leukres.2017.08.008DOI Listing
October 2017

Extreme assay sensitivity in molecular diagnostics further unveils intratumour heterogeneity in metastatic colorectal cancer as well as artifactual low-frequency mutations in the KRAS gene.

Br J Cancer 2017 Jul 15;117(3):358-366. Epub 2017 Jun 15.

Department of Medical Sciences, University of Turin, via Santena 7, Turin 10126, Italy.

Background: Gene mutations in the RAS family rule out metastatic colorectal carcinomas (mCRCs) from anti-EGFR therapies.

Methods: We report a retrospective analysis by Sequenom Massarray and fast COLD-PCR followed by Sanger sequencing on 240 mCRCs.

Results: By Sequenom, KRAS and NRAS exons 2-3-4 were mutated in 52.9% (127/240) of tumours, while BRAF codon 600 mutations reached 5% (12/240). Fast COLD-PCR found extra mutations at KRAS exon 2 in 15/166 (9%) of samples, previously diagnosed by Sequenom as wild-type or mutated at RAS (exons 3-4) or BRAF genes. After UDG digestion results were reproduced in 2/12 analysable subclonally mutated samples leading to a frequency of true subclonal KRAS mutations of 1.2% (2.1% of the previous Sequenom wild-type subgroup). In 10 out of 12 samples, the subclonal KRAS mutations disappeared (9 out of 12) or turned to a different sequence variant (1 out of 12).

Conclusions: mCRC can harbour coexisting multiple gene mutations. High sensitivity assays allow the detection of a small subset of patients harbouring true subclonal KRAS mutations. However, DNA changes with mutant allele frequencies <3% detected in formalin-fixed paraffin-embedded samples may be artifactual in a non-negligible fraction of cases. UDG pre-treatment of DNA is mandatory to identify true DNA changes in archival samples and avoid misinterpretation due to artifacts.
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http://dx.doi.org/10.1038/bjc.2017.170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5537488PMC
July 2017

Peripheral ENO1-specific T cells mirror the intratumoral immune response and their presence is a potential prognostic factor for pancreatic adenocarcinoma.

Int J Oncol 2016 Jul 16;49(1):393-401. Epub 2016 May 16.

Department of Experimental and Clinical Medicine, University of Florence, and Department of Biomedicine, Azienda Ospedaliera Universitaria Careggi (AOUC), I-50134 Florence, Italy.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with an average survival of 4-6 months following diagnosis. Surgical resection is the only treatment with curative intent, but resectable PDAC patients are in the minority. Also, unlike other neoplasms, PDAC is resistant to conventional and targeted chemotherapy. Innovative treatments, such as immunotherapy, can be very important and the study of the immune response is fundamental. We previously demonstrated that PDAC patients show tumor-infiltrating T cells specific to α-enolase (ENO1), a glycolytic enzyme over-expressed by pancreatic tumor cells, which plays an important role in promoting cell migration and cancer metastasis. In the present study, we evaluate the functional anticancer proprieties of ENO1-specific T cells isolated from the peripheral blood of PDAC patients. Furthermore, comparing the T cell receptor repertoire of ENO1-specific peripheral and infiltrating tumor T cells from the same patient suggests that ENO1-specific T cells, despite having a different functional profile, can recirculate from the tumor to the periphery. Finally, of clinical relevance, the presence of peripheral ENO1-specific T cells has a prognostic value and significantly correlates with a longer survival.
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http://dx.doi.org/10.3892/ijo.2016.3524DOI Listing
July 2016

Myelodysplastic syndrome with del (5q) and JAK2(V617F) mutation transformed to acute myeloid leukaemia with complex karyotype.

Ann Hematol 2016 Feb 11;95(3):525-7. Epub 2016 Jan 11.

Department of Molecular Biotechnology and Health Sciences, Section of Pathology, University of Turin, Via Santena 7, I-10126, Turin, Italy.

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http://dx.doi.org/10.1007/s00277-015-2584-8DOI Listing
February 2016

Novel CALR somatic mutations in essential thrombocythaemia.

Br J Haematol 2016 06 7;173(5):797-801. Epub 2015 Sep 7.

Section of Pathology, AO Città della Salute e della Scienza, Torino, Italy.

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http://dx.doi.org/10.1111/bjh.13638DOI Listing
June 2016

Immunohistochemical and molecular profiling of histologically defined apocrine carcinomas of the breast.

Hum Pathol 2015 Sep 5;46(9):1350-9. Epub 2015 Jun 5.

Department of Medical Sciences, University of Turin, Turin 10126, Italy. Electronic address:

Despite the marked improvement in the understanding of molecular mechanisms and classification of apocrine carcinoma, little is known about its specific molecular genetic alterations and potentially targetable biomarkers. In this study, we explored immunohistochemical and molecular genetic characteristics of 37 invasive apocrine carcinomas using immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), and next-generation sequencing (NGS) assays. IHC revealed frequent E-cadherin expression (89%), moderate (16%) proliferation activity [Ki-67, phosphohistone H3], infrequent (~10%) expression of basal cell markers [CK5/6, CK14, p63, caveolin-1], loss of PTEN (83%), and overexpression of HER2 (32%), EGFR (41%), cyclin D1 (50%), and MUC-1 (88%). MLPA assay revealed gene copy gains of MYC, CCND1, ZNF703, CDH1, and TRAF4 in 50% or greater of the apocrine carcinomas, whereas gene copy losses frequently affected BRCA2 (75%), ADAM9 (54%), and BRCA1 (46%). HER2 gain, detected by MLPA in 38% of the cases, was in excellent concordance with HER2 results obtained by IHC/FISH (κ = 0.915, P < .001). TOP2A gain was observed in one case, while five cases (21%) exhibited TOP2A loss. Unsupervised hierarchical cluster analysis revealed two distinct clusters: HER2-positive and HER2-negative (P = .03 and .04, respectively). NGS assay revealed mutations of the TP53 (2 of 7, 29%), BRAF/KRAS (2 of 7, 29%), and PI3KCA/PTEN genes (7 of 7, 100%). We conclude that morphologically defined apocrine carcinomas exhibit complex molecular genetic alterations that are consistent with the "luminal-complex" phenotype. Some of the identified molecular targets are promising biomarkers; however, functional studies are needed to prove these observations.
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http://dx.doi.org/10.1016/j.humpath.2015.05.017DOI Listing
September 2015

Flexible lab-tailored cut-offs for suitability of formalin-fixed tumor samples for diagnostic mutational analyses.

PLoS One 2015 6;10(4):e0121815. Epub 2015 Apr 6.

Department of Medical Sciences; University of Torino, Torino, Italy.

The selection of proper tissues from formalin-fixed and paraffin-embedded tumors before diagnostic molecular testing is responsibility of the pathologist and represents a crucial step to produce reliable test results. The international guidelines suggest two cut-offs, one for the percentage and one for the number of tumor cells, in order to enrich the tumor content before DNA extraction. The aim of the present work was two-fold: to evaluate to what extent a low percentage or absolute number of tumor cells can be qualified for somatic mutation testing; and to determine how assay sensitivities can guide pathologists towards a better definition of morphology-based adequacy cut-offs. We tested 1797 tumor specimens from melanomas, colorectal and lung adenocarcinomas. Respectively, their BRAF, K-RAS and EGFR genes were analyzed at specific exons by mutation-enriched PCR, pyrosequencing, direct sequencing and real-time PCR methods. We demonstrate that poorly cellular specimens do not modify the frequency distribution of either mutated or wild-type DNA samples nor that of specific mutations. This observation suggests that currently recommended cut-offs for adequacy of specimens to be processed for molecular assays seem to be too much stringent in a laboratory context that performs highly sensitive routine analytical methods. In conclusion, new cut-offs are needed based on test sensitivities and documented tumor heterogeneity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0121815PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4386759PMC
April 2016

Core binding factor acute myeloid leukaemia and c-KIT mutations.

Oncol Rep 2013 May 5;29(5):1867-72. Epub 2013 Mar 5.

Department of Molecular Biotechnology and Health Sciences, Section of Pathology, University of Turin, Turin, Italy.

Core binding factor (CBF) acute myeloid leukaemia (AML) represents 5-8% of all AMLs and has a relatively favourable prognosis. However, activating c-KIT mutations are reported to be associated with higher risk of relapse and shorter survival. To verify the incidence and prognostic value of c-KIT mutations in CBF AML, we retrospectively analysed bone marrow samples of 23 consecutive adult patients with de novo CBF AML [14 inv(16) and 9 t(8;21)] treated at a single institution from 2000 to 2011. All patients received standard induction chemotherapy with cytarabine, idarubicin and etoposide; 13 underwent allogeneic stem cell transplantation. c-KIT mutations in exons 8, 9, 10, 11, 13, 14 and 17 were assessed by PCR amplification in combination with direct sequencing. c-KIT mutations (3 in exon 10 and 4 in exon 17) were detected in 7/23 (30.4%) patients, 3 with t(8;21) and 4 with inv(16). No difference in c-KIT mutation status was observed between cases with inv(16) or t(8;21) alone and cases with additional cytogenetic abnormalities. No association between gender, age, white blood cell and platelet count, peripheral blood and bone marrow blast cells at diagnosis, achievement of complete remission, cytogenetic risk groups and Wilms tumour gene 1 (WT1) levels was found. On the contrary, lactate dehydrogenase (LDH) values were higher in mutated than in non-mutated patients (p=0.01). Overall survival (OS) rates were longer in CBF compared to the other types of AML and disease-free survival (DFS) was longer in inv(16) than in t(8;21) AML. OS and DFS were similar in mutated and non-mutated CBF AML patients. Our results confirm a better prognosis for CBF AML than all other AML categories, and for inv(16) than t(8;21) AML. However, no prognostic value for c-KIT mutational status was found in our series. The association between LDH levels and c-KIT mutation would indicate a more active proliferation for mutated CBF AML.
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http://dx.doi.org/10.3892/or.2013.2328DOI Listing
May 2013

Recurrent SETBP1 mutations in atypical chronic myeloid leukemia.

Nat Genet 2013 Jan 9;45(1):18-24. Epub 2012 Dec 9.

Department of Health Sciences, University of Milano-Bicocca, Monza, Italy.

Atypical chronic myeloid leukemia (aCML) shares clinical and laboratory features with CML, but it lacks the BCR-ABL1 fusion. We performed exome sequencing of eight aCMLs and identified somatic alterations of SETBP1 (encoding a p.Gly870Ser alteration) in two cases. Targeted resequencing of 70 aCMLs, 574 diverse hematological malignancies and 344 cancer cell lines identified SETBP1 mutations in 24 cases, including 17 of 70 aCMLs (24.3%; 95% confidence interval (CI) = 16-35%). Most mutations (92%) were located between codons 858 and 871 and were identical to changes seen in individuals with Schinzel-Giedion syndrome. Individuals with mutations had higher white blood cell counts (P = 0.008) and worse prognosis (P = 0.01). The p.Gly870Ser alteration abrogated a site for ubiquitination, and cells exogenously expressing this mutant exhibited higher amounts of SETBP1 and SET protein, lower PP2A activity and higher proliferation rates relative to those expressing the wild-type protein. In summary, mutated SETBP1 represents a newly discovered oncogene present in aCML and closely related diseases.
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http://dx.doi.org/10.1038/ng.2495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3588142PMC
January 2013

Stereotyped B-cell receptors in one-third of chronic lymphocytic leukemia: a molecular classification with implications for targeted therapies.

Blood 2012 May 13;119(19):4467-75. Epub 2012 Mar 13.

Institute of Agrobiotechnology, Center for Research and Technology Hellas, Thessaloniki, Greece.

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.
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http://dx.doi.org/10.1182/blood-2011-11-393694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3392073PMC
May 2012

Flow cytometric detection and quantification of CD56 (neural cell adhesion molecule, NCAM) expression in diffuse large B cell lymphomas and review of the literature.

Histopathology 2012 Feb;60(3):452-9

Flow Cytometry Unit, Molinette Hospital, Turin, Italy.

Aim: To report unusual CD56 (neural cell adhesion molecule, NCAM) expression on diffuse large B cell lymphoma (DLBCL).

Methods And Results: CD56 expression was first detected and quantified on tissues obtained from five cases of DLBCL by flow cytometry (FC), then confirmed by immunohistochemistry. The CD56 expression pattern was heterogeneous among the cases [the molecular equivalent of soluble fluorochrome (MESF) level ranged from 2214 to 133 466]. All were CD10 and Bcl-6 positive, suggesting their germinal centre origin; one was also CD5 positive. An extranodal presentation occurred in three of five cases.

Conclusions: CD56 expression in B cell lymphoma is a rare occurrence. FC is able to identify aberrant immunophenotypes that can be useful in the identification and monitoring of B cell lymphoma subtypes. The presence of CD56 reported by the literature on certain DLBCL with extranodal presentation might be related to mechanisms involved in growth and expansion.
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http://dx.doi.org/10.1111/j.1365-2559.2011.04098.xDOI Listing
February 2012

FBXO11 targets BCL6 for degradation and is inactivated in diffuse large B-cell lymphomas.

Nature 2012 Jan;481(7379):90-3

Department of Pathology, NYU Cancer Institute, New York University School of Medicine, New York, New York 10016, USA.

BCL6 is the product of a proto-oncogene implicated in the pathogenesis of human B-cell lymphomas. By binding specific DNA sequences, BCL6 controls the transcription of a variety of genes involved in B-cell development, differentiation and activation. BCL6 is overexpressed in the majority of patients with aggressive diffuse large B-cell lymphoma (DLBCL), the most common lymphoma in adulthood, and transgenic mice constitutively expressing BCL6 in B cells develop DLBCLs similar to the human disease. In many DLBCL patients, BCL6 overexpression is achieved through translocation (~40%) or hypermutation of its promoter (~15%). However, many other DLBCLs overexpress BCL6 through an unknown mechanism. Here we show that BCL6 is targeted for ubiquitylation and proteasomal degradation by a SKP1–CUL1–F-box protein (SCF) ubiquitin ligase complex that contains the orphan F-box protein FBXO11 (refs 5, 6). The gene encoding FBXO11 was found to be deleted or mutated in multiple DLBCL cell lines, and this inactivation of FBXO11 correlated with increased levels and stability of BCL6. Similarly, FBXO11 was either deleted or mutated in primary DLBCLs. Notably, tumour-derived FBXO11 mutants displayed an impaired ability to induce BCL6 degradation. Reconstitution of FBXO11 expression in FBXO11-deleted DLBCL cells promoted BCL6 ubiquitylation and degradation, inhibited cell proliferation, and induced cell death. FBXO11-deleted DLBCL cells generated tumours in immunodeficient mice, and the tumorigenicity was suppressed by FBXO11 reconstitution. We reveal a molecular mechanism controlling BCL6 stability and propose that mutations and deletions in FBXO11 contribute to lymphomagenesis through BCL6 stabilization. The deletions/mutations found in DLBCLs are largely monoallelic, indicating that FBXO11 is a haplo-insufficient tumour suppressor gene.
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http://dx.doi.org/10.1038/nature10688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3344385PMC
January 2012

Simple genetic diagnosis of hairy cell leukemia by sensitive detection of the BRAF-V600E mutation.

Blood 2012 Jan 25;119(1):192-5. Epub 2011 Oct 25.

Institute of Hematology, University of Perugia, Perugia, Italy.

Hairy cell leukemia (HCL) is a distinct clinicopathologic entity that responds well to purine analogs but is sometimes difficult to differentiate from HCL-like disorders (e.g., splenic marginal zone lymphoma and HCL variant). We recently identified the BRAF-V600E mutation as the disease-defining genetic event in HCL. In this study, we describe a new, simple, and inexpensive test for genetics-based diagnosis of HCL in whole-blood samples that detects BRAF-V600E through a sensitive allele-specific PCR qualitative assay followed by agarose-gel electrophoresis. This approach detected BRAF-V600E in all 123 leukemic HCL samples investigated containing as few as 0.1% leukemic cells. BRAF-V600E was detected at different time points during the disease course, even after therapy, pointing to its pivotal role in HCL pathogenesis and maintenance of the leukemic clone. Conversely, 115 non-HCL chronic B-cell neoplasms, including 79 HCL-like disorders, were invariably negative for BRAF-V600E. This molecular assay is a powerful tool for improving the diagnostic accuracy in HCL.
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http://dx.doi.org/10.1182/blood-2011-08-371179DOI Listing
January 2012

Description of a novel Janus kinase 3 P132A mutation in acute megakaryoblastic leukemia and demonstration of previously reported Janus kinase 3 mutations in normal subjects.

Leuk Lymphoma 2011 Sep 23;52(9):1742-50. Epub 2011 May 23.

Center for Experimental Research and Medical Studies, University of Turin, Turin, Italy.

Gain-of-function (GOF) mutations of Janus kinase 2 (JAK2) are frequently seen in myeloproliferative disorders (MPDs). Meanwhile, JAK3 activating substitutions have been found in a few megakaryocytic cell lines and in primary myeloid leukemia (AMKL). Here, we sought to discover novel leukemogenetic mutations in de novo acute myeloid leukemia of non-Down syndrome (N-DS) by DNA sequencing. A total of 191 normal Caucasian individuals were studied to define single nucleotide polymorphisms (SNPs) within the JH2 and JH6 domains. Although known activating substitutions were observed in rare cases of acute myeloid leukemia (AML) (V722I [2/134] or P132T [1/119]), all samples were wild-type (WT) for the oncogenic A572V (119/119). Interestingly, a novel homozygous mutation (P132A) was discovered in a patient with acute megakaryoblastic leukemia and in vivo studies demonstrated that its ectopic expression was oncogenic in a mouse xenotransplant model. This study defines a novel JAK3 mutation among patients with N-DS AML and demonstrates that normal individuals can also display germline JAK3 substitutions, previously proven to have oncogenic properties, in vitro and in vivo. The discovery of these substitutions in normal donors encourages future studies to define new risk factors among patients with MPDs.
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http://dx.doi.org/10.3109/10428194.2011.574757DOI Listing
September 2011

Usefulness of multiparametric flow cytometry in detecting composite lymphoma: study of 17 cases in a 12-year period.

Am J Clin Pathol 2011 Apr;135(4):541-55

Flow Cytometry Unit, Pathology Department, Centre for Experimental Research and Medical Studies, Molinette Hospital, University of Turin, Italy.

Composite lymphoma (CL) is a rare occurrence of 2 or more morphologically and immunophenotypically distinct lymphoma clones in a single anatomic site. A retrospective analysis of 1,722 solid tissue samples clinically suggestive of lymphoma was carried out in our institute during a 12-year period to evaluate the efficacy of flow cytometry (FC) in identifying CL. We report 17 CL cases. A strong correlation between morphologic findings and FC was observed in 13 cases (76%). In the 4 cases diagnosed as non-Hodgkin lymphoma plus Hodgkin lymphoma, although FC did not detect Reed-Sternberg cells, it accurately identified the neoplastic B- or T-cell component. In 3 cases, FC indicated the need to evaluate an additional neoplastic component that was not morphologically evident. Our data demonstrate that FC immunophenotyping of tissues may enhance the performance of the diagnostic morphologic evaluation of CL. To the best of our knowledge, this is the first report in the literature of a wide series of CL studied also by FC.
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http://dx.doi.org/10.1309/AJCPQKE25ADCFZWNDOI Listing
April 2011

TCRgamma-chain gene rearrangement by GeneScan: incidence and significance of clonal heterogeneity in Sézary syndrome.

J Invest Dermatol 2010 Sep 22;130(9):2312-9. Epub 2010 Apr 22.

Section of Dermatology, Department of Biomedical Sciences and Human Oncology, University of Turin, Turin, Italy.

GeneScan (GS) analysis is a highly sensitive method for the early detection of cutaneous T-cell lymphoma (CTCL) and allows the identification of clonal heterogeneity, defined as the coexistence of two or more different T-cell clones in multiple samples from the same patient. We analyzed by GS the incidence and the significance of long-lived oligoclonal expansions in multiple skin and blood samples from 24 Sézary syndrome (SS) patients, and tried to correlate them with the clinical outcome. A skin clonal heterogeneity with additional reproducible TCRgamma-gene rearrangements (TCRgamma-GRs) was detected at diagnosis in 19/24 patients, 13 of whom had a constant prevalence of pathological TCRgamma-GRs in both skin and blood (dominant clonal pattern). During follow-up, an increase in oligoclones that were present at diagnosis or the appearance of new oligoclones was observed in 10 patients; all of them achieved a clinical response to treatment with extracorporeal photochemotherapy (ECP). The TCRgamma pattern (homogeneity or heterogeneity) in the skin at diagnosis showed a relevant prognostic value, and patients with an oligoclonal pattern had a significantly longer survival than those with a homogeneous pattern. In conclusion, multiple-sample approach GS analysis allows the identification of clonal heterogeneity and could also help in identifying SS patients with a potential higher response to therapy.
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http://dx.doi.org/10.1038/jid.2010.97DOI Listing
September 2010

NPM-ALK oncogenic tyrosine kinase controls T-cell identity by transcriptional regulation and epigenetic silencing in lymphoma cells.

Cancer Res 2009 Nov 3;69(22):8611-9. Epub 2009 Nov 3.

Department of Biomedical Sciences and Human Oncology, University of Torino, Center for Experimental Research and Medical Studies, ASO San Giovanni Battista, Torino, Italy.

Transformed cells in lymphomas usually maintain the phenotype of the postulated normal lymphocyte from which they arise. By contrast, anaplastic large cell lymphoma (ALCL) is a T-cell lymphoma with aberrant phenotype because of the defective expression of the T-cell receptor and other T-cell-specific molecules for still undetermined mechanisms. The majority of ALCL carries the translocation t(2;5) that encodes for the oncogenic tyrosine kinase NPM-ALK, fundamental for survival, proliferation, and migration of transformed T cells. Here, we show that loss of T-cell-specific molecules in ALCL cases is broader than reported previously and involves most T-cell receptor-related signaling molecules, including CD3epsilon, ZAP70, LAT, and SLP76. We further show that NPM-ALK, but not the kinase-dead NPM-ALK(K210R), downregulated the expression of these molecules by a STAT3-mediated gene transcription regulation and/or epigenetic silencing because this downregulation was reverted by treating ALCL cells with 5-aza-2-deoxycytidine or by knocking down STAT3 through short hairpin RNA. Finally, NPM-ALK increased the methylation of ZAP70 intron 1-exon 2 boundary region, and both NPM-ALK and STAT3 regulated the expression levels of DNA methyltransferase 1 in transformed T cells. Thus, our data reveal that oncogene-deregulated tyrosine kinase activity controls the expression of molecules that determine T-cell identity and signaling.
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http://dx.doi.org/10.1158/0008-5472.CAN-09-2655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2784121PMC
November 2009

Interleukin-3 promotes expansion of hemopoietic-derived CD45+ angiogenic cells and their arterial commitment via STAT5 activation.

Blood 2008 Jul 6;112(2):350-61. Epub 2008 May 6.

Department of Internal Medicine, University of Torino, Torino, Italy.

Interleukin-3 (IL-3) released by infiltrating inflammatory cells in different pathologic settings contributes to organ and tumor angiogenesis. Here we demonstrate that IL-3 expands a subset of CD45+ circulating angiogenic cells clonally derived from the hemopoietic progenitors. Moreover, CD45+ cells exposed to IL-3 acquire arterial specification and contribute to the formation of vessels in vivo. Depletion of signal transducer and activator of transcription 5 (STAT5) provides evidence that IL-3-mediated cell expansion and arterial morphogenesis rely on STAT5 activation. In addition, by means of Tie2-transgenic mice, we demonstrate that STAT5 also regulates IL-3-induced expansion and arterial specification of bone marrow-derived CD45+ cells. Thus, our data provide the first evidence that, in inflammatory microenvironments containing IL-3, angiogenic cells derived from hemopoietic precursors can act as adult vasculogenic cells. Moreover, the characterization of the signaling pathway regulating these events provides the rationale for therapeutically targeting STAT5 in these pathologic settings.
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http://dx.doi.org/10.1182/blood-2007-12-128215DOI Listing
July 2008

TCRgamma-chain gene rearrangement by PCR-based GeneScan: diagnostic accuracy improvement and clonal heterogeneity analysis in multiple cutaneous T-cell lymphoma samples.

J Invest Dermatol 2008 Apr 8;128(4):1030-8. Epub 2007 Nov 8.

Section of Dermatology, Department of Medical Sciences and Human Oncology, University of Turin, Turin, Italy.

Cutaneous T-cell lymphomas are a heterogeneous group of lymphomas where the tumor population emerges within a multiple subclone pattern ("clonal heterogeneity"). PCR analysis has been shown to be useful in the diagnosis of mycosis fungoides (MF) and Sézary Syndrome (SS). Focusing the attention on clonal heterogeneity, the efficacy of the multiplex/heteroduplex (HD) PCR and the GeneScan (GS) capillary electrophoresis analysis was compared in the early diagnosis of MF/SS, using a multiple sample approach. Indeed, GS demonstrated TCRgamma gene rearrangement (GR) in all the 57 SS (100%) and in 123/146 (84%) of the MF samples, whereas the multiplex/HD PCR was less sensitive. An increase in clonality was observed in connection with both a worsening of the cutaneous disease (79% T1/T2; 100% T3/T4) and an increase in the histopathological score (HS < 5, 76%; HS > or = 5, 94%). Clonal heterogeneity with adjunctive reproducible skin TCRgamma-GRs was also observed. "Clonal instability," with different GRs, was present in a small percentage of patients. Therefore, it can be concluded that GS analysis in TCRgamma-GR is able to improve diagnosis in MF/SS patients and the multiple sample approach is helpful for a correct interpretation of clonal patterns in skin lesions, especially in early-stage MF and in SS skin/blood samples.
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http://dx.doi.org/10.1038/sj.jid.5701109DOI Listing
April 2008

The usefulness of flow cytometric CD10 detection in the differential diagnosis of peripheral T-cell lymphomas.

Am J Clin Pathol 2007 Nov;128(5):854-64

Flow Cytometry Unit, Laboratory of Pathology, Department of Pathology, Molinette Hospital, Turin, Italy.

We studied the histologic and multiparameter flow cytometry (MFC) features of 12 cases of angioimmunoblastic T-cell lymphoma (AITL), 13 of mature T-cell lymphoma, and 25 control cases of reactive lymphoid hyperplasia to evaluate the role of CD10 in the differential diagnosis of peripheral T-cell lymphomas (PTCLs). A characteristic immunophenotypic profile (CD2+/CD4+) with recurrent phenotypic aberrancies (eg, CD3 and CD7 loss) was identified in most AITL cases; MFC documented CD10 coexpression on T cells in 10 (83%). Mature T-cell lymphoma showed a more heterogeneous altered immunophenotypic pattern, and 2 cases of PTCL, unspecified, had clear evidence of aberrant CD10 expression on T cells. A small physiologic CD3+/CD4+/CD10+ T-cell population was detected by MFC in all control cases tested (range, 0.28%-4.71%), suggesting that a normal subset of peripheral CD10+ T cells exists. CD10 was a highly sensitive but incompletely specific phenotypic marker for diagnosing AITL; the differential diagnosis of PTCL, unspecified, must be related with traditional histologic features. A small number of CD10+ T cells in reactive lymph nodes suggests that this subpopulation may be the normal counterpart of neoplastic T cells in AITL. The biologic role of CD10+ T cells should be studied further.
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http://dx.doi.org/10.1309/MC7QRGPTV0LRR98XDOI Listing
November 2007

Prognostic value of quantitative analysis of WT1 gene transcripts in adult acute lymphoblastic leukemia.

Haematologica 2006 Feb;91(2):270-1

We quantified Wilm's tumor gene (WT1) using a real time quantitative polymerase chain reaction in 20 adult patients with acute lymphoblastic leukemia at presentation. A WT1 level greater than 906 (median value for the whole series) was a significant predictor of a poor disease-free and overall survival in uni- and multivariate analyses.
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February 2006

Telomere length correlates with histopathogenesis according to the germinal center in mature B-cell lymphoproliferative disorders.

Blood 2004 Jun 26;103(12):4644-9. Epub 2004 Feb 26.

Cattedra di Ematologia, Dipartimento di Medicina ed Oncologia Sperimentale, Università di Torino, Azienda Ospedaliera San Giovanni Battista, Via Genova 3, 10126 Turin, Italy.

In this study we investigated telomere restriction fragment (TRF) length in a panel of mature B-cell lymphoproliferative disorders (MBCLDs) and correlated this parameter with histology and histopathogenesis in relation to the germinal center (GC). We assessed 123 MBCLD samples containing 80% or more tumor cells. TRF length was evaluated by Southern blot analysis using a chemiluminescence-based assay. GC status was assessed through screening for stable and ongoing somatic mutations within the immunoglobulin heavy-chain genes. Median TRF length was 6170 bp (range, 1896-11 200 bp) and did not correlate with patient age or sex. TRF length was greater in diffuse large cell lymphoma, Burkitt lymphoma, and follicular lymphoma (medians: 7789 bp, 9471 bp, and 7383 bp, respectively) than in mantle cell lymphoma and chronic lymphocytic leukemia (medians: 3582 bp and 4346 bp, respectively). GC-derived MBCLDs had the longest telomeres, whereas those arising from GC-inexperienced cells had the shortest (P < 10(-9)). We conclude that (1) TRF length in MBCLD is highly heterogeneous; (2) GC-derived tumors have long telomeres, suggesting that minimal telomere erosion occurs during GC-derived lymphomagenesis; and (3) the short TRF lengths of GC-inexperienced MBCLDs indicates that these neoplasms are good candidates for treatment with telomerase inhibitors, a class of molecules currently the subject of extensive preclinical evaluation.
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http://dx.doi.org/10.1182/blood-2003-12-4412DOI Listing
June 2004