Armando Bartolazzi

Application of an immunodiagnostic method for improving preoperative diagnosis of nodular thyroid lesions

BACKGROUND Thyroid cancer is the most common endocrine malignant disease, but preoperative diagnosis remains a challenge. Fine-needle aspiration cytology has greatly improved the clinical management of thyroid nodules, but the preoperative characterisation of follicular lesions is very difficult. Many patients are thus referred to surgery more for diagnosis than for therapeutic necessity. We undertook an international multicentre study to assess the usefulness of immunohistocytochemical staining for two potential markers of malignant thyrocytes. METHODS Expression of galectin-3 and CD44v6 was tested on 1009 thyroid lesions (tissue specimens and cytological cell-blocks) and 226 fresh cytological samples obtained preoperatively by ultrasound-guided fine-needle aspiration of thyroid nodules (prospective analysis). The test used monoclonal antibodies specific for CD44v6 and galectin-3, the indirect avidin-biotin complex immunoperoxidase method, and 3-amino-9-ethyl-carbazole as substrate. FINDINGS The sensitivity, specificity, positive predictive value, and diagnostic accuracy of this test method (for coexpression of the two markers) in the prospective analysis were 88%, 98%, 91%, and 97%, respectively. The sensitivity and specificity of galectin-3 immunodetection alone in discriminating benign from malignant thyroid lesions were more than 99% and 98% respectively, and the positive predictive value and diagnostic accuracy were 92% and 99%. INTERPRETATION The integration of galectin-3 immunostaining with conventional cytomorphological and clinical diagnostic procedures represents a sensitive and reliable diagnostic approach for preoperative identification of thyroid carcinomas. This test method improves the diagnostic accuracy of conventional cytology and provides the molecular basis for a new nosological assignation of the not yet classified thyroid neoplasms of indeterminate malignant behaviour.

Cyclolignans as Inhibitors of the Insulin-Like Growth Factor-1 Receptor and Malignant Cell Growth

The insulin-like growth factor-1 receptor (IGF-1R) plays a pivotal role in transformation, growth, and survival of malignant cells, and has emerged as a general and promising target for cancer treatment. However, no fully selective IGF-1R inhibitors have thus far been found. This is explained by the fact that IGF-1R is highly homologous to the insulin receptor, coinhibition of which may cause diabetic response. The receptors are both tyrosine kinases, and their ATP binding sites are identical, implying that ATP inhibitors cannot discriminate between them. Therefore, the current strategy has been to identify compounds interfering with receptor autophosphorylation at the substrate level. In this study we investigated the effects of cyclolignans and related molecules on IGF-1R activity. We report that certain cyclolignans are potent and selective inhibitors of tyrosine phosphorylation of the IGF-1R. Of particular interest was picropodophyllin (PPP), which is almost nontoxic (LD50 >500 mg/kg in rodents). PPP efficiently blocked IGF-1R activity, reduced pAkt and phosphorylated extracellular signal regulated kinase 1 and 2 (pErk1/2), induced apoptosis in cultured IGF-1R-positive tumor cells, and caused complete tumor regression in xenografted and allografted mice. PPP did not affect the insulin receptor or compete with ATP in an in vitro kinase assay, suggesting that it may inhibit IGF-1R autophosphorylation at the substrate level. This is also in agreement with our molecular model of how the cyclolignans may act on the IGF-1R kinase. Our results open the possibility to use PPP or related compounds with inhibitory effects on IGF-1R as lead compounds in development of anticancer agents.

Galectin-3 and HBME-1 expression in well-differentiated thyroid tumors with follicular architecture of uncertain malignant potential

Well-differentiated encapsulated tumors of the thyroid gland with a follicular architecture may cause diagnostic difficulties. Questionable vascular or capsular penetration may raise the possibility of a follicular carcinoma, while focal nuclear clearing and grooves may suggest a diagnosis of papillary carcinoma. A proposal has recently been made to designate cases showing suggestive but not conclusive morphological evidence of malignancy along these lines as well-differentiated or follicular tumors of uncertain malignant potential. The aim of the present study was to investigate the expression and diagnostic role in well-differentiated or follicular tumors of uncertain malignant potential of Galectin-3 and HBME-1, two malignancy-related markers. A total of 21 tumors fulfilling the criteria of well-differentiated or follicular tumors of uncertain malignant potential were collected from two institutions, including eight cases with questionable vascular and/or capsular invasion and 13 cases with some degree of nuclear changes in the form of clearing, grooves, and/or pseudoinclusions. Tumors in the first group expressed HBME-1 and Galectin-3 focally (less than 25% of tumor cells) in 5/8 and 3/8 cases, respectively, with 62.5% of cases reacting for at least one marker. Cases in the second category expressed HBME-1 and Galectin-3 in 9/13 and 10/13 cases, respectively, with 92.3% of cases having at least one marker expressed. These findings indicate that HBME-1 and Galectin-3 are heterogeneously distributed in these borderline tumors, but that a strong and diffuse expression of HBME-1 and to a lower extent of Galectin-3 was preferentially observed in the group characterized by nuclear changes which were similar but less developed than those of conventional papillary carcinoma. The relationship found between the markers investigated and these nuclear changes suggests that the tumors containing them are pathogenetically linked to papillary carcinomas.

Pivotal Role of KARAP/DAP12 Adaptor Molecule in the Natural Killer Cell–mediated Resistance to Murine Cytomegalovirus Infection

Natural killer (NK) cells are major contributors to early defense against infections. Their effector functions are controlled by a balance between activating and inhibiting signals. To date, however, the involvement of NK cell activating receptors and signaling pathways in the defense against pathogens has not been extensively investigated. In mice, several NK cell activating receptors are coexpressed with and function through the immunoreceptor tyrosine-based activation motif (ITAM)-bearing molecule KARAP/DAP12. Here, we have analyzed the role of KARAP/DAP12 in the early antiviral response to murine cytomegalovirus (MCMV). In KARAP/DAP12 mutant mice bearing a nonfunctional ITAM, we found a considerable increase in viral titers in the spleen (30–40-fold) and in the liver (2–5-fold). These effects were attributed to NK cells. The formation of hepatic inflammatory foci appeared similar in wild-type and mutant mice, but the latter more frequently developed severe hepatitis with large areas of focal necrosis. Moreover, the percentage of hepatic NK cells producing interferon γ was reduced by 56 ± 22% in the absence of a functional KARAP/DAP12. This is the first study that shows a crucial role for a particular activating signaling pathway, in this case the one induced through KARAP/DAP12, in the NK cell–mediated resistance to an infection. Our results are discussed in relation to recent reports demonstrating that innate resistance to MCMV requires the presence of NK cells expressing the KARAP/DAP12-associated receptor Ly49H.

The Loss of the p53 Activator HIPK2 Is Responsible for Galectin-3 Overexpression in Well Differentiated Thyroid Carcinomas

Background Galectin-3 (Gal-3) is an anti-apoptotic molecule involved in thyroid cells transformation. It is specifically overexpressed in thyroid tumour cells and is currently used as a preoperative diagnostic marker of thyroid malignancy. Gal-3 expression is downregulated by wt-p53 at the transcriptional level. In well-differentiated thyroid carcinomas (WDTCs) there is an unexplained paradoxical concomitant expression of Gal-3 and wt-p53. HIPK2 is a co-regulator of different transcription factors, and modulates basic cellular processes mainly through the activation of wt-p53. Since we demonstrated that HIPK2 is involved in p53-mediated Gal-3 downregulation, we asked whether HIPK2 deficiency might be responsible for such paradoxical Gal-3 overexpression in WDTC. Methodology/Principal Findings We analyzed HIPK2 protein and mRNA levels, as well as loss of heterozygosity (LOH) at the HIPK2 locus (7q32-34), in thyroid tissue samples. HIPK2 protein levels were high in all follicular hyperplasias (FHs) analyzed. Conversely, HIPK2 was undetectable in 91.7% of papillary thyroid carcinomas (PTCs) and in 60.0% of follicular thyroid carcinomas (FTCs). HIPK2 mRNA levels were upregulated in FH compared to normal thyroid tissue (NTT), while PTC showed mean HIPK2 mRNA levels lower than FH and, in 61.5% of cases, also lower than NTT. We found LOH at HIPK-2 gene locus in 37.5% of PTCs, 14.3% of FTCs and 18.2% of follicular adenomas. To causally link these data with Gal-3 upregulation, we performed in vitro experiments, using the PTC-derived K1 cells, in which HIPK2 expression was manipulated by RNA interference (RNAi) or plasmid-mediated overexpression. HIPK2 RNAi was associated with Gal-3 upregulation, while HIPK2 overexpression with Gal-3 downregulation. Conclusions/Significance Our results indicate that HIPK2 expression and function are impaired in WDTCs, in particular in PTCs, and that this event explains Gal-3 overexpression typically observed in these types of tumours. Therefore, HIPK2 can be considered as a new tumour suppressor gene for thyroid cancers.

Repression of the Antiapoptotic Molecule Galectin-3 by Homeodomain-Interacting Protein Kinase 2-Activated p53 Is Required for p53-Induced Apoptosis

ABSTRACT Galectin 3 (Gal-3), a member of the β-galactoside binding lectin family, exhibits antiapoptotic functions, and its aberrant expression is involved in various aspects of tumor progression. Here we show that p53-induced apoptosis is associated with transcriptional repression of Gal-3. Previously, it has been reported that phosphorylation of p53 at Ser46 is important for transcription of proapoptotic genes and induction of apoptosis and that homeodomain-interacting protein kinase 2 (HIPK2) is specifically involved in these functions. We show that HIPK2 cooperates with p53 in Gal-3 repression and that this cooperation requires HIPK2 kinase activity. Gene-specific RNA interference demonstrates that HIPK2 is essential for repression of Gal-3 upon induction of p53-dependent apoptosis. Furthermore, expression of a nonrepressible Gal-3 prevents HIPK2- and p53-induced apoptosis. These results reveal a new apoptotic pathway induced by HIPK2-activated p53 and requiring repression of the antiapoptotic factor Gal-3.

Large needle aspiration biopsy and galectin-3 determination in selected thyroid nodules with indeterminate FNA-cytology.

Thyroid fine-needle aspiration biopsy (FNA)-cytology is widely used for the preoperative characterisation of thyroid nodules but this task is difficult for follicular lesions, which often remain undefined. We propose a strategy for improving the preoperative characterisation of selected follicular thyroid proliferations, which is based on large needle aspiration biopsy (LNAB) and galectin-3 expression analysis. Eighty-five thyroid specimens were obtained by LNAB (20-gauge needles) from thyroid nodules with indeterminate follicular FNA-cytology. Aspirated material was processed as a tissue microbiopsy to obtain cell blocks for both cyto/histo-morphological evaluation and galectin-3 expression analysis, by using a purified monoclonal antibody to galectin-3 and a biotin-free immunoperoxidase staining method. Preoperative diagnosis was compared to the final histology. LNAB and cell-block technique allow a preliminary distinction between nodules with a homogeneous microfollicular/trabecular structure, as frequently observed in tumours, and lesions with mixed normo–micro–macrofollicular architecture, as observed in goitre. Furthermore, LNAB provides optimal substrates for galectin-3 expression analysis. Among 85 cases tested, 14 galectin-3-positive cases were discovered preoperatively (11 thyroid cancers and three adenomas confirmed at the final histology), whereas galectin-3-negative cases were 71 (one carcinoma and 70 benign proliferations at the final histology). Sensitivity, specificity and diagnostic accuracy of this integrated morphologic and phenotypic diagnostic approach were 91.6, 97.2 and 95.3%, respectively. In conclusion, LNAB plus galectin-3 expression analysis when applied preoperatively to selected thyroid nodules candidate to surgery can potentially reduce unnecessary thyroid resections.

Cloning and characterization of spliced fusion transcript variants of synovial sarcoma: SYT/SSX4, SYT/SSX4v, and SYT/SSX2v. Possible regulatory role of the fusion gene product in wild type SYT expression

The synovial sarcoma translocation t(X;18)(p11.2; q11.2) results in the fusion of the SYT gene on chromosome 18 to exon 5 of either SSX1 or SSX2 genes on chromosome X. We recently reported that the SSX4 gene is also involved in such a translocation. In the present investigation we cloned and sequenced the full-length cDNA of SYT/SSX1, SYT/SSX2 and SYT/SSX4 from synovial sarcoma tissues. We isolated a novel fusion transcript type variant involving the fusion of SYT with exon 6 of the SSX4 gene (SYT/SSX4v). The SYT/SSX4 and SYT/SSX2 open reading frame also differed from previously reported SYT/SSX sequences by an in-frame addition of 93bp exon located in the junction between exon 7 and 8 of the SYT. This exon is identical to that reported for the murine SYT but has not been previously found in the human transcript. Two SYT transcripts, with and without the 93 bp exon, were co-expressed in mouse NIH3T3 cells, human malignant cells and human testis tissue, but not in human normal fibroblasts. Stable transfection of an SYT/SSX4 expression vector into human and murine cell lines correlated with a down-regulation of SYT transcripts. This was also observed in a synovial sarcoma tumor expressing SYT/SSX4. This suggests that the SYT/SSX fusion gene may regulate SYT expression from the normal allele and as such alter the normal function of SYT.

Galectin-3 and HBME-1 expression in oncocytic cell tumors of the thyroid.

Oncocytic cell tumors (OCTs) of the thyroid include oncocytic cell adenomas (OCAs) and oncocytic cell carcinomas (OCCs). Oncocytic variant of papillary carcinoma (OVPC) has also been described. These tumors may present similar diagnostic problems as their non-oncocytic counterparts, in both conventional histology and fine-needle aspiration biopsies. Several markers were shown able to distinguish benign from malignant thyroid follicular tumors, galectin-3 and HBME-1 being the most promising ones. Controversial data have been reported on their discriminatory potential in the small series of OCTs so far analyzed. We aimed to assess the role of galectin-3 and HBME-1 in a large series of 152 OCTs (including 50 OCAs, 70 OCCs and 32 OVPCs). The expression of PPARγ protein was also evaluated. Using a biotin-free detection system, the sensitivity of galectin-3 was 95.1%, while that for HBME-1 was nearly 53%. The combination of galectin-3 and HBME-1 increased the sensitivity up to 99%. However, for both markers, the specificity was 88%, lower than that reported for non-oncocytic follicular tumors. PPARγ protein overexpression was absent in all OCAs tested and present in only 10% of OCCs, confirming previous reports on the low prevalence of PAX8-PPARγ translocations in OCT and ruling out its role as a potential diagnostic marker of malignancy.

Structural Variability of CD44v Molecules and Reliability of Immunodetection of CD44 Isoforms Using mAbs Specific for CD44 Variant Exon Products

CD44 can be considered structurally and functionally one of the most variable surface molecules. Alternative splicing of variant exons as well as posttranslational modifications of the molecule (differences in glycosylation) generate a rich repertoire of CD44 isoforms (CD44v), some of which seem to play a key role in tumor growth and progression. Immunodetection of CD44 isoforms in vivo, using mAbs specific for CD44 variant exon products, is largely used to identify those CD44 molecules involved in tumor growth and progression and to interfere with CD44-mediated processes. In the present work we demonstrate that the immunoreactivity of some mAbs directed to CD44 exon-specific epitopes can be impaired by the structural variability of the molecule. Our findings demonstrate that (1) specific exon assortment and/or posttranslational modifications of CD44v molecules can mask CD44 exon-specific epitopes; (2) glycosaminoglycan side chains, carried by some CD44v isoforms of high molecular weight, may play a critical role in determining the exact conformation of the molecule, which is necessary for the detection of CD44 variant epitopes by specific mAbs; and (3) in a panel of stable transfectants expressing CD44 N-glycosylation site-specific mutants, generated in the constant region of CD44 extracellular domain, asparagine-isoleucine substitution is sufficient per se to impair the immunoreactivity of several mAbs to pan-CD44. Thus, conformational changes due to the alternative splicing of CD44 variant exons and/or posttranslational modifications of the molecule (different degree of glycosylation), which are cell type-specific, are likely to generate CD44 variants that elude immunodetection. These findings strongly suggest that immunohistochemical analysis of CD44 expression in vitro and in vivo, using mAbs specific for CD44 variant exon epitopes, can potentially be impaired by a large number of false negative results.