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Massi D.,University of Florence | Simi L.,University of Florence | Sensi E.,Laboratory of Molecular Pathology | Baroni G.,University of Florence | And 8 more authors.
Modern Pathology | Year: 2015

Testing for NRAS is now integral part in the assessment of metastatic melanoma patients because there is evidence that NRAS-mutated patients may be sensitive to MEK inhibitors, and RAS mutation is a common mechanism of acquired resistance during treatment with BRAF inhibitors. This study evaluated the sensitivity and specificity of immunohistochemical analysis using an N-Ras (Q61R) antibody to detect the presence of the NRASQ61R mutation in melanoma patients. A total of 98 primary cutaneous melanomas that have undergone examination of NRAS mutation were retrieved from a multicentric database. Formalin-fixed and paraffin-embedded melanoma tissues were analyzed for BRAF and NRAS mutations by independent, blinded observers using both conventional DNA molecular techniques and immunohistochemistry with the novel anti-human N-Ras (Q61R) monoclonal antibody (clone SP174). The antibody showed a sensitivity of 100% (14/14) and a specificity of 100% (83/83) for detecting the presence of an NRASQ61R mutation. Of the NRAS-mutated cases, none of the non-Q61R cases stained positive with the antibody (0/7). There were three cases with discordant NRAS mutational results. Additional molecular analysis confirmed the immunohistochemically obtained NRAS result in all cases, suggesting that a multiple analytical approach can be required to reach the correct sample classification. The reported immunohistochemical method is an accurate, rapid, and cost-effective method for detecting NRASQ61R mutation in melanoma patients, and represents a valuable supplement to traditional mutation testing. If validated in further studies, genetic testing would only be required for immunohistochemistry-negative patients to detect non-Q61R mutations.


PubMed | Friedrich Miescher Institute for Biomedical Research, Laboratory of Molecular Pathology, SM Annunziata Hospital, Papa Giovanni XXIII Hospital and 2 more.
Type: Journal Article | Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc | Year: 2015

Testing for NRAS is now integral part in the assessment of metastatic melanoma patients because there is evidence that NRAS-mutated patients may be sensitive to MEK inhibitors, and RAS mutation is a common mechanism of acquired resistance during treatment with BRAF inhibitors. This study evaluated the sensitivity and specificity of immunohistochemical analysis using an N-Ras (Q61R) antibody to detect the presence of the NRASQ61R mutation in melanoma patients. A total of 98 primary cutaneous melanomas that have undergone examination of NRAS mutation were retrieved from a multicentric database. Formalin-fixed and paraffin-embedded melanoma tissues were analyzed for BRAF and NRAS mutations by independent, blinded observers using both conventional DNA molecular techniques and immunohistochemistry with the novel anti-human N-Ras (Q61R) monoclonal antibody (clone SP174). The antibody showed a sensitivity of 100% (14/14) and a specificity of 100% (83/83) for detecting the presence of an NRASQ61R mutation. Of the NRAS-mutated cases, none of the non-Q61R cases stained positive with the antibody (0/7). There were three cases with discordant NRAS mutational results. Additional molecular analysis confirmed the immunohistochemically obtained NRAS result in all cases, suggesting that a multiple analytical approach can be required to reach the correct sample classification. The reported immunohistochemical method is an accurate, rapid, and cost-effective method for detecting NRASQ61R mutation in melanoma patients, and represents a valuable supplement to traditional mutation testing. If validated in further studies, genetic testing would only be required for immunohistochemistry-negative patients to detect non-Q61R mutations.


Yan Z.-X.,Shanghai JiaoTong University | Yan Z.-X.,Laboratory of Molecular Pathology | Wu L.-L.,Shanghai JiaoTong University | Xue K.,Fudan University | And 11 more authors.
Leukemia | Year: 2014

MicroRNAs (miRs) are involved in tumorigenesis by regulating tumor suppressor genes and/or oncogenes. MiR187 was overexpressed in peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) and associated with high Ki67 expression, elevated lactate dehydrogenase, advanced International Prognostic Index and poor prognosis of patients. In vitro, ectopic expression of miR187 in T-lymphoma cell lines accelerated tumor cell proliferation, whereas treatment with miR187 inhibitor reduced cell growth. MiR187 downregulated tumor suppressor gene disabled homolog-2 (Dab2), decreased the interaction of Dab2 with adapter protein Grb2, resulting in Ras activation, phosphorylation/activation of extracellular signal-regulated kinase (ERK) and AKT, and subsequent stabilization of MYC oncoprotein. MiR187-overexpressing cells were resistant to chemotherapeutic agents like doxorubicin, cyclophosphamide, cisplatin and gemcitabine, but sensitive to the proteasome inhibitor bortezomib. Bortezomib inhibited T-lymphoma cell proliferation by downregulating miR187, dephosphorylating ERK and AKT and degrading MYC. In a murine xenograft model established with subcutaneous injection of Jurkat cells, bortezomib particularly retarded the growth of miR187-overexpressing tumors, consistent with the downregulation of miR187, Ki67 and MYC expression. Collectively, these findings indicated that miR187 was related to tumor progression in PTCL-NOS through modulating Ras-mediated ERK/AKT/MYC axis. Although potentially oncogenic, miR187 indicated the sensitivity of T-lymphoma cells to bortezomib. Cooperatively targeting ERK and AKT could be a promising clinical strategy in treating MYC-driven lymphoid malignancies. © 2014 Macmillan Publishers Limited.


Shi W.-Y.,Shanghai Rui Jin Hospital | Xiao D.,Shanghai Rui Jin Hospital | Wang L.,Shanghai Rui Jin Hospital | Wang L.,Laboratory of Molecular Pathology | And 9 more authors.
Cell Death and Disease | Year: 2012

Adenosine monophosphate-activated protein kinase (AMPK) acts as a major sensor of cellular energy status in cancers and is critically involved in cell sensitivity to anticancer agents. Here, we showed that AMPK was inactivated in lymphoma and related to the upregulation of the mammalian target of rapamycin (mTOR) pathway. AMPK activator metformin potentially inhibited the growth of B- and T-lymphoma cells. Strong antitumor effect was also observed on primary lymphoma cells while sparing normal hematopoiesis ex vivo. Metformin-induced AMPK activation was associated with the inhibition of the mTOR signaling without involving AKT. Moreover, lymphoma cell response to the chemotherapeutic agent doxorubicin and mTOR inhibitor temsirolimus was significantly enhanced when co-treated with metformin. Pharmacologic and molecular knock-down of AMPK attenuated metformin-mediated lymphoma cell growth inhibition and drug sensitization. In vivo, metformin induced AMPK activation, mTOR inhibition and remarkably blocked tumor growth in murine lymphoma xenografts. Of note, metformin was equally effective when given orally. Combined treatment of oral metformin with doxorubicin or temsirolimus triggered lymphoma cell autophagy and functioned more efficiently than either agent alone. Taken together, these data provided first evidence for the growth-inhibitory and drug-sensitizing effect of metformin on lymphoma. Selectively targeting mTOR pathway through AMPK activation may thus represent a promising new strategy to improve treatment of lymphoma patients. © 2012 Macmillan Publishers Limited All rights reserved.


Zhao W.-L.,Shanghai JiaoTong University | Zhao W.-L.,Laboratory of Molecular Pathology
Leukemia | Year: 2010

T-cell malignancies, mainly known as T-cell acute lymphoblastic leukemia (T-ALL) and T-cell non-Hodgkins lymphoma (T-NHL), are aggressive tumors. Although the clinical outcome of the patients has improved dramatically with combination chemotherapy, significant challenges remain, including understanding of the factors that contribute to the malignant behavior of these tumor cells and developing subsequently optimal targeted therapy. Aberrant cell signal transduction is generally involved in tumor progression and drug resistance. This review describes the pathogenetic role of multiple cellular signaling pathways in T-cell malignancies and the potential therapeutic strategies based on the modulation of these key signaling networks. © 2010 Macmillan Publishers Limited All rights reserved.


Dong L.H.,Shanghai JiaoTong University | Cheng S.,Shanghai JiaoTong University | Zheng Z.,Shanghai JiaoTong University | Wang L.,Shanghai JiaoTong University | And 7 more authors.
Journal of Hematology and Oncology | Year: 2013

Background: Burkitt leukemia/lymphoma is a major subtype of aggressive B-cell lymphoma. Biological targeted therapies on this disease need to be further investigated and may help to improve the clinical outcome of the patients. Methods. This study examined the anti-tumor activity of the histone deacetylases (HDAC) inhibitor valproic acid (VPA) combined with the mammalian target of rapamycin (MTOR) inhibitor temsirolimus in Burkitt leukemia/lymphoma cell lines, as well as in primary tumor cells and a murine xenograft model. Results: Co-treatment of VPA and temsirolimus synergistically inhibited the tumor cell growth and triggered the autophagic cell death, with a significant inhibition of MTOR signaling and MYC oncoprotein. Functioned as a class I HDAC inhibitor, VPA potentiated the effect of temsirolimus on autophagy through inhibiting HDAC1. Molecular silencing of HDAC1 using small interfering RNA (siRNA) attenuated VPA-mediated regulation of CDKN1A, CDKN1B and LC3-I/II, regression of tumor cell growth and induction of autophagy. Meanwhile, VPA counteracted temsirolimus-induced AKT activation via HDAC3 inhibition. HDAC3 siRNA abrogated the ability of VPA to modulate AKT phosphorylation, to suppress tumor cell growth and to induce autophagy. Strong antitumor effect was also observed on primary tumor cells while sparing normal hematopoiesis ex vivo. In a murine xenograft model established with subcutaneous injection of Namalwa cells, dual treatment efficiently blocked tumor growth, inhibited MYC and induced in situ autophagy. Conclusions: These findings confirmed the synergistic effect of the HDAC and MTOR inhibitors on Burkitt leukemia/lymphoma, and provided an insight into clinical application of targeting autophagy in treating MYC-associated lymphoid malignancies. © 2013 Dong et al.; licensee BioMed Central Ltd.


Xu P.-P.,Shanghai JiaoTong University | Xu P.-P.,Laboratory of Molecular Pathology | Wang Y.,Shanghai JiaoTong University | Shen Y.,Shanghai JiaoTong University | And 4 more authors.
Medical Oncology | Year: 2012

T/natural killer-cell lymphoma (T/NKCL) is a heterogeneous group of lymphoma and has a higher incidence in Asia than in Western countries. T/NKCL is presented with various clinicopathologic features, and in general, associated with a poor clinical outcome. This study aims to analyze the clinical prognostic factors in patients with T/NKCL. From January 1999 to December 2009, a total of 170 patients with T/NKCL, except mycosis fungoides, were included in this retrospective study. The diagnosis was established according to World Health Organization classification. The clinical characteristics and prognostic factors were evaluated. Of the 170 patients, mainly peripheral T-cell lymphoma-unspecified (65 cases), precursor T-lymphoblastic lymphoma/leukemia (31 cases) and nasal NK/T-cell lymphoma (NKTCL, 19 cases), advanced disease (Ann Arbor stages III-IV) was presented in 68.8% and extranodal involvement was in 71.2% of the patients. According to the international prognostic index (IPI), 77 cases were categorized as high/intermediate or high-risk group. Using the prognostic index for peripheral T-cell lymphoma-unspecified (PIT), 87 cases were classified as group 3 or 4. Most of the initial regimens were CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone)-based chemotherapy (87.6%). Cumulative probability of overall survival at 5 years was 43%, and the median survival time was 44.5 months. Univariate analysis revealed that factors associated with a poor outcome were poor performance status (ECOG > 1) (P = 0.001), advanced disease (P = 0.009), the presence of B symptom (P = 0.001), multiple extranodal involvement (P = 0.005), bone marrow involvement (P = 0.003), elevated lactic dehydrogenase level (P = 0.019), IPI (P < 0.001), PIT (P < 0.001), abnormal white blood cell count (P = 0.016), decreased platelet count (P = 0.005) and serum Epstein-Barr virus (EBV) IgA positivity (P = 0.016). In the multivariate analysis, PIT (P < 0.001; relative risk, 3.221; 95% CI = 2.115-4.907) and EBV serum IgA (P = 0.049; relative risk, 1.901; 95% CI = 1.002-3.606) remained independent factors predictive for overall survival. The PIT may therefore be a useful index for risk stratification in patients with T/NKCL. The serum EBV antibody test could be a simple and quick marker to predict the outcome of the patients. Copyright © Springer Science+Business Media, LLC 2011.


Vescovo V.D.,University of Trento | Cantaloni C.,S Chiara Hospital | Cucino A.,S Chiara Hospital | Girlando S.,Laboratory of Molecular Pathology | And 14 more authors.
American Journal of Surgical Pathology | Year: 2011

Accurate classification of nonsmall cell lung cancers is of paramount clinical relevance, as novel chemotherapeutic agents show different efficacy in adenocarcinomas (ADCs) compared with squamous cell carcinomas (SQCCs). Cyto and histomorphology may sometimes be insufficient for this distinction and immunohistochemistry may improve diagnostic accuracy. The measurement of miR-205 may be another tool for the distinction between ADC and SQCC. The aim of our study was to compare morphologic and immunohistochemical classification with the relative quantification of miR-205 and miR-21 insurgically resected and well-characterized lung tumors (25 ADCs, 24 SQCCs, 1 adenosquamous). The miR-21 relative levels were similar in SQCC and ADC, whereas the miR-205 relative levels were lower in ADC (P<0.0001). The miR-205 sample score value, determined according to Lebanony et al, was higher in ADC (range, 2.8 to 9.08) compared with SQCC (range, -4.17 to 2.445) (P<0.0001). Accordingly, 22 tumors were classified as ADC and 28 tumors as SQCC, although 8 cases (2 SQCCs and 6 ADCs) were in the range of "near cutoff values." Four cases classified as SQCC (according to the sample score method) corresponded to cases classified as ADC on the basis of morphoimmunohistochemical evaluation. In conclusion, the relative quantification of miR-205 and miR-21 seems to be a promising diagnostic tool. However, the molecular approach is still not completely satisfactory as it may misclassify a non-negligible percentage of cases. Therefore, it cannot be used as a substitute of accurate morphologic and immunophenotypical characterization of tumors, but could be used as an adjunctive diagnostic criterion in selected cases. Copyright © 2011 by Lippincott Williams & Wilkins.


Pietronave S.,University of Piemonte Orientale | Forte G.,University of Rome Tor Vergata | Locarno D.,University of Piemonte Orientale | Merlin S.,University of Piemonte Orientale | And 5 more authors.
American Journal of Physiology - Heart and Circulatory Physiology | Year: 2010

Hepatocyte growth factor (HGF), a pleiotropic cytokine with mitogenic, motogenic, morphogenic, and antiapoptotic effects in various cell types, is a cardioprotective growth factor that can counteract the loss of cardiomyocytes usually observed in cardiac diseases. HGF is a quite unstable molecule in its biologically active heterodimeric form. Since all HGF-induced biological responses are mediated by its high-affinity tyrosine kinase receptor (Met/HGF-R) encoded by the Met gene, we asked whether a monoclonal antibody (MAb) that displays receptor full agonist activity could protect cardiac muscle cell lines from hydrogen peroxide-induced apoptosis. We report that the MAb efficiently inhibited hydrogen peroxide-induced cell shrinkage, DNA fragmentation, annexin V positivity, mitochondrial translocation of bax, and caspase activation. The MAb was thus able to counteract apoptosis evaluated by both morphological and biochemical criteria. The agonist activity of the MAb was mediated by Met/HGF-R, since a Met/HGF-R-specific short hairpin RNA (shRNA) inhibited both activation of transduction pathways and motility triggered by MAb DO-24. The protective antiapoptotic effect of MAb DO-24 was dependent on activation of the ras-MAPK Erk1/2 and phosphatidylinositol 3-kinase (PI3-kinase)-Akt transduction pathways, since it was abrogated by treatments with their specific pharmacological inhibitors, PD-98059 and wortmannin. Moreover, the MAb induced a motogenic, but not mitogenic, response in these cells, mimicking in all aspects the natural ligand HGF but displaying a significant higher stability than HGF in culture. This MAb may thus be a valuable substitute for HGF, being more easily available in a biologically active, highly stable, and purified form. Copyright © 2010 the American Physiological Society.


PubMed | Laboratory of Molecular Pathology, Cystic Fibrosis Center and Azienda Ospedaliera Universitaria
Type: | Journal: NeuroImage. Clinical | Year: 2015

Shwachman-Diamond syndrome is a rare recessive genetic disease caused by mutations in SBDS gene, at chromosome 7q11. Phenotypically, the syndrome is characterized by exocrine pancreatic insufficiency, bone marrow dysfunction, skeletal dysplasia and variable cognitive impairments. Structural brain abnormalities (smaller head circumference and decreased brain volume) have also been reported. No correlation studies between brain abnormalities and neuropsychological features have yet been performed. In this study we investigate neuroanatomical findings, neurofunctional pathways and cognitive functioning of Shwachman-Diamond syndrome subjects compared with healthy controls. To be eligible for inclusion, participants were required to have known SBDS mutations on both alleles, no history of cranial trauma or any standard contraindication to magnetic resonance imaging. Appropriate tests were used to assess cognitive functions. The static images were acquired on a 30T magnetic resonance scanner and blood oxygen level-dependent functional magnetic resonance imaging data were collected both during the execution of the Stroop task and at rest. Diffusion tensor imaging was used to assess brain white matter. The Tract-based Spatial Statistics package and probabilistic tractography were used to characterize white matter pathways. Nine participants (5 males), half of all the subjects aged 9-19years included in the Italian Shwachman-Diamond Syndrome Registry, were evaluated and compared with nine healthy subjects, matched for sex and age. The patients performed less well than norms and controls on cognitive tasks (p=0.0002). Overall, cortical thickness was greater in the patients, both in the left (+10%) and in the right (+15%) hemisphere, significantly differently increased in the temporal (left and right, p=0.04), and right parietal (p=0.03) lobes and in Brodmann area 44 (p=0.04) of the right frontal lobe. The greatest increases were observed in the left limbic-anterior cingulate cortex (43%, p<0.0004). Only in Brocas area in the left hemisphere did the patients show a thinner cortical thickness than that of controls (p=0.01). Diffusion tensor imaging showed large, significant difference increases in both fractional anisotropy (+37%, p<0.0001) and mean diffusivity (+35%, p<0.005); the Tract-based Spatial Statistics analysis identified six abnormal clusters of white matter fibres in the fronto-callosal, right fronto-external capsulae, left fronto-parietal, right pontine, temporo-mesial and left anterior-medial-temporal regions. Brain areas activated during the Stroop task and those active during the resting state, are different, fewer and smaller in patients and correlate with worse performance (p=0.002). Cognitive impairment in Shwachman-Diamond syndrome subjects is associated with diffuse brain anomalies in the grey matter (verbal skills with BA44 and BA20 in the right hemisphere; perceptual skills with BA5, 37, 20, 21, 42 in the left hemisphere) and white matter connectivity (verbal skills with alterations in the fronto-occipital fasciculus and with the inferior-longitudinal fasciculus; perceptual skills with the arcuate fasciculus, limbic and ponto-cerebellar fasciculus; memory skills with the arcuate fasciculus; executive functions with the anterior cingulated and arcuate fasciculus).

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