Publications by authors named "Fabrizio Tondat"

8 Publications

  • Page 1 of 1

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0121815PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4386759PMC
April 2016

MGMT promoter methylation in plasma of glioma patients receiving temozolomide.

J Neurooncol 2014 Apr 12;117(2):347-57. Epub 2014 Feb 12.

Unit of Cancer Epidemiology - CERMS, Department of Medical Sciences, University of Turin, Città della Salute e della Scienza Hospital, Via Santena 7, 10126, Turin, Italy,

Promoter methylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene plays a role in cellular response to alkylating agents. In the present study aimed to: (i) evaluate the concordance between MGMT promoter methylation status in tumor tissue and plasma; (ii) monitor MGMT promoter methylation status in plasma taken before and during temozolomide treatment; (iii) explore the value of MGMT promoter methylation status in plasma as a prognostic/predictive biomarker in glioma patients. We enrolled 58 patients with histologically confirmed glioma at different grades of malignancy. All patients underwent surgical resection and temozolomide treatment. Paraffin-embedded tumor tissue was available for 48 patients. Blood samples were collected from all patients before temozolomide treatment (baseline) and at each MRI examination for a 12-month period. MGMT promoter methylation status was assessed in both sample types by real time PCR with a specific probe. The frequency of MGMT promoter methylation was 60.4 % in tumor tissue and 41.38 % in plasma. MGMT promoter methylation status was concordant in the two sample types (Kappa = 0.75, 95 % confidence interval (CI) 0.57-0.93; p value <0.001). Overall and progression-free survival were longer in patients with methylated MGMT promoter. Mortality was higher in patients with unmethylated MGMT promoter, whether in tumor tissue [hazard ratio (HR) 2.21; 95 % CI 0.99-4.95] or plasma (HR 2.19; 95 % CI 1.02-4.68). Progression-free survival was shorter in patients with unmethylated MGMT promoter, whether in tissue (HR 2.30; 95 % CI 1.19-4.45) or plasma (HR 1.77; 95 % CI 0.95-3.30). The cumulative incidence of unmethylated MGMT promoter in plasma at baseline was 58 %, and reached virtually 100 % at 12 months. In conclusion MGMT promoter methylation status in tumor tissue and plasma was highly concordant, and both were associated with longer survival, supporting the role of the detection of methylated MGMT promoter in predicting treatment response. However we suggest caution in using plasma as a surrogate of tumor tissue due to possible false-negative results.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11060-014-1395-4DOI Listing
April 2014

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3109/10428194.2011.574757DOI Listing
September 2011

KIT and PDGFRA mutations and PDGFRA immunostaining in gastrointestinal stromal tumors.

Mol Med Rep 2011 Jan-Feb;4(1):3-8. Epub 2010 Nov 30.

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

In the present study, we investigated the association of PDGFRA and KIT mutations as well as PDGFRA immunohistochemical expression with clinicopathologic features and prognosis in a series of gastrointestinal stromal tumors (GISTs). Tumor DNA from 40 GISTs was sequenced for the presence of mutations in KIT exons 9, 11, 13 and 17, and in PDGFRA exons 12 and 18. Tissue sections were stained with polyclonal anti-PDGFRA antibody. KIT mutations occurred in 26 cases. There were 13 deletions, 6 substitutions, 3 deletion-substitutions, 3 duplications and 1 insertion. Tumors with KIT deletions/insertion were large with a high mitotic index (MI), and were associated with a high rate of symptoms at diagnosis, invasion into adjacent organs, distant metastasis, relapse and a short disease-free survival (DFS). PDGFRA mutations occurred in 6 gastric GISTs. There were 4 deletions and 2 substitutions. Tumors with PDGFRA mutations were small, with a low MI and Ki67 score, and were associated with a very low rate of symptoms at diagnosis, invasion into adjacent organs and distant metastasis. PDGFRA immunopositivity was found in 23 cases: a peculiar 'dotlike' staining was found in 5 out of 6 PDGFRA mutated cases. Patients with positive PDGFRA immunostaining had a longer DFS than those with negative staining. Our data confirm that the type of KIT mutation is associated with various clinicopathologic features of GISTs, and indicate that PDGFRA mutations are associated with rather indolent tumors. PDGFRA immunopositivity reflects PDGFRA mutational status and is associated with a favorable outcome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3892/mmr.2010.399DOI Listing
August 2011

CD4-positive diffuse large B cell lymphoma identified by flow cytometry: two case reports.

Int J Hematol 2010 Jul 25;92(1):198-203. Epub 2010 Jun 25.

Laboratory of Pathology, Department of Pathology, Molinette Hospital, via Santena 7, Turin, Italy.

We report two cases of diffuse large B cell lymphoma (DLBCL), both occurring in the small bowel, which coexpress PAX5, weak or no CD20 and the CD4 antigen. The CD4 was initially identified by flow cytometry and then confirmed by immunohistochemistry. CD4 is a representative marker for helper T-lymphocytes and is present on a subset of thymocytes, peripheral T cells and monocytes or macrophages. Unlike CD2 and CD5, no B cell fractions are known to express CD4. It might be hypothesized that the deregulated control of gene expression in malignant B cells, in particular PAX5, leads to the activation of some silent or repressed genes of T cell differentiation. Although lineage infidelity is described in some B lymphomas, it remains as an uncommon phenomenon; to our knowledge, cases reported here are the first two cases of DLBCL of the gastrointestinal tract coexpressing the CD4 antigen to be described to date.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12185-010-0631-8DOI Listing
July 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-09-2655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2784121PMC
November 2009

Epidermal growth factor receptor gene in primary tumor and metastatic sites from non-small cell lung cancer.

J Thorac Oncol 2009 Jun;4(6):684-8

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

Introduction: The majority of patients with non-small cell lung cancer (NSCLC) develop distant metastases. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are capable of reducing brain and adrenal metastases. However, the EGFR status may be discordant between primary NSCLC and the corresponding metastases.

Methods: Using fluorescence in situ hybridization (FISH) analysis, the EGFR gene status was evaluated in a series of 38 cerebral or adrenal metastases collected from two institutions and in the corresponding primary tumors. Also, EGFR mutational analysis was performed using direct sequencing on the cerebral metastases.

Results: EGFR FISH was positive in 28% of the primary tumors and in 45% of the metastases (p < 0.05). Among the seven cases FISH-positive at the metastatic site but negative in the primary tumor, six were brain metastases, and one was an adrenal metastasis; all were polysomic for chromosome 7, none were amplified. No EGFR mutations have been found in the cerebral metastases.

Conclusion: Because the molecular asset of EGFR may change during the metastatic progression of NSCLC to brain (but not to adrenal), the selection of patients with brain metastasis for specific targeted therapies by EGFR FISH analysis should be performed on metastatic lesions rather than on their corresponding primary tumors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/JTO.0b013e3181a52359DOI Listing
June 2009