Alessandra Franzoni

Nucleophosmin is overexpressed in thyroid tumors

Nucleophosmin (NPM) is a protein that contributes to several cell functions. Depending on the context, it can act as an oncogene or tumor suppressor. No data are available on NPM expression in thyroid cells. In this work, we analyzed both NPM mRNA and protein levels in a series of human thyroid tumor tissues and cell lines. By using immunohistochemistry, NPM overexpression was detected in papillary, follicular, undifferentiated thyroid cancer, and also in follicular benign adenomas, indicating it as an early event during thyroid tumorigenesis. In contrast, various levels of NPM mRNA levels as detected by quantitative RT-PCR were observed in tumor tissues, suggesting a dissociation between protein and transcript expression. The same behavior was observed in the normal thyroid FRTL5 cell lines. In these cells, a positive correlation between NPM protein levels, but not mRNA, and proliferation state was detected. By using thyroid tumor cell lines, we demonstrated that such a post-mRNA regulation may depend on NPM binding to p-Akt, whose levels were found to be increased in the tumor cells, in parallel with reduction of PTEN. In conclusion, our present data demonstrate for the first time that nucleophosmin is overexpressed in thyroid tumors, as an early event of thyroid tumorigenesis. It seems as a result of a dysregulation occurring at protein and not transcriptional level related to an increase of p-Akt levels of transformed thyrocytes.

Prohibitin Is Overexpressed in Papillary Thyroid Carcinomas Bearing the BRAFV600E Mutation

BACKGROUND Prohibitin (PHB) is a multifunctional protein that is localized in different intracellular sites. PHB may exert different roles in tumorigenesis, having either a permissive action on tumor growth or an oncosuppressor role, depending on the cellular context. The objective of this study was to evaluate PHB expression in normal thyroid tissues, thyroid follicular adenomas (FAs), and papillary thyroid carcinomas (PTCs). METHODS PHB expression was analyzed by immunohistochemistry, Western blot, and quantitative reverse transcription polymerase chain reaction (RT-PCR). Transfections in the BCPAP and TPC-1 thyroid cancer cell lines were used to evaluate the PHB promoter activity. RESULTS In terms of protein and mRNA levels, normal tissues from patients with serum thyrotropin (TSH) values >0.8mU/L had PHB levels that were significantly reduced compared to specimens from patients with serum TSH values <0.5mU/L, suggesting that TSH exerts an inhibitory effect on PHB expression. Consistent with this was the finding that the presence of TSH was associated with low PHB levels in normal FRTL5 thyroid cells. Immunohistochemical analysis showed relatively low and high PHB expression in FAs and PTCs, respectively. PHB mRNA and protein overexpression, as assessed by quantitative RT-PCR and Western blot, was noted only in PTCs bearing the BRAF(V600E) mutation. Notably, cell transfection experiments suggested that presence of the BRAF(V600E) mutation may be associated to increase of the PHB promoter activity. CONCLUSIONS PHB is overexpressed in PTCs bearing the BRAF(V600E) mutation. We postulate that the presence of the BRAF(V600E) mutation increases PHB promoter activity and therefore potentially mediates effects of this mutation on the behavior of BRAF(V600E) positive PTCs.

Nucleophosmin Delocalization in Thyroid Tumour Cells

Nucleophosmin (NPM) is a multifunctional nucleolar protein that, depending on the context, can act as oncogene or tumour suppressor. Mutations of the NPM1 gene induce delocalization of NPM in acute myeloid leukaemia. Differently, in solid tumours, only NPM overexpression, but not delocalization, has been so far reported. Here, NPM localization in thyroid tumours was investigated. By using immunohistochemistry, we show increase of NPM cytoplasmic localization in follicular adenomas and papillary carcinomas compared to normal thyroid tissue (p = 0.0125 and <0.0001, respectively). NPM1 mutations commonly found in human leukaemia are not present in thyroid tumours. Immunofluorescence in cultured cell lines was utilized to discriminate between nucleolar and nuclear localization. We show that in thyroid cancer cell lines NPM localizes both in the nucleolus and in nucleus, while in non-tumorigenic thyroid cell lines localizes only in nucleolus. Either presence of the histone deacetylase inhibitor trichostatin A or absence of thyroid-stimulating hormone induces NPM nuclear localization in non-tumorigenic thyroid cell lines.

BAALC overexpression retains its negative prognostic role across all cytogenetic risk groups in acute myeloid leukemia patients

Overexpression of brain and acute leukemia cytoplasmic (BAALC) gene confers poor prognosis in cytogenetically normal acute myeloid leukemia (AML) patients, while less defined is its role in AML with abnormal karyotype. We evaluated the effect of BAALC overexpression on outcome of 175 adult AML patients with different cytogenetic risks. Karyotype was favorable in 13, intermediate in 117 and unfavorable in 45 patients, respectively. Quantitative BAALC expression was determined by real‐time PCR, with cut off value set at 50th percentile. BAALC was overexpressed in 87/175 (50%) patients, without association with cytogenetic status. High BAALC was associated with unmutated NPM (P = 0.006) and CD34 positivity (P < 0.0001). Complete remission (CR) was attained in 111 patients (63%), and was maintained at 5 years in 52 ± 7%. BAALC overexpression had a negative impact on CR achievement (P = 0.04), while did not influence relapse probability. Median survival was 22 months with a 5‐years overall survival (OS) of 35%. Factors with a negative impact on OS were older age (P = 0.0001), unfavorable cytogenetic (P = 0.005), ABCG2 overexpression (P = 0.03) and high BAALC levels (P = 0.01). We observed a worse outcome in patients with high BAALC expression through all cytogenetic risk categories: 5‐years OS was 100% vs. 71% in patients with favorable cytogenetics (P = 0.05), 55% vs. 40% in cases with intermediate karyotype (P = 0.04) and 34% vs. 23% in unfavorable cytogenetic subgroup (P = 0.02). BAALC overexpression identified AML patients with poor prognosis in all cytogenetic groups. Though relatively rare, BAALC positivity in patients with favorable or unfavorable karyotype significantly worsened survival. Am. J. Hematol. 88:848–852, 2013.

Q141K polymorphism of ABCG2 protein is associated with poor prognosis in adult acute myeloid leukemia treated with idarubicin-based chemotherapy

Over-expression of multidrug resistance protein ABCG2 has been associated to chemotherapy failure in solid and hematologic tumors.[1][1]–[3][2] More than 40 ‘synonymous’ and ‘non-synonymous’ single nucleotide polymorphisms (SNPs) of ABCG2 have been identified.[4][3] Among them, the 421C>A

A new germline VHL gene mutation in three patients with apparently sporadic pheochromocytoma

Germline mutations in four genes (RET, VHL, SDHB and SDHD) are detected in about 17% of patients with apparently sporadic pheochromocytoma. Thus, genetic screening of all patients with this disease is suggested for a rational diagnostic approach and management.

Cyclic AMP–Response Element Modulator Inhibits the Promoter Activity of the Sodium Iodide Symporter Gene in Thyroid Cancer Cells

BACKGROUND Comprehension of the regulatory mechanism involved in the sodium iodide symporter (NIS) expression is of great relevance for thyroid cancer. In fact, restoration of NIS expression would be a strategy to treat undifferentiated thyroid cancer. Previous in vitro findings suggest that the cyclic AMP-response element (CRE) modulator (CREM) is involved in control of NIS expression. In this work, we examined the expression of CREM in a series of thyroid cancer tissues and its action on NIS promoter in human thyroid cancer cells. METHODS Expression of mRNA levels for CREM, PAX8 and NIS was measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in 6 normal thyroid tissues, 22 papillary, 12 follicular and 4 anaplastic thyroid cancers. The effect of CREM on transcriptional activity of the NIS promoter was investigated by transient transfection of human thyroid cell lines. RESULTS Compared to normal tissues, NIS and PAX8 mRNA levels were significantly reduced in all types of thyroid cancer. As expected, the maximal decrease was detected in anaplastic thyroid cancer. Conversely, CREM mRNA levels were increased in all types of thyroid cancer, reaching statistical significance for follicular and anaplastic thyroid carcinoma (p=0.0157 and 0.0045, respectively). Transfection experiments showed an inhibitory effect of CREM on NIS promoter activity in various thyroid cancer cell lines. CONCLUSIONS These data demonstrate that CREM expression is increased in thyroid cancer tissue and may play a role in the downregulation of NIS expression in thyroid cancer acting at the transcriptional level.

Unexpected phenotype of a typical NPM1 mutant

NPM1 mutations are the most frequently known gene alterations in cytogenetically normal acute myeloid leukaemia patients (CN-AML), and are considered as positive prognostic factors (Mrozek et al, 2007). All of the NPM1 mutations identified so far in CN-AML patients generate a Nuclear Export Signal (NES) and cause the loss of one or two tryptophan residues. This feature is believed to be necessary for the mutated npm protein to play a role in leukaemia (Falini et al, 2006a; Liso et al, 2008). NPM1 mutations were investigated in 103 AML patients referred to the Haematology Unit of Udine University Hospital. RNA extraction from bone marrow and peripheral blood mononucleated cells was performed by RNABlood Mini Kit (Qiagen, Hilden, Germany); cDNA was generated by standard techniques and amplified to obtain NPM1 polymerase chain reaction (PCR) products, as previously described (Noguera et al, 2002; Pitiot et al, 2007). Exons 11 and 12 of NPM1 were amplified together using the following primers: 5¢-6FAM-GGTGGTTCTCTTCCCAAAGT-3¢ and 5¢-AACATT TATCAAACACGGTA-3¢; the amplified products were analysed by DNA sequencing, using standard procedures. Two novel NPM1 mutations were identified in two patients; one was in exon 11 and the other was in exon 12. Patient 1 was diagnosed with AML in June 2007. Bone marrow aspirate was diagnostic for AML, French-AmericanBritish classification subtype M2. Cytogenetic and molecular analysis showed a normal karyotype, internal tandem duplication (ITD) mutation of FLT3 and overexpression of BAALC. This patient was heterozygous for a double mutation in NPM1, namely an insertion (976_985insGGAATCTCCG) and a deletion (976_977delTC) in exon 11, that caused a frameshift, leading to the creation of a truncated protein (274 aa instead of 294 aa). However, as shown in Fig 1, the mutant allele may be due to a double insertion (i.e. 976_979insGGAA and 982_985insTCCG). The sizing of PCR fragments showed that the two mutations were in cis because of the presence of only two alleles, wild-type (243 bp) and mutant (251 bp) (data not shown). The mutant protein was characterized by a putative NES motif with the VxxxFxxLxI sequence at its C-terminal (Albiero et al, 2007), according to the nesbase version 1.1 programme (http://www.cbs.dtu.dk prediction server). This mutation differed from the other two that have been identified so far in exon 11 (Albiero et al, 2007; Pitiot et al, 2007), but produced a protein with an amino acid sequence identical to the one that has already been described (Albiero et al, 2007), with the exception of two amino acids (Fig 1). Patient 2 was also diagnosed with AML in June 2007. Cytogenetic analysis displayed a normal chromosomal asset. The patient was positive for the FLT3 ITD mutation and overexpression of BAALC. This patient was heterozygous for a novel insertion (1023_1026insAGGC) in exon 12, which determined the loss of W290 (but retention of W288) and created a putative NES motif with the LxxxLxxVxL sequence, which was different to that observed in Patient 1 (Fig 1). This type of NES motif has already been described and, as in our case, has been found in association with W288 (Bolli et al, 2007). Both patients are currently in complete remission with no NPM1 mutation (data not shown). In order to detect the effects of mutations on the npm phenotype, npm localization in mutant blasts was analysed by immunofluorescence. As expected, a sample from an AML patient without the npm mutation was characterized by complete npm nucleolar localization, while blasts from an AML patient carrying the npm type ‘A’ mutation (the most frequent NPM1 mutation in AML-CN patients) showed the typical npm cytoplasmic localization. While the blasts of Patient 2 displayed a nuclear/nucleolar localization, in addition to cytoplasmic npm, the sample from Patient 1 showed a complete nucleolar localization of npm (Fig 2A). The latter phenotype was unexpected on the basis of current models of npm localization (Mrozek et al, 2007). To better investigate the results obtained from the patients’ blasts, we used a transfection approach. Mutant constructs were generated using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) and the plasmid template that was adopted for this purpose was pcDNAHANPM, containing full length NPM1 cDNA, with the HA flag at the N-terminal. HeLa cells were transfected with wild-type and mutant constructs using the calcium-phosphate method. Localization of npm proteins produced by transfected constructs was performed by immunofluorescence using an anti-HA flag antibody. Different types of mutants were generated: (i) Mutant 1 from Patient 1’s mutation; (ii) Mutant 2 from Patient 2’s mutation; (iii) Mutant 3 consisting of mutant 1 reverted to an already described NPM1 mutant (Albiero et al, 2007); and (iv) Mutant A, containing the well-known NPM1 type ‘A’ mutation, used as positive control for npm delocalization (Mrozek et al, 2007). All these mutant constructs were transfected in HeLa cells and npm localization was detected by immunofluorescence (Fig 2B, C). As expected, the wild-type protein had correspondence

Screening of PAX6 gene in Italian congenital aniridia patients revealed four novel mutations

ABSTRACT Purpose: To uncover underlying mutations in a cohort of Italian patients with aniridia, a rare congenital panocular condition with an incidence ranging from 1:64,000 to 1:100,000. The disease may be found isolated or in association with other syndromes characterized by partial or complete absence of the iris and iris hypoplasia. Methods: We analyzed the PAX6 gene in 11 patients with aniridia fulfilling the following inclusion criteria: partial or complete absence of the iris and age < 18 years at the time of diagnosis. DNA sequence analysis was integrated with Multiple Ligation Probe Assay (MLPA) analysis. Results: We identified seven PAX6 mutations, including four novel ones. The majority of mutations lie in the DNA-binding domain and all produce a truncated protein. All tested patients did not have WT1 gene deletions thus excluding the WAGR syndrome. We present the clinical findings in the four cases harboring novel mutations. We were unable to identify mutations in four cases with complete aniridia thus indicating that other gene/s could be involved in the disease. Conclusions: It is important to establish the molecular diagnosis early to avoid repeated and long-term screening for Wilms tumor. Our work further emphasizes that a wide range of ocular phenotypes are associated with loss of function PAX6 mutations. In addition to the possibility of stochastic variations, other genetic variations could play a role as modifier genes, thus giving rise to the observed different ocular phenotypes.

KRAS Codons 12 and 13 Mutation Analysis: A Comparative Study between Direct Sequencing and a New Sensitive Real-Time PCR Assay

KRAS somatic mutations are found in 30–40% of colorectal cancer (CRC). Seven mutations in codons 12 and 13 of KRAS (95% of the observed human mutations) preclude the efficacy of anti-EGFR therapy for the treatment of CRC. Assessment of KRAS mutational status has become a standard procedure in the management of patients with CRC. Technically, KRAS mutation testing can be performed with different methods, characterized by distinct sensitivities and specificities. The present study analyzed KRAS in 182 CRC histological samples by using direct sequencing and a new kit based on a Real-Time Sequence-Specific Primers-PCR technology. The kit allowed to recover as positive 17 samples that were negative or unclear by sequencing, with a recovery rate equal to 13.82%. This study proposes a fast, sensitive, and high-throughput system to identify such seven described mutations of KRAS gene in CRC samples.