Galectin-3 is a marker of favorable prognosis and a biologically relevant molecule in neuroblastic tumors.

Childhood neuroblastic tumors are characterized by heterogeneous clinical courses, ranging from benign ganglioneuroma (GN) to highly lethal neuroblastoma (NB). Although a refined prognostic evaluation and risk stratification of each tumor patient is becoming increasingly essential to personalize treatment options, currently only few biomolecular markers (essentially MYCN amplification, chromosome 11q status and DNA ploidy) are validated for this purpose in neuroblastic tumors. Here we report that Galectin-3 (Gal-3), a ß-galactoside-binding lectin involved in multiple biological functions that has already acquired diagnostic relevance in specific clinical settings, is variably expressed in most differentiated and less aggressive neuroblastic tumors, such as GN and ganglioneuroblastoma, as well as in a subset of NB cases. Gal-3 expression is associated with the INPC histopathological categorization (P<0.001) and Shimada favorable phenotype (P=0.001), but not with other prognostically relevant features. Importantly, Gal-3 expression was associated with a better 5-year overall survival (P=0.003), and with improved cumulative survival in patient subsets at worse prognosis, such as older age at diagnosis, advanced stages or NB histopathological classification. In vitro, Gal-3 expression and nuclear accumulation accompanied retinoic acid-induced cell differentiation in NB cell lines. Forced Gal-3 overexpression increased phenotypic differentiation and substrate adherence, while inhibiting proliferation. Altogether, these findings suggest that Gal-3 is a biologically relevant player for neuroblastic tumors, whose determination by conventional immunohistochemistry might be used for outcome assessment and patient's risk stratification in the clinical setting.

HIPK2 phosphorylates ΔNp63α and promotes its degradation in response to DNA damage.

Homeodomain-interacting protein kinase 2 (HIPK2) is an emerging player in cell response to genotoxic agents that senses damage intensity and contributes to the cell's choice between cell cycle arrest and apoptosis. Phosphorylation of p53 at S46, an apoptosis-specific p53 posttranslational modification, is the most characterized HIPK2 function in response to lethal doses of ultraviolet (UV), ionizing radiation or different anticancer drugs, such as cisplatin, roscovitine and doxorubicin (DOX). Indeed, like p53, HIPK2 has been shown to contribute to the effectiveness of these treatments. Interestingly, p53-independent mechanisms of HIPK2-induced apoptosis were described for UV and tumor growth factor-ß treatments; however, it is unknown whether these mechanisms are relevant for the responses to anticancer drugs. Because of the importance of the so-called 'p53-independent apoptosis and drug response' in human cancer chemotherapy, we asked whether p53-independent factor(s) might be involved in HIPK2-mediated chemosensitivity. Here, we show that HIPK2 depletion by RNA interference induces resistance to different anticancer drugs even in p53-null cells, suggesting the involvement of HIPK2 targets other than p53 in response to chemotherapy. In particular, we found that HIPK2 phosphorylates and promotes proteasomal degradation of ΔNp63α, a prosurvival ΔN isoform of the p53 family member, p63. Indeed, effective cell response to different genotoxic agents was shown to require phosphorylation-induced proteasomal degradation of ΔNp63α. In DOX-treated cells, we show that HIPK2 depletion interferes with ΔNp63α degradation, and expression of a HIPK2-resistant ΔNp63α-Δ390 mutant induces chemoresistance. We identify T397 as the ΔNp63α residue phosphorylated by HIPK2, and show that the non-phosphorylatable ΔNp63α-T397A mutant is not degraded in the face of either HIPK2 overexpression or DOX treatment. These results indicate ΔNp63α as a novel target of HIPK2 in response to genotoxic drugs.

HIPK2 is involved in cell proliferation and its suppression promotes growth arrest independently of DNA damage.

INTRODUCTION/OBJECTIVES: The serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) is a co-regulator of an increasing number of transcription factors and cofactors involved in DNA damage response and development. We and others have cloned HIPK2 as an interactor of the p53 oncosuppressor, and have studied the role of this interaction in cell response to stress. Nevertheless, our original cloning of HIPK2 as a p53-binding protein, was aimed at discovering partners of p53 involved in cell differentiation and development, still controversial p53 functions. To this aim, we used p53 as bait in yeast two-hybrid screening of a cDNA library from mouse embryo (day 11 postcoitus) when p53 is highly expressed. METHODS AND RESULTS: In this study, we directly explored whether HIPK2 and p53 cooperate in cell differentiation. By measuring HIPK2 expression and activity in skeletal muscle and haemopoietic differentiation, we observed inverse behaviour of HIPK2 and p53--excluding cooperation activity of these two factors in this event. However, by HIPK2 depletion experiments, we showed that drastic HIPK2 suppression promotes cell-cycle arrest by induction of the cyclin-dependent kinase inhibitor p21(Waf-1/Cip-1). HIPK2 activity is independent of DNA damage and takes place in cell-cycle-arresting conditions, such as terminal differentiation, growth factor deprivation, and G(0) resting. CONCLUSIONS: HIPK2 was found to be involved in cell-cycle regulation dependent on p21(Waf-1/Cip-1) and independent of DNA damage.

Frizzled-1 is down-regulated in follicular thyroid tumours and modulates growth and invasiveness.

The mechanisms of follicular thyroid carcinoma (FTC) transformation and progression are not well understood. Previously, we detected LOH at 7q21 in all FTCs examined, indicating that loss of genetic material in that region is a common trait in these lesions. To analyse the effects of LOH on gene expression, we performed an analysis of the mRNA expression levels of six different genes, located at 7q21.1-7q21.3. A total of 23 lesions, including eight follicular hyperplasias (FHs), eight follicular adenomas (FAs), two FTCs and five papillary thyroid carcinomas (PTCs) were analysed. The Frizzled-1 (FZD-1) gene, located at 7q21.13, showed the lowest levels of mRNA expression. Down-regulation of FZD-1 expression was also confirmed in an independent series of 69 follicular neoplastic lesions compared to 25 PTCs, analysed by quantitative RT-PCR. In vitro studies showed that FZD-1 expression was also markedly reduced at both protein and mRNA levels in three FTC-derived cell lines (FRO, WRO and FTC-133), while it was normal in the three PTC-derived cell lines (Ca300, Ca301 and K1) examined. We demonstrated that over-expression of FZD-1 in 3 FTC-derived cells decreased invasiveness and proliferation rate, indicating a possible pathogenetic role. In addition, FZD-1 RNA interference in the PTC-derived cell line K1 increased invasiveness. Our data indicated that FZD-1 is involved in growth of follicular tumours and may be considered as a novel marker of this type of tumour.

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.

Detection and molecular characterisation of thyroid cancer precursor lesions in a specific subset of Hashimoto's thyroiditis.

Hashimoto's thyroiditis (HT) represents the most common cause of hypothyroidism and nonendemic goiter, but its clinical and pathological heterogeneity opens the question if this disease should be more properly considered as a spectrum of different thyroid conditions rather than as a single nosological entity. In this study, we analysed 133 cases of HT for the expression of galectin-3, a lectin molecule involved in malignant transformation, apoptosis and cell cycle control. An unexpected expression of galectin-3 was demonstrated in a subset of HT together with the presence of HBME-1, c-met and cyclin-D1 that are also involved in malignant transformation and deregulated cell growth. Furthermore, a loss of allelic heterozygosity in a specific cancer-related chromosomal region was demonstrated in some HT harbouring galectin-3-positive follicular cells, by using laser capture microdissection. On the basis of the morphological and molecular findings we identified four subsets of HT: (a) HT with classic features of chronic autoimmune thyroiditis; (b) HT associated to hyperplastic/adenomatous lesions; (c) HT harbouring thyroid cancer precursors; (d) HT associated to unequivocal thyroid microcarcinomas. Our findings provide a well-substantiated morphological and molecular demonstration that HT may include a spectrum of different thyroid conditions ranging from chronic autoimmune thyroiditis to thyroiditis triggered by specific immune-response to cancer-related antigens.

[Galectin-3 in differential diagnosis and prognosis of follicular tumors of the thyroid gland]. / Galektin-3 v differentsial'noi diagnostike i prognozirovanii follikuliarnykh opukholei shchitovidnoi zhelezy.

Differential diagnosis between follicular thyroid tumours (FTT) is difficult both at cytological and histological levels. Infiltrative growth is authentic criterion of malignancy. Immunohistochemical reaction (PAP-method) with galectin-3 antibodies was used. The results allow to make differential diagnosis of thyroid tumours before the surgery. Immunohistochemical identification of galectin-3 in the tissue of follicular adenomas with grave dysplasia and follicular carcinoma is an unfavourable prognostic sign.

CD44s adhesive function spontaneous and PMA-inducible CD44 cleavage are regulated at post-translational level in cells of melanocytic lineage.

Adhesion between the CD44s receptor and hyaluronic acid plays an important role in cell migration, tumour growth and progression. Although the alternative splicing of CD44 variant exons represents the principal regulatory mechanism of CD44-mediated functions, CD44v spliced variants are scantily expressed in melanoma cells. For this reason, we have investigated the possibility that post-translational modifications of the CD44 standard receptor could play a pivotal role in regulating CD44-mediated functions in melanoma. Using metabolic inhibitors of N- and O-glycosylation, as well as melanoma transfectants expressing CD44s O-glycosylation site-specific mutants, we performed structural and functional analysis of N- and O-deglycosylated CD44s molecules expressed in melanoma cells. We discovered that complete N- and O-glycosylation is not required by CD44s to be correctly expressed on the melanoma cell surface. Indeed, variably glycosylated and functionally different CD44s molecules were constitutively expressed in primary and metastatic lesions. Furthermore, we observed that changes in N- and O-glycosylation of CD44s could modulate its cleavage. In fact, spontaneous CD44s shedding was dependent on the presence of partial or complete O-glycosylation of four serine-glycine motifs localized in the membrane-proximal CD44 ectodomain. Mutation of these serine residues, as well as an extensive metabolic O-deglycosylation, strongly impaired spontaneous CD44 shedding. Furthermore, an O-glycosylation-independent mechanism of CD44 cleavage has been identified. This alternative mechanism of receptor cleavage is phorbol 12-myristate-13-acetate (PMA) inducible, mediated by metalloproteinase and requires the presence of N-linked sugar residues. Our findings demonstrate that the post-translational modification of CD44s represents the principal regulatory mechanism of CD44s-mediated functions in melanoma.

Association of HLA class I and class II antigen expression and mortality in uveal melanoma.

PURPOSE: Malignant transformation of cells is frequently associated with abnormalities in human leukocyte antigen (HLA) expression. These abnormalities may play a role in the clinical course of the disease, because HLA antigens mediate interactions of tumor cells with T cells and NK cells. Uveal melanoma is a highly malignant tumor of the eye and is characterized by a hematogenic spread to the liver. Little is known about the role of HLA expression in progression of this malignant disease. METHODS: In the present study HLA class I antigen, beta(2)-microglobulin (beta(2)-m), and HLA class II antigen expression was analyzed in primary uveal melanoma lesions by immunoperoxidase staining with monoclonal antibodies of 65 archival clinical samples. The results were correlated with the clinical course of the disease. RESULTS: HLA class I antigen expression and beta(2)-m expression were downregulated in 40 and 35 lesions, respectively. HLA class II antigens were expressed in 30 lesions. Patients with high HLA class I, including beta(2)-m, and HLA class II antigen expression in their primary melanoma lesions had a significantly decreased survival (P = 0.009, P < 0.001, and P = 0.006, respectively). CONCLUSIONS: The findings argue against a major role of cytotoxic T-lymphocyte (CTL)-mediated control of tumor growth in the clinical course of uveal melanoma and are compatible with a potential role of NK-cell-mediated control of hematogenic metastatic spread.

Extraskeletal osteosarcoma in a cat.

A 7-year-old spayed female domestic shorthair cat was referred for evaluation of a localized growing mass on the left flank. Cytologic and histologic findings suggested that the mass was an extraskeletal osteosarcoma. Radiography failed to reveal any association between the lesion and the axial or appendicular skeleton. Because of the large size of the tumor, the cat was treated with carboplatin prior to and after surgery (hemipelvectomy) to ensure that surgical margins were free of neoplastic cells and to prevent systemic dissemination of malignant cells. The tumor has not recurred during a 2-year follow-up period.

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.

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.

Increased expression of insulin-like growth factor I receptor in malignant cells expressing aberrant p53: functional impact.

We investigated the functional impact of p53 on insulin-like growth factor I receptor (IGF-IR) expression in malignant cells. Using the BL-41tsp53-2 cell line, a transfectant carrying temperature-sensitive (ts) p53 and endogenous mutant p53 (codon 248), we demonstrated a drastic down-regulation of plasma membrane-bound IGF-IRs on induction of wild-type p53. However, a similar response was obtained by treatment of BL-41tsp53-2 cells expressing mutant ts p53 with a p53 antisense oligonucleotide. Thus, even if the negative effect of wild-type p53 predominates under a competitive condition, these data indicate that mutant p53 may be important for up-regulation of IGF-IR. To further elucidate this issue, three melanoma cell lines (BE, SK-MEL-5, and SK-MEL-28) that overexpressed p53 were investigated. The BE cell line has a "hot spot" mutation (codon 248) and expresses only codon 248-mutant p53. SK-MEL-28 has a point mutation at codon 145. SK-MEL-5 cells did not exhibit any p53 mutations, but the absence of p21Waf1 expression suggested functionally aberrant p53. Our data suggest that interaction with Mdm-2 may underlie p53 inactivation in these cells. Using p53 antisense oligonucleotides, we demonstrated a substantial down-regulation of cell surface expression of IGF-IR proteins in all melanoma cell lines after 24 h. This was paralleled by decreased tyrosine phosphorylation of IGF-IR and growth arrest, and, subsequently, massive cell death was observed (this was also seen in BL-41tsp53-2 cells with mutant conformation of ts p53). Taken together, our results suggest that up-regulation of IGF-IR as a result of expression of aberrant p53 may be important for the growth and survival of malignant cells.

Seminoma with cutaneous metastases in a dog.

A 10-year-old Great Pyrenees was presented for anorexia and weight loss. On physical examination, the dog was emaciated and showed a large ulcerated lesion on the right lower lip in addition to an enlarged right testicle. Fine-needle aspiration biopsy of the testicle and surgical biopsy of the lip lesion were performed; the histopathological report was consistent with metastatic seminoma. The diagnostic and therapeutic approach in this unusual metastatic seminoma is presented and compared to the previous literature. A multimodality therapy consisting of surgery and chemotherapy is proposed for the clinical management of metastatic seminoma in dogs.

Overexpression of p53 and bcl-2 proteins and the presence of HPV infection are independent events in head and neck cancer.

The expression of p53 and bcl-2 proteins by immunohistochemistry and the identification of human papillomavirus (HPV) infection by a non-isotopic polymerase chain reaction (PCR)based method were investigated in 30 patients with head and neck cancer. Ten cases were HPV-positive (33%), mostly as double or multiple infections by high- or intermediate-risk types. Twenty-one patients were p53-positive (70%), 9/10 with HPV-positive tumours and 12/20 with HPV-negative tumours; this difference was not statistically significant. Only four cases were bcl-2-positive, irrespective of the presence of either HPV or p53. No correlation was found between these biological factors and tumour stage, differentiation grade, and alcohol or tobacco use. Our findings indicate that p53 is involved in the majority of cases, bcl-2 is rare, and high-risk HPV could play a key role, especially in tumours of tongue and tonsil. In conclusion p53 and bcl-2 protein expression and the presence of HPV infection are independent events in these malignancies.

Brachial plexus neuropathy as unusual onset of diffuse neurolymphomatosis.

We present a patient with a large B cell gastric lymphoma in total remission who, after 4 months, developed a fatal progressive peripheral neuropathy with an unusual early involvement of the right brachial plexus. No evidence of lymphoma was found at whole body computed tomography, magnetic resonance imaging of the head, cervical spine and right brachial plexus, bone marrow biopsy or repeated lumbar punctures. The diagnosis of neurolymphomatosis was made only at postmortem examination.