Silvia Uccella

Genomic and expression profiling identifies the B‐cell associated tyrosine kinase Syk as a possible therapeutic target in mantle cell lymphoma

Among B‐cell lymphomas mantle cell lymphoma (MCL) has the worst prognosis. By using a combination of genomic and expression profiling (Affymetrix GeneChip Mapping 10k Xba131 and U133 set), we analysed 26 MCL samples to identify genes relevant to MCL pathogenesis and that could represent new therapeutic targets. Recurrent genomic deletions and gains were detected. Genes were identified as overexpressed in regions of DNA gain on 3q, 6p, 8q, 9q, 16p and 18q, including the cancer genes BCL2 and MYC. Among the transcripts with high correlation between DNA and RNA, we identified SYK, a tyrosine kinase involved in B‐cell receptor signalling. SYK was amplified at DNA level, as validated by fluorescence in situ hybridisation (FISH) analysis, and overexpressed at both RNA and protein levels in the JeKo‐1 cell line. Low‐level amplification, with protein overexpression of Syk was demonstrated by FISH in a small subset of clinical samples. After treatment with low doses of the Syk inhibitor piceatannol, cell proliferation arrest and apoptosis were induced in the cell line overexpressing Syk, while cells expressing low levels of Syk were much less sensitive. A combination of genomic and expression profiling suggested Syk inhibition as a new therapeutic strategy to be explored in lymphomas.

Improved histologic and clinicopathologic criteria for prognostic evaluation of pancreatic endocrine tumors

Currently used histopathologic criteria for the diagnosis of pancreatic endocrine tumors are still under discussion as far as to their capacity to identify prognostically different tumor subsets, which are potentially helpful for patient management. A recently developed TNM staging system and a variety of proposed histologic and clinicopathologic parameters still need to be fully validated. One hundred fifty-five pancreatic endocrine tumors encompassing all the main histologic types and stages, operated with intention to cure and then followed up for a median 126 months, were carefully investigated histologically to identify prognostically informative parameters at univariable, bivariable, and multivariable analysis. Ki67 index, mitotic rate, neuroinvasion with or without vascular, peritumoral or stromal infiltrative patterns, as well as tumor size, and association with endocrine syndromes other than insulinoma proved effective in predicting recurrence and disease-specific death among well-differentiated tumors. Poorly differentiated histologic features, more than 10 mitoses/10 high power fields, and necrosis were helpful in the identification of high-grade cancers with an invariably poor prognosis. The TNM system proved to be highly predictive of patient outcome and easy to combine with histologic and clinicopathologic parameters to classify pancreatic endocrine tumors into groups of increasing malignant potential.

Genome wide DNA-profiling of marginal zone lymphomas identifies subtype-specific lesions with an impact on the clinical outcome

Marginal zone B-cell lymphomas (MZLs) have been divided into 3 distinct subtypes (extranodal MZLs of mucosa-associated lymphoid tissue [MALT] type, nodal MZLs, and splenic MZLs). Nevertheless, the relationship between the subtypes is still unclear. We performed a comprehensive analysis of genomic DNA copy number changes in a very large series of MZL cases with the aim of addressing this question. Samples from 218 MZL patients (25 nodal, 57 MALT, 134 splenic, and 2 not better specified MZLs) were analyzed with the Affymetrix Human Mapping 250K SNP arrays, and the data combined with matched gene expression in 33 of 218 cases. MALT lymphoma presented significantly more frequently gains at 3p, 6p, 18p, and del(6q23) (TNFAIP3/A20), whereas splenic MZLs was associated with del(7q31), del(8p). Nodal MZLs did not show statistically significant differences compared with MALT lymphoma while lacking the splenic MZLs-related 7q losses. Gains of 3q and 18q were common to all 3 subtypes. del(8p) was often present together with del(17p) (TP53). Although del(17p) did not determine a worse outcome and del(8p) was only of borderline significance, the presence of both deletions had a highly significant negative impact on the outcome of splenic MZLs.

BCL2, BCL6, MYC, MALT 1, and BCL10 rearrangements in nodal diffuse large B-cell lymphomas: a multicenter evaluation of a new set of fluorescent in situ hybridization probes and correlation with clinical outcome ☆

Diffuse large B-cell lymphoma is the most common type of non-Hodgkin lymphoma. Although it is a curable disease, fewer than half of patients are cured with conventional chemotherapy. The highly variable outcome reflects a heterogeneous group of tumors, with different genetic abnormalities and responses to therapy. We analyzed 74 cases of diffuse large B-cell lymphoma using interphase fluorescent in situ hybridization with commercially available probes for split-signal targeting BCL-2, BCL-6, MYC, BCL-10, and MALT-1. Gene rearrangements were identified in 48 (65%) of 74 cases. BCL-6 was the most rearranged gene (45%), followed by BCL-2 (21%), BCL-10 (18%), and MYC (16%). No MALT-1 rearrangements were found. When diffuse large B-cell lymphoma cases were subdivided into germinal-center B-cell-like and activated B-cell-like groups, an inverse pattern of BCL-2 and BCL-6 rearrangements was observed. Of interest, the presence of chromosome rearrangements was associated with a worse prognosis. The pattern of cytogenetic abnormalities highlighted the fact not only that diffuse large B-cell lymphoma is a heterogeneous entity but also that even individual cases may contain subclones bearing different chromosomal rearrangements. The relevance and the clinical implication of minor clones showing gene rearrangements are poorly understood; however, this first observation suggests that different rearrangements may be involved in the progression of the disease. The fluorescent in situ hybridization analysis with the panel used in this study is useful to detect the heterogeneity of diffuse large B-cell lymphomas and identify alterations with prognostic implications.

Microallelotyping defines the monoclonal or the polyclonal origin of mixed and collision endocrine-exocrine tumors of the gut.

Mixed endocrine-exocrine tumors of the gut are a heterogeneous group of neoplasms with uncertain histogenesis showing different morphologic and clinical features. The aim of this work is to clarify the histogenesis of these tumors by studying the genetic profile of both the endocrine and exocrine components. We performed an allelotyping analysis of five mixed endocrine-exocrine tumors (two gastric and three colonic) and one rectal collision tumor, using 35 polymorphic microsatellite markers covering a total of six chromosomes, including 3, 5q, 6, 11, 17, and 18. The loss of heterozygosity (LOH) analysis showed concurrent losses of the same allele in both the endocrine and exocrine components in all of the five mixed tumors composed by a poorly differentiated endocrine carcinoma or a well differentiated endocrine carcinoma associated with adenocarcinoma or adenoma. Among these tumors an identical LOH pattern was frequently found on chromosomes 17p, 18q, and 5q. Additional allelic losses restricted to the poorly differentiated endocrine carcinoma were often observed. On the contrary, in the only collision tumor composed by a well differentiated endocrine carcinoma associated with adenocarcinoma, completely different allelotypes between the two components were detected. These findings confirm a close genetic relationship between the two distinct histologic components within mixed endocrine-exocrine tumors, supporting the hypothesis that a monoclonal mechanism of tumorigenesis is the most frequent genetic event in mixed exocrine-endocrine tumors. The clonal divergence observed in the only collision tumor, composed by a well differentiated endocrine carcinoma associated with an adenocarcinoma, confirms the existence of double tumors growing next to each other coincidentally but showing different histogenesis and different tumorigenetic pathways.

CDX2 as a marker of intestinal EC-cells and related well-differentiated endocrine tumors

Gastroenteropancreatic (GEP) endocrine tumors (ETs) are neoplasms showing different hormonal profiles and different clinical and prognostic features, which depend consistently on the site of origin. Histological features and general endocrine markers do not differentiate tumors in relation to their location, making it difficult to establish the site of origin of a GEP ET that has metastasized to the liver or lymph nodes. A site-specific marker would be particularly useful in the examination of small specimens where there is not sufficient material for an extensive study of the hormonal expression. CDX2 is a transcription factor that has been recently proposed as a marker of intestinal adenocarcinomas. Our aim was to evaluate the immunohistochemical expression of CDX2 in normal tissues and in 184 formalin-fixed and paraffin-embedded ETs to verify whether it could be used to identify intestinal ETs with a high degree of sensitivity and specificity. Of these cases, 154 were primary tumors (99 GEP and 55 non-GEP tumors), 101 were well-differentiated endocrine tumors, and 53 were poorly differentiated endocrine carcinomas (PDECs). Of the cases, 30 were metastases from differently located ETs. Nuclear CDX2 immunoreactivity was found in all EC-cells (serotonin-producing cells), in about 10% of G-cells (gastrin-producing cells), in about 30% of GIP-cells (gastric inhibitory peptide cells) and in a few motilin-positive cells of the normal intestinal mucosa, while other gastrointestinal endocrine cell types were CDX2 negative. All midgut EC-cell tumors, their metastases, and two of three pancreatic EC-cell ETs were diffusely and intensely CDX2 positive. The other GEP ETs, their metastases, as well as the non-GEP ETs, were all CDX2 negative, with the exception of four PDECs, five gastrinomas and one pheochromocytoma, which were only focally positive. We conclude that CDX2 may be considered a sensitive and specific marker of midgut EC-cells and EC-cell tumors, and its expression may be useful in the diagnosis of metastases from occult ETs.

Prognostic and biological significance of cytokeratin 19 in pancreatic endocrine tumours.

Aim:  The prognosis of well‐differentiated pancreatic endocrine tumours (PETs) is difficult to establish on a histological basis. The expression of cytokeratin (CK19) has recently been proposed as an indicator of unfavourable outcome. However, this finding still needs to be verified and to be compared with more frequently used prognosticators such as proliferative indices, vascular and/or perineural invasion. The aim of this study was to evaluate the prognostic value of CK19 expression in PETs.

Different Molecular Profiles Characterize Well-Differentiated Endocrine Tumors and Poorly Differentiated Endocrine Carcinomas of the Gastroenteropancreatic Tract

Purpose: The molecular pathogenesis of gastroenteropancreatic endocrine tumors (ETs) is still largely unknown. The purpose of this work was a molecular characterization of 38 gastroenteropancreatic ETs with respect to the primary site and to the morphofunctional profile, pointing out useful diagnostic or prognostic molecular markers. Experimental Design: Twenty-four well-differentiated ETs or carcinomas (WDET/Cs; 11 pancreatic, 3 gastric, and 10 intestinal) and 14 poorly differentiated endocrine carcinomas (1 pancreatic, 6 gastric, and 7 colorectal) were microallelotyped using 38 polymorphic microsatellite markers covering chromosomes 1, 3, 5q, 6, 11, 17, and 18. Results: Regardless of the primary site, a significantly higher percentage of allelic imbalances (AIs) was observed in poorly differentiated endocrine carcinomas than in WDET/Cs (P = 0.012), except for 3 of 8 nonfunctioning pancreatic endocrine tumors and 1 colorectal WDEC, exhibiting multiple AIs on chromosomes 1, 3, 6, and 11. A strong positive correlation between AI percentage and Ki-67 proliferation index was detected considering both the whole series of ETs (P = 0.004) and the group of WDET/Cs alone (P = 0.011). The survival analysis showed a positive correlation between low percentage of AI and longer survival (P = 0.01). No recurrent AIs at specific chromosomal regions were identifiable with respect to the primary site. Conclusions: The malignant progression of endocrine tumors seems to be associated with complex allelotypes and chromosomal instability. Although no specific molecular markers of malignancy can be defined with certainty, the ploidy status and the degree of chromosomal derangements appear to be the most informative genetic factors with prognostic significance.

Chromogranin-A as a serum marker for neuroendocrine tumors: comparison with neuron-specific enolase and correlation with immunohistochemical findings.

Background Chromogranin-A (Cg-A) is a 439-amino-acid protein contained in secretory granules of neuroendocrine cells, in addition to specific hormone peptides or neuropeptides. Since Cg-A is co-released with peptide hormones its serum concentration can be used as a marker of neuroendocrine tumors. Aim Evaluation of the analytical performance of a new IRMA method for Cg-A assay and of the clinical value of serum Cg-A and neuron-specific enolase (NSE) in neuroendocrine tumors. In addition, we compared the diagnostic usefulness of both Cg-A and NSE serum levels and their relationship to tissue expression. Patients and methods Initially we evaluated the analytical performance (intra- and interassay imprecision, dilution test and detection limit) of the Cg-A RIACT method (CIS Bio-International, Gifsur-Yvette, France). We selected 50 patients affected by various histologically confirmed neuroendocrine tumors (NETs): 111In-pentetreotide scan and helical computed tomography were employed to assess tumor extent. Cg-A and NSE were measured before surgery in serum samples of patients and 50 age-matched controls by IRMA methods. After surgery immunohistochemical stains for Cg-A and NSE were performed on surgical specimens of tumor tissue. Results Cg-A levels were significantly higher (p<0.0001) in patients with NETs than in healthy controls and we found a positive correlation between serum and tissue expression (p<0.05). Serum levels of Cg-A were also related to tumor extent (p<0.05) but in some cases we observed significant elevation of serum Cg-A in small, intensely immunoreactive NETs. ROC curve analysis showed better accuracy for serum Cg-A compared to NSE in the diagnosis of NETs, while no significant relationship was found between serum expression and immunostaining for NSE. Discussion Our results confirmed the biological and clinical significance of circulating Cg-A as an expression of granular content in neuroendocrine tissues and supported the complementary usefulness of serum Cg-A in the diagnosis and evaluation of NETs together with imaging modalities.

Detection of gonadotropin-releasing hormone receptor in normal human pituitary cells and pituitary adenomas using immunohistochemistry

Abstract Gonadotropin-releasing hormone (GnRH), which is a well-known regulator of gonadotroph function, has recently been considered to be a paracrine factor involved in the control of somatotroph, lactotroph, and corticotroph cells. GnRH action is initiated by binding to a specific cell surface receptor, the gonadotropin-releasing hormone receptor (GnRHR), which is expressed by follicle-stimulating hormone/luteinizing hormone (FSH/LH) cells. Using in situ hybridization techniques, GnRHR messenger ribonucleic acid (mRNA) has recently been detected in normal human anterior pituitary gland and in various pituitary adenomas, including FSH/LH-cell, growth hormone (GH)-cell, adrenocorticotropic hormone (ACTH)-cell, and null-cell adenomas. However, immunohistochemical studies indicating the specific cell distribution of GnRHR in normal pituitary cells have never been reported. The aim of the present investigation was to evaluate the immunohistochemical expression of GnRHR in different types of normal pituitary cells and related tumors. Using double-label immunohistochemical techniques on formalin-fixed and paraffin-embedded tissues and specific antibodies directed against pituitary hormones and GnRHR, we found GnRHR immunoreactivity not only in FSH/LH cells, but also in GH- and thyroid-stimulating hormone (TSH) cells. GnRHR was detected in FSH/LH-cell, GH-cell, mixed GH- and prolactin (PRL)-cell, and α-subunit (α-SU)/null-cell adenomas. The findings of this study suggest that the interaction between GnRH and GnRHR may play a role in paracrine/autocrine regulation of different types of normal pituitary cells and pituitary adenomas.